Apparatus and method for analyzing living cells

Abstract

An analytical instrument is disclosed having a sensing system, a stage adapted to receive a sample carrier, a motion actuator assembly for positioning the sensing system and / or the stage, a temperature control element, and a signal processing module. A method for analyzing a cell sample using the analytical instrument is also disclosed.

Description

[Technical field]

[0001] Aspects and embodiments disclosed herein relate generally to the measurement of constituents (analytes) of the extracellular medium surrounding living cells.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 306,142, filed February 3, 2022, and U.S. Provisional Patent Application No. 63 / 311,838, filed February 18, 2022, the contents of which are incorporated by reference in their entireties. [Background technology]

[0003] Rates such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are important indicators of mitochondrial respiration and glycolysis, and these measurements provide a systems-level overview of cellular metabolic function in cultured cells and ex vivo samples.

[0004] Despite technological advances in this field, there remains a need to develop new devices and methods for analyzing living cells. Summary of the Invention

[0005] According to one embodiment, an analytical device is provided. The analytical device detects a first analyte, e.g., O 2The sensing system may include an array of sensor units configured to generate a first signal in response to at least one test component proportional to the content and a second signal in response to at least one test component proportional to a second test component, e.g., a pH value, wherein each sensor unit of the array of sensors is positioned to correspond to a corresponding well on a sample carrier comprising an array of wells; a stage configured to receive the sample carrier; a motion actuator assembly configured to position the stage and / or the sensing system (i.e., the stage or the sensing system or both) relative to each other in one or more of the x-axis, z-axis, and y-axis; a sample temperature control element, e.g., a heating element, configured to control the temperature of a sample in at least one well (e.g., each well) of the sample carrier to be within a predetermined amount of a sample in another well of the sample carrier; and a signal processing module operably connected to the sensing system, e.g., configured to receive and / or amplify (i.e., receive or amplify or both) the first signal and the second signal.

[0006] In some embodiments, the sensing system, stage, motion actuator assembly, sample control element, and signal processing module are contained within a housing, hi some embodiments, the housing includes an opening in a sidewall dimensioned to allow passage of the stage and sample carrier.

[0007] In some embodiments, the apparatus further comprises a sample carrier, hi some embodiments, the sample carrier is disposed on the stage.

[0008] In some embodiments, the motion actuator assembly includes at least one axis actuator assembly, for example at least one x-axis actuator assembly.

[0009] In some embodiments, the at least one axis actuator assembly, e.g., the x-axis actuator assembly, is configured to position the stage relative to the sensing system along at least one axis, e.g., the x-axis, e.g., to align at least one well (e.g., each well) of a sample carrier disposed on the stage with a corresponding sensor unit along the x-axis. In some embodiments, the at least one axis actuator assembly, e.g., the x-axis actuator assembly, is configured to position the stage relative to the housing along at least one axis, e.g., the x-axis, e.g., within the housing or external to the housing through an opening.

[0010] In some embodiments, the movement actuator assembly includes at least one y-axis actuator assembly. In some embodiments, the at least one axis actuator assembly, e.g., the y-axis actuator assembly, is configured to position the stage relative to the sensing system along at least one axis, e.g., the y-axis, e.g., to align at least one well (e.g., each well) of a sample carrier disposed on the stage with a corresponding sensor unit along the y-axis.

[0011] In some embodiments, the movement actuator assembly includes at least one z-axis actuator assembly, hi some embodiments, the at least one axis actuator assembly, e.g., the z-axis actuator assembly, is configured to position the sensing system relative to the stage on at least one axis, e.g., the z-axis, and configured to position each sensor unit in fluid communication with a corresponding well.

[0012] In some embodiments, the sensing system is integrated into or on the cartridge.

[0013] In some embodiments, the analytical instrument further comprises a dispensing system including at least one injector configured to dispense at least one target agent into one or more wells of the sample carrier, hi some embodiments, the instrument may further comprise an injector movement actuator assembly positioned to actuate the at least one injector to dispense the at least one target agent across multiple wells of the sample carrier.

[0014] In some embodiments, the dispensing system can include an array of injectors configured to dispense at least one target agent, each injector positioned to correspond to a corresponding well on the sample carrier.

[0015] In some embodiments, the array of injectors includes two or more injectors, for example at least two, three, four, five, six, seven, eight, nine, or ten injectors positioned to correspond to at least two wells (e.g., each well) on the sample carrier.

[0016] In some embodiments, the analytical instrument further comprises a manifold temperature control element, e.g., a heating element, configured to control the temperature of the dispensing system, e.g., the target agent, the sensing system, and / or the cartridge (i.e., the dispensing system or the cartridge or both). In some embodiments, the manifold temperature control element and the sample temperature control element are configured to independently control temperature. In some embodiments, the manifold temperature control element is configured to control the temperature of the target agent and / or the sensing system and / or the cartridge, and the sample temperature control element is configured to control the temperature of the sample in the array of wells of the sample carrier to within 3° C., e.g., within 2° C., within 1° C., within 0.6° C., within 0.5° C., within 0.4° C., within 0.3° C., within 0.2° C., or within 0.1° C., of the temperature of the corresponding target agent and / or the corresponding sensor unit.

[0017] In some embodiments, the sample temperature control elements and / or manifold temperature control elements are configured to control evaporation of the sample within the array of wells to less than 25%, e.g., less than 20%, less than 15%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0018] In some embodiments, at least one axial actuator assembly, e.g., a z-axis actuator assembly, is configured to position the dispensing system relative to the stage on at least one axis, e.g., the z-axis, e.g., configured to position each injector in communication, e.g., fluid communication, with a corresponding well to enable delivery of the target agent to the sample.

[0019] In some embodiments, at least one axial actuator assembly, e.g., a z-axis actuator assembly, is configured to position the dispensing system relative to the stage on at least one axis, e.g., the z-axis, and is configured to position each injector, e.g., to communicate with a corresponding sensor unit of the sensing system.

[0020] In some embodiments, at least one injector or each injector in the array of injectors is configured to dispense the same target agent.

[0021] In some embodiments, each injector is configured to independently dispense a selected targeted agent, e.g., a first injector configured to dispense a first targeted agent, a second injector configured to dispense a second targeted agent, optionally a third injector configured to dispense a third targeted agent, a fourth injector configured to dispense a fourth targeted agent, and an nth injector configured to dispense an nth targeted agent. In some embodiments, a plurality or array of injectors is configured to independently dispense two or more targeted agents into at least one well of a sample carrier.

[0022] In some embodiments, the sensing system and the dispensing system are integrated into or on the cartridge.

[0023] In some embodiments, the analytical instrument further comprises at least one target agent loaded into the dispensing system.

[0024] In some embodiments, the sample temperature control element and / or the manifold temperature control element (i.e., the sample temperature control element or the manifold temperature control element or both) are formed of a temperature conductive material. In some embodiments, the sample temperature control element is fixed to the stage. In some embodiments, the sample temperature control element is configured to be in close proximity or direct contact with the sample carrier. In some embodiments, the manifold temperature control element is configured to be in close proximity or direct contact with the pipetting system.

[0025] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g. each well) of the sample carrier to be between 0°C and 70°C above ambient temperature, for example between 8°C and 20°C above ambient temperature, for example 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 30°C, 40°C, 50°C, 60°C, or 70°C above ambient temperature.

[0026] In some embodiments, the sample temperature control element is configured to maintain the temperature of the sample in at least one well (eg, each well) of the sample carrier within a predetermined range.

[0027] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier so that a sensor signal corresponding to the level, production, or consumption of a target test component, e.g., a first test component or a second test component, does not differ by more than a predetermined amount between two identical or substantially identical samples.

[0028] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier so that a sensor signal corresponding to the level, production, or consumption of a target test component, e.g., a first test component or a second test component, does not differ significantly between two identical or substantially identical samples.

[0029] In some embodiments, the target analyte is O 2 and the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) such that a sensor signal responsive to the level, production, or consumption of a target analyte differs by no more than 10% between two identical or substantially identical samples, e.g., no more than 5%, 3%, 1%, or 0.1%.

[0030] In some embodiments, the target analyte is a analyte proportional to a pH value, and the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) such that a sensor signal corresponding to the level, production, or consumption of the target analyte does not differ by more than 10% between two identical or substantially identical samples, e.g., by more than 5%, 3%, 1%, or 0.1%.

[0031] In some embodiments, the sample temperature control element is configured to control the temperature of a sample in at least one well (e.g. each well) of the sample carrier to be within 3°C, e.g. within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​a sample in another well of the sample carrier.

[0032] In some embodiments, the manifold temperature control element is configured to control the temperature of a sensor to within 3°C of another sensor, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C.

[0033] In some embodiments, the sample temperature control element and the manifold temperature control element are configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier to within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the corresponding sensor.

[0034] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in a first well to be within a predetermined amount of the sample in a second well, where the first well is a border well and the second well is an internal well of the sample carrier, the border well being a well with no other wells disposed between the border well and an edge or border of the sample carrier.

[0035] In some embodiments, the manifold temperature control element is configured to control the temperature of a sensor corresponding to a first well to be within a predetermined amount of a sensor corresponding to a second well, where the first well is a boundary well and the second well is an interior well of the sample carrier, where the boundary well is a well with no other wells disposed between the boundary well and an edge or boundary of the sample carrier.

[0036] In some embodiments, the sample temperature control element and / or the manifold temperature control element are configured to maintain the temperature of the sample in the first well and the second well and / or the sensors corresponding to the first well and the second well within a predetermined range.

[0037] In some embodiments, the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well, such that, e.g., when the samples are analyzed under the same or substantially the same conditions, a sensor signal corresponding to the level, production, or consumption of a target analyte, e.g., a first analyte or a second analyte, does not differ by more than a predetermined amount between each sample, e.g., does not differ significantly between each sample.

[0038] In some embodiments, the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well, such that, e.g., when the samples are analyzed under the same or substantially the same conditions, the sensor signal corresponding to the level, production, or consumption of a target analyte, e.g., a first analyte or a second analyte, does not differ significantly between each sample.

[0039] In some embodiments, the target analyte is O 2 and the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well, such that, for example, when the samples are analyzed under the same or substantially the same conditions, a sensor signal responsive to the level, production, or consumption of a target analyte differs between each sample by no more than 10%, e.g., no more than 5%, 3%, 1%, or 0.1%.

[0040] In some embodiments, the target analyte is a analyte proportional to a pH value, and the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well, such that, e.g., when the samples are analyzed under the same or substantially the same conditions, a sensor signal corresponding to the level, production, or consumption of the target analyte does not differ by more than 10% between each sample, e.g., by more than 5%, 3%, 1%, or 0.1%.

[0041] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in the first well to be within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the sample in the second well, and / or the manifold temperature control element is configured to control the temperature of the sensor corresponding to the first well to be within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the sensor corresponding to the second well.

[0042] In some embodiments, the first well is a boundary well and the second well is an interior well of a sample carrier having one or more wells, for example 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384 or more wells.

[0043] In some embodiments, the sample temperature control element comprises a heating element.

[0044] In some embodiments, the sample temperature control element forms a controlled temperature zone comprising an array of wells of the sample carrier. In some embodiments, the controlled temperature zone does not include the headspace of the housing. In some embodiments, the volume of the controlled temperature zone does not exceed the volume of the sample carrier by more than 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×. In some embodiments, the volume of the controlled temperature zone does not exceed 10% of the volume of the housing, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the temperature of components outside the controlled temperature zone does not change substantially, e.g., does not increase or decrease, upon actuation of the sample temperature control element.

[0045] In some embodiments, the sample temperature control element is configured to bring the temperature of the sample in at least one well (e.g., each well) of the sample carrier within a predetermined range of the target temperature within about 5 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, or 1 minute of activation of the sample temperature control element and / or introduction of the sample carrier into the controlled temperature zone.

[0046] In some embodiments, the sample temperature control element and / or the manifold temperature control element are configured to control the temperature to control, e.g., reduce, limit, or inhibit, the diffusion of gas in the controlled temperature zone. In some embodiments, the sample temperature control element and / or the manifold temperature control element are configured to control the temperature to reduce, limit, or inhibit, the diffusion of gas in the controlled temperature zone such that the composition of the gas in the controlled temperature zone does not change significantly, e.g., by no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20%.

[0047] In some embodiments, the signal processing module is capable of operating at elevated relative humidity (RH), for example at least 75% RH, 85% RH, 95% RH, or 99% RH.

[0048] In some embodiments, the signal processing module is configured to simultaneously receive and amplify the first signal and the second signal.

[0049] In some embodiments, the signal processing module is configured to receive and amplify the first and second signals separately, eg, sequentially.

[0050] In some embodiments, the signal processing module is configured to detect one or more of the first signal and the second signal using time-based detection, such as decay rate, phase shift, or anisotropy detection.

[0051] In some embodiments, the signal processing module is configured to detect one or more of the first signal and the second signal using intensity-based detection, optionally including ratiometric measurement.

[0052] In some embodiments, the signal processing module includes a printed circuit assembly formed from an insulating material having a high dielectric constant.

[0053] In some embodiments, the signal processing module includes a printed circuit assembly having a transimpedance amplifier that includes a grounded guard trace.

[0054] In some embodiments, the signal processing module includes a printed circuit assembly formed from surface mount components, eg, substantially free of secondary hand soldered high gain components.

[0055] In some embodiments, the signal processing module includes a printed circuit assembly including a thermally conductive excitation source, optionally in thermal communication with, e.g., in thermal contact with, a thermal sink. In some embodiments, the sensing system does not include a reference signal detector.

[0056] In some embodiments, the signal processing module is configured to operate with reduced parasitic currents, e.g., reduced interference, dark current, or noise, associated with the detection and / or amplification of at least one of the first and second signals.

[0057] In some embodiments, when the movement actuator assembly is deployed to position each sensor unit in fluid communication with the sample in a corresponding well, the sensing system is constructed and arranged to form a measurement chamber between the sample-facing surface of each sensor unit and the sensor-facing surface of at least one well (e.g., each well), such that sample evaporation, sample run-off, or diffusion of components of the sample, e.g., analyte components, out of the measurement chamber is reduced. In some embodiments, the sample temperature control element is configured to control the temperature of the sample in each measurement chamber.

[0058] In some embodiments, the sensing system includes one or more optical sensors, such as photoluminescence sensors.

[0059] In some embodiments, the sensing system includes one or more electrochemical sensors.

[0060] In some embodiments, the sensing system detects O in the sample. 2 The device is configured to generate a signal in response to a rate of change of the analyte that is proportional to its content, for example, a signal that is proportional to the oxygen consumption rate (OCR) of the sample.

[0061] In some embodiments, the sensing system is configured to generate a signal in response to a rate of change of the analyte that is proportional to the pH value of the sample, e.g., generating a signal proportional to the extracellular acidification rate (ECAR) and / or the proton efflux rate (PER) of the sample.

[0062] In some embodiments, the sensing system is configured to generate a signal in response to one or more electrochemical properties of the sample, such as impedance.

[0063] In some embodiments, the signal processing module is operably connected to a computing network or computer device programmed to calculate one or more of mitochondrial respiration, glycolysis, adenosine triphosphate (ATP) production rate, and a mitochondrial toxicity (mitotox) index value of the sample in response to one or more of the first signal and the second signal. In some embodiments, the signal processing module is operably connected to a cloud-based computing network. In some embodiments, the signal processing module is operably connected to a data storage module that stores historical values ​​of the first signal and the second signal. In some embodiments, the data storage module is a local memory storage device. In some embodiments, the data storage module is a cloud-based memory storage device.

[0064] In some embodiments, the array of sensor units is X s1 Contains X sensor units s1 is equal to or greater than 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, or 384.

[0065] In some embodiments, the array of injectors is X s2 Includes x injectors s2 is equal to or greater than 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384, 768, or 1536.

[0066] In some embodiments, the device has a sensor unit ratio of 1:1 to 1:384, e.g., 1:1, 1:2, 1:3, 1:4, 1:8, 1:12, 1:24, 1:36, 1:48, 1:64, 1:72, 1:96, 1:192, or 1:384. s1 and the injector X s2 The ratio is:

[0067] In some embodiments, the device is between 1:1 and 1:384, e.g., 1:1, 1:2, 1:3, 1:4, 1:8, 1:12, 1:24, 1:36, 1:48, 1:64, 1:72, 1:96, 1:192, or 1:384 injector. s2 and the sensor unit X s1 The ratio is:

[0068] In some embodiments, each sensor unit of the array of sensor units is configured to independently generate one or more of the first signal and the second signal.

[0069] In some embodiments, each sensor unit of the array of sensor units is configured to simultaneously generate a first signal and a second signal.

[0070] In some embodiments, the analytical instrument further comprises a light source, e.g., a fluorescent lamp, a light emitting diode (LED), or a laser, configured to excite the sensor of the sensor unit to generate one or more of the first and second signals. In some embodiments, the light source is configured to generate a reference signal, and fluctuations in intensity from the light source are corrected in proportion to the drift by monitoring the reference signal generated by the light source. In some embodiments, the light source is positioned on a thermally conductive printed circuit assembly configured to minimize drift from the light source, and optionally, the thermally conductive printed circuit assembly is formed of a material configured to minimize drift caused by thermally induced fluctuations from the light source by at least 20%, e.g., at least 15%, 10%, 5%, or 1%.

[0071] In some embodiments, the analytical instrument further comprises an electric motor configured to actuate a motion actuator assembly, for example one or more of the x-axis actuator assembly, the z-axis actuator assembly, and the y-axis actuator assembly.

[0072] In some embodiments, the analytical instrument further comprises a stall sensing module programmed to generate a notification signal, and optionally pause the protocol, e.g., stop motor movement, if a given protocol step is not completed within a given time interval.

[0073] In some embodiments, the analytical instrument further comprises a proximity sensor configured to generate a notification signal, and optionally pause the protocol, when a component is positioned within a predetermined distance from another component, e.g. when a sensor unit is positioned within a predetermined distance from a corresponding well of a sample carrier.

[0074] In some embodiments, the analytical instrument further comprises a proximity sensor configured to generate a notification signal, and optionally pause the protocol, when the opening in the side wall of the housing is ajar and / or when external light is detected within the housing.

[0075] In some embodiments, the analytical instrument has an OCR detection range of 2000 pmol / min to 0.01 pmol / min, such as 700 pmol / min to 0.01 pmol / min, for example 50 pmol / min to 0.01 pmol / min.

[0076] In some embodiments, the analytical device has a lower OCR detection limit of less than 50 pmol / min, e.g., less than 40 pmol / min, less than 30 pmol / min, less than 20 pmol / min, less than 10 pmol / min, less than 5 pmol / min, less than 3 pmol / min, less than 1 pmol / min, less than 0.1 pmol / min, or less than 0.01 pmol / min.

[0077] In some embodiments, the analytical instrument further comprises an optical module positioned to image or scan one or more samples in the array of wells of the sample carrier, hi some embodiments, the optical module is operably connected to a computer, optionally configured to display and / or record images or scans of the samples in real time.

[0078] In some embodiments, the analytical instrument further comprises a transfer module formed of multiplexed fiber optic material configured to transfer optical signals from the array of sensor units to the signal processing module. In some embodiments, the transfer module is configured to transfer one or more of the excitation optical signal, the reference optical signal, and the luminescence optical signal. In some embodiments, the transfer module is configured to directly interface with one or more sensor units. In some embodiments, the sensing system includes a homogenized fiber optic waveguide optically connected to the transfer module, optionally configured to distribute light uniformly to the one or more sensor units.

[0079] In some embodiments, the instrument further comprises an environmental control module configured to control the environment of the sample in at least one well (e.g., each well) of the sample carrier, e.g., configured to control the environmental gas and / or relative humidity (RH). In some embodiments, the environmental control module controls the N 2 Concentration, O 2 concentration, and CO 2 In some embodiments, the environmental control module is configured to control one or more of the concentrations of a gas, such as N, in fluid connection with the sample carrier. 2 , O 2 , and CO 2 In some embodiments, the environmental control module forms a controlled environmental zone comprising an array of wells in the sample carrier. In some embodiments, the controlled environmental zone is formed within a sealed container, e.g., a hermetically sealed container.

[0080] In some embodiments, the device is configured for use in a gas-controlled environment.

[0081] According to another aspect, there is provided a method of using an analytical instrument comprising loading into the analytical instrument a sample carrier containing one or more cell samples, each sample disposed in a corresponding well of the sample carrier.

[0082] In some embodiments, the cellular sample comprises live cells.In some embodiments, loading the sample carrier comprises fixing the sample carrier on a stage.

[0083] According to yet another aspect, a method for analyzing a cell sample is provided, the method including providing an analytical instrument, loading into the analytical instrument a sample carrier containing one or more cell samples, each sample disposed in a corresponding well of the sample carrier, and detecting a first analyte, e.g., O. 2 The method includes obtaining a first plurality of values ​​from a signal responsive to at least one test component proportional to its content, each signal being generated by a corresponding sensor unit of the sensing system; optionally obtaining a second plurality of values ​​from a signal responsive to at least one test component proportional to a second test component, e.g., a pH value, each signal being generated by a corresponding sensor unit of the sensing system; processing the first plurality of values; and, optionally, processing the second plurality of values.

[0084] In some embodiments, the method further comprises controlling the temperature of the sample in at least one well (eg, each well) of the sample carrier to be within a predetermined amount of the sample in another well of the sample carrier.

[0085] In some embodiments, the sample carrier is loaded into a controlled temperature zone and / or controlling the temperature of the sample comprises forming a controlled temperature zone.

[0086] In some embodiments, the method further comprises controlling a temperature of the sensing system.

[0087] In some embodiments, the method further comprises dispensing a target agent into each sample in the array of wells of the sample carrier. In some embodiments, the method further comprises loading the target agent into a dispensing system of the analytical instrument. In some embodiments, the method further comprises controlling a temperature of the target agent.

[0088] In some embodiments, the same sample is present in at least one well (eg, each well) of the array of wells of the sample carrier.

[0089] In some embodiments, a first sample is present in a first well of the array of wells of the sample carrier and a second sample is present in a second well of the array of wells of the sample carrier, hi some embodiments, the first sample is a test sample and the second sample is a control.

[0090] In some embodiments, the sample comprises living cells.

[0091] In some embodiments, the sample comprises one or more of free cells, cell constructs, free tissues, tissue constructs, cell organelles, enzymes, cell products or by-products, and conditioned medium.

[0092] In some embodiments, the sample comprises mammalian cells or tissue. In some embodiments, the sample comprises non-mammalian cells or tissue. In some embodiments, the sample comprises a single-cell organism, e.g., a microorganism. In some embodiments, the sample comprises whole animal model tissue, e.g., zebrafish, C. elegans, Drosophila. In some embodiments, the sample comprises whole plant model tissue or whole plant model cells.

[0093] In some embodiments, the first test component is O 2 Proportional to the content.

[0094] In some embodiments, the second test component is proportional to the pH value.

[0095] In some embodiments, the first value and the second value are obtained independently.

[0096] In some embodiments, the first value and the second value are obtained simultaneously.

[0097] In some embodiments, the method further comprises obtaining an image or scan of the sample during or after the analysis.

[0098] In some embodiments, the method further comprises measuring one or more electrochemical properties of the sample, such as impedance, during or after the analysis.

[0099] In some embodiments, the method further comprises obtaining or calculating a mitochondrial toxicity (mitotox) index value for the sample during or after the analysis.

[0100] In some embodiments, the method further comprises controlling the environment of the sample in at least one well (e.g., each well) of the sample carrier, e.g., controlling the environmental gas and / or relative humidity (RH). In some embodiments, controlling the environment comprises controlling the N 2 Concentration, O 2 concentration, and CO 2 The present invention relates to a method for controlling one or more of the concentrations of the

[0101] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like reference numeral. For clarity, every component may not be labeled in every drawing. [Brief description of the drawings]

[0102] [Figure 1] FIG. 1 is a front perspective view of an apparatus for analyzing live cells, according to one embodiment. [Diagram 2] FIG. 1 is a rear perspective view of an apparatus for analyzing live cells, according to one embodiment. [Diagram 3] FIG. 1 is a side view of an apparatus for analyzing live cells, according to one embodiment. [Figure 4] FIG. 1 is a top view of an apparatus for analyzing live cells, according to one embodiment. [Diagram 5] FIG. 2 is a side view of selected components of an apparatus for analyzing live cells, according to one embodiment. [Figure 6] FIG. 1 is a schematic diagram of a system for analyzing live cells, according to one embodiment. [Figure 7] FIG. 1 is a schematic diagram of a system for analyzing live cells, according to one embodiment. [Figure 8] 1 is a schematic diagram of selected components of a system for analyzing live cells, according to one embodiment. [Figure 9] 1 is a schematic diagram of selected components of an apparatus for analyzing live cells, according to one embodiment. [Figure 10A] FIG. 10 is a graph showing the oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 10A is an OCR reading from an analytical instrument disclosed herein and a comparative analytical instrument, showing improved and stable performance from 0 minutes to 15 minutes by the analytical instrument compared to the comparative instrument. [Figure 10B] FIG. 10B is a graph showing the oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein according to one embodiment, in particular FIG. 10B is an OCR reading from an analytical instrument disclosed herein and a comparative analytical instrument, showing improved and stable performance from 0 minutes to 15 minutes by the present analytical instrument compared to the comparative instrument. [Figure 10C]10A-10C are graphs showing the oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 10C is the ECAR reading from an analytical instrument disclosed herein and a comparative analytical instrument. [Figure 10D] FIG. 10C is a graph showing the oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 10D is the ECAR readings from the analytical instrument disclosed herein and a comparative analytical instrument. [Figure 11A] 11A-11C are graphs showing the oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 11A is an OCR reading from an analytical instrument disclosed herein and a comparative analytical instrument, illustrating results with significantly lower variability, particularly with metformin-treated cells (bottom line). [Figure 11B] FIG. 11B is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein according to one embodiment; in particular, FIG. 11B is an OCR reading from an analytical instrument disclosed herein and a comparative analytical instrument, illustrating results with significantly lower variability, particularly with metformin-treated cells (bottom line). [Figure 12] 1 is a table showing the average evaporation of material (e.g., culture medium) contained in a sample carrier analyzed by the method disclosed herein, according to one embodiment. [Figure 13A] This is a comparison diagram of spheroids. [Figure 13B] FIG. 1 is a comparison diagram of spheroids. [Figure 14] FIG. 1 is a block diagram illustrating a multi-detection system according to an embodiment. [Figure 15] FIG. 1 is a block diagram illustrating a multi-detection system according to an embodiment. [Figure 16] FIG. 1 is a block diagram illustrating a multi-detection system according to an embodiment. [Figure 17] FIG. 1 is a block diagram illustrating a multi-detection system according to an embodiment. [Figure 18] FIG. 2 illustrates a spinning disk according to an embodiment. [Figure 19] FIG. 2 illustrates a confocal disk imaging module according to an embodiment. [Figure 20] FIG. 2 illustrates a disc changing mechanism and a disc focusing mechanism according to an embodiment. [Figure 21] FIG. 2 is a diagram of a non-imaging analysis subsystem according to an embodiment. [Figure 22] FIG. 2 illustrates an injection subsystem according to an embodiment. [Figure 23] FIG. 1 illustrates a multi-detection system according to an embodiment. [Figure 24A] FIG. 2 is a perspective view of an environmental control subsystem according to an embodiment. [Figure 24B] FIG. 2 is a rear view illustrating an environmental control subsystem according to an embodiment. [Figure 24C] FIG. 2 is a front view illustrating an environmental control subsystem according to an embodiment. [Diagram 25] FIG. 2 is a functional block diagram illustrating control of device modalities according to an embodiment. [Figure 26] 1 is a flow diagram of a method for controlling a multi-detection system in accordance with an exemplary embodiment. [Figure 27A] FIG. 1 is a first diagram showing an immersion objective lens according to an embodiment. [Figure 27B] FIG. 2 is a second diagram showing an immersion objective lens according to an embodiment. [Figure 28] FIG. 1 illustrates a fluid pump system according to an embodiment. [Figure 29] FIG. 2 illustrates an objective lens combination according to an embodiment. [Figure 30A] FIG. 1 is a perspective view showing an immersion objective lens according to a first embodiment. [Figure 30B] FIG. 1 is a top view showing an immersion objective lens according to a first embodiment. [Figure 30C]FIG. 30C is a first cross-sectional view taken along line AA in FIG. 30B, showing the immersion objective according to the first embodiment with a liquid bulb provided thereon; [Figure 30D] A second cross-sectional view taken along the line AA in FIG. 30B shows the immersion objective according to the first embodiment with a sample carrier (eg a microplate) provided thereon. [Figure 31A] FIG. 4 is a top view showing an immersion objective lens according to a second embodiment. [Figure 31B] FIG. 31B is a first cross-sectional view taken along the line BB in FIG. 31A, showing the immersion objective according to the second embodiment provided with a liquid sphere. [Figure 31C] A second cross-sectional view taken along the line BB in FIG. 31A, showing an immersion objective according to a second embodiment with a sample carrier (eg, a microplate) provided thereon. [Figure 32A] FIG. 11 is a top view showing an immersion objective lens according to a third embodiment. [Figure 32B] FIG. 32B is a first cross-sectional view taken along the line CC in FIG. 32A, showing the immersion objective according to the third embodiment provided with a liquid sphere. [Figure 32C] FIG. 32B is a second cross-sectional view taken along the line CC in FIG. 32A, showing an immersion objective according to a third embodiment with a sample carrier (eg, a microplate) provided thereon. [Figure 33A] FIG. 13 is a top view showing an immersion objective lens according to a fourth embodiment. [Figure 33B] FIG. 33B is a first cross-sectional view taken along the line DD in FIG. 33A, showing the immersion objective according to the fourth embodiment provided with a liquid sphere. [Figure 33C] FIG. 33B is a second cross-sectional view taken along the line DD in FIG. 33A, showing an immersion objective according to a fourth embodiment with a sample carrier (eg, a microplate) provided thereon. [Figure 34A]FIG. 1 is a first diagram of a multi-detection system of a laser point-scanning confocal modality according to an embodiment. [Figure 34B] FIG. 2 is a second diagram of a multi-detection system for wide-field or spinning disk confocal modality according to an embodiment. [Diagram 35] FIG. 2 is a diagram of an exemplary user interface according to an embodiment. [Diagram 36] FIG. 2 is a schematic diagram illustrating a cross-sectional view of a transfer module, according to one embodiment. [Figure 37] FIG. 1 is a drawing of a side view of a transfer module, according to one embodiment. [Figure 38] 1 includes a graph illustrating the accuracy of measurements made with an instrument having a thermal conductivity excitation source, according to one embodiment. [Figure 39A] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 39B] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 39C] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 40A] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Figure 40B] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Figure 40C] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Diagram 41] 1 is an exemplary MTI detection graph. [Diagram 42] 1 is a graph showing dynamic dose-response OCR data for three test compounds. [Diagram 43] 1 is a graph showing Z' values ​​achieved using the MitoTox assay and MTI-based analysis, according to one embodiment. [Diagram 44] FIG. 1 is a schematic diagram of a 96-well plate with shaded circles indicating which wells were plated at each cell density for use in measuring oxygen consumption rates of live cells analyzed by the methods disclosed herein, according to one embodiment. [Figure 45A]Graphs showing oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 45A shows three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), notably showing results with significantly less variability in lower OCR rates when cells are plated at lower densities or after cells are treated with respiratory inhibitors (oligomycin or rotenone + antimycin A) compared to the comparative instrument. [Figure 45B] FIG. 45B is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein according to one embodiment; in particular, FIG. 45B is three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), in particular, the analytical instrument shows results with significantly less variability in lower OCR rates when cells are plated at lower densities or after cells are treated with respiratory inhibitors (oligomycin or rotenone + antimycin A) compared to the comparative instrument. [Figure 45C] FIG. 4C is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein according to one embodiment; in particular, FIG. 4CA shows three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), in particular, the analytical instrument shows results with significantly less variability in lower OCR rates when cells are plated at lower densities or after cells are treated with respiratory inhibitors (oligomycin or rotenone + antimycin A) compared to the comparative instrument. [Figure 46A] Graphs of the same data showing basal oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein, according to one embodiment; in particular, FIG. 46A shows basal OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), notably showing that compared to the comparative instrument, the analytical instrument shows results with significantly less variability in the lower OCR rate when cells are plated at low density. [Figure 46B] FIG. 46B is a graph of the same data showing basal oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment; in particular, FIG. 46B shows basal OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), notably showing that compared to the comparative instrument, the analytical instrument shows results with significantly less variability in the lower OCR rate when cells are plated at low density. [Figure 47] FIG. 46 is a graph showing the standard deviation of basal oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment, over three experimental replicates. In particular, FIG. 46 shows the standard deviation of basal OCR readings from an analytical instrument disclosed herein (grey) and a comparative analytical instrument (black), with the present analytical instrument showing results with significantly less variability compared to the comparative instrument. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] Bioenergetic capacity is the driving force behind cellular biological processes, and cellular metabolism is a central indicator of biological function and cellular health. The devices and methods disclosed herein can be used to measure cellular metabolic pathways using high-throughput screening techniques. Thus, the devices and methods disclosed herein can be employed to determine and / or quantify key indicators of healthy cellular function, prediction of cellular performance in in vitro disease models, drug / compound / substance discovery through modulation of metabolic targets, signaling, and substrates, with the aim of better understanding disease states and enabling insights into appropriate therapeutic approaches to alter disease states, healthy phenotypes, and / or optimize and enhance cellular performance.

[0104] The devices and methods disclosed herein can be employed to measure two major metabolic pathways, mitochondrial respiration and glycolysis, in real time for living cells to provide a functional kinetic measurement of the bioenergetic capacity of cells.

[0105] The devices and methods disclosed herein can be provided for testing disease models and key cellular processes, including activation, proliferation, differentiation, cell death, cell homeostasis, and / or disease progression, to facilitate therapeutic discovery by uncovering and validating targets for potential therapeutics / compounds / substances, and to optimize the engineering and manufacturing of cell therapies.

[0106] In one embodiment, mitochondrial respiration, glycolytic activity and / or metabolic poise are temporal measurements of cellular activity independent of the medium / buffer that surrounds the cells. Creating microchambers allows highly sensitive measurements of cellular activity to be detected. Changes in mitochondrial respiration and / or glycolytic activity of cells can be correlated with changes in O2 levels in the immediate environment surrounding the cells. 2 , CO 2 This results in minute changes in lactate in real time in the immediate environment, which are detected by the device via OCR, ECAR, and / or PER measurements.

[0107] In another embodiment, mitochondrial respiration and / or glycolysis activity is a temporal measurement of cellular activity that is affected by the medium / buffer surrounding the cells, by the addition of gases, therapeutic targets, or drugs that affect cellular activity, such as an ATP synthase inhibitor, a mitochondrial uncoupling agent, or an ETC inhibitor, to the medium that affects the cells.

[0108] In particular, the devices and methods disclosed herein can be employed to measure oxygen consumption rate (OCR), extracellular acidification rate (ECAR), proton efflux rate (PER), adenosine triphosphate (ATP) production rate, and other parameters of multiple cell samples in a sample carrier (e.g., a multi-well plate). OCR and ECAR or PER can be used to determine mitochondrial respiration and glycolysis as well as ATP production rate. Measurements obtainable by the devices and methods disclosed herein can provide a comprehensive overview of cellular metabolic function in cultured cell samples and ex vivo samples.

[0109] It should be noted that the cell samples described herein can include free cells, cell constructs, free tissues, tissue constructs samples. The cell samples can be or include organelles, enzymes, cell products or by-products, and / or conditioned media. Parameters for each cell sample (each well) can be independently and selectively measured. In certain embodiments, live cell samples can be tested, for example, without significant loss of cell viability. The devices and methods described herein can provide lower dissolved oxygen or OCR detection limits, higher precision consistency, improved temperature control, and improved automation compared to conventional devices and methods.

[0110] Conventional systems are susceptible to moisture and contamination caused by factors such as laboratory environment, storage, and manufacturing processes, tend to experience motion errors over time including inconsistencies in debris motion / accumulation, and are susceptible to evaporation, end-well temperature gradients, and long warm-up times caused by environmental heating techniques. The devices and methods disclosed herein include components that overcome these shortcomings of conventional systems, resulting in improved measurement performance and, surprisingly, lower O 2 This provides a lower detection limit and improved measurement precision.

[0111] The combination of hardware and analytical software provided in the instruments disclosed herein allows for real-time monitoring of live cells using rare cells, ex vivo cells, and genetically engineered cells in fields such as immunology and disease, and builds better disease models. The enhancements disclosed herein improve measurement performance. These enhancements generally facilitate identifying novel drug / compound / substance targets, validating target effects on cell function, optimizing disease models, and determining the safety and anti-tumor potential of T cell therapy drugs / compounds / substances moving from the laboratory to pharmaceutical therapeutic development and toxicology programs.

[0112] The instruments disclosed herein can provide better accuracy at low oxygen consumption rates (OCR), allowing analysts to confidently interrogate more immune cell types, even those with reduced bioenergetics.

[0113] The devices and methods disclosed herein provide the ability to analyze live cells over an extended temperature range, for example, temperature control elements and controlled temperature zones smaller than the headspace of the housing, which represent improvements over conventional devices.

[0114] The devices and methods disclosed herein provide greater uniformity in the heating of temperature control elements, which can improve cell biology sensing with consistent temperature and instrument sensors, and reduce whole-body edge effects.

[0115] The devices and methods disclosed herein may provide temperature control with a faster start up time than conventional devices.

[0116] The devices and methods disclosed herein include electro-optical substrates capable of functioning at humidity levels as high as 95%. Previous equipment often performs suboptimally at 70%-80% humidity. Thus, the present equipment can be shipped, stored, or used in areas with high humidity.

[0117] The devices and methods disclosed herein provide improved performance and detection at low levels of OCR that previously appeared as noise, allowing the analysis of damaged or deteriorated immune cells, thereby expanding the types of cells that can be analyzed by the instrument.

[0118] In some embodiments, the devices described herein measure OCR and ECAR / PER of live cells in a 96-well format. Without wishing to be bound by theory, it is believed that in some embodiments, the devices described herein feature better OCR accuracy at lower rates, verified functional performance and reproducibility specifications, optimized temperature control, and are automatable.

[0119] [Definition] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0120] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0121] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0122] "Acquire" or "obtaining" as the term is used herein refers to taking possession of a physical entity or value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" that physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another person or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a material, separating or purifying a material, combining two or more separate entities into a mixture, and performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Obtaining a value directly includes performing a process involving a physical change of a sample or another substance, for example performing an analytical process (also referred to herein as "physical analysis") involving a physical change of a substance, e.g., a sample, a test component, or a reagent, and also includes performing an analytical method, e.g., a method that includes one or more of: separating or purifying a substance, e.g., a test component, or a fragment or other derivative thereof, from other substances; combining a test component, or a fragment or other derivative thereof, with another substance, e.g., a buffer, a solvent, or a reactant; or altering the structure of a test component, or a fragment or other derivative thereof, for example by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the test component; or altering the structure of a reagent, or a fragment or other derivative thereof, for example by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent. In an embodiment, directly obtaining includes direct measurement. In an embodiment, indirectly obtaining includes inference.

[0123] "Obtaining a sample" as the term is used herein refers to obtaining possession of a sample, such as a sample described herein, by "directly obtaining" or "indirectly obtaining" the sample. "Obtaining a sample directly" means performing a process to obtain the sample (e.g., performing a physical method such as surgery or extraction). "Obtaining a sample indirectly" refers to receiving a sample from another person or source (e.g., a third-party laboratory that directly obtained the sample). Obtaining a sample directly includes performing a process that involves a physical change in a starting material, such as a physical substance, e.g., tissue, e.g., tissue of a human patient or tissue previously isolated from a patient. Exemplary changes include making a physical entity from a starting material, cutting or scraping tissue, separating or purifying a substance, combining two or more separate entities into a mixture, and performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.

[0124] "Ambient temperature" as the term is used herein refers to the air temperature of the environment or immediate surroundings. Ambient temperature may also be referred to as the baseline temperature, or the temperature of a device or object before a temperature control is activated. In certain embodiments, the ambient temperature may be between 1°C and 60°C. In certain embodiments, the ambient temperature may be between 18°C ​​and 25°C. In certain embodiments, the ambient temperature may be between 1°C and 5°C. In certain embodiments, the ambient temperature may be between 32°C and 60°C.

[0125] As used herein, the term "basal mitochondrial ATP production rate" refers to the rate of ATP production by mitochondria in a cell sample before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal mitochondrial ATP production rate is calculated by subtracting the minimum oxygen consumption rate (oligo OCR) from a measurement of the oxygen consumption rate (e.g., the last measurement or the average of several measurements) before the initial contact of the cell sample with either an ATP synthase inhibitor, a mitochondrial uncoupler, or an ETC inhibitor (basal OCR) and multiplying by a constant (called the P / O ratio)*2 (to convert oxygen atoms to oxygen molecules) between 2.45 and 2.86. In an embodiment, the constant is 2.75.

[0126] "Bioenergetic capacity" as the term is used herein refers to the level of increased glycolysis and / or mitochondrial activity that a cell can affect, utilize, and / or induce. In embodiments, bioenergetic capacity is determined in response to increased energy demand and / or in response to inhibition / perturbation of energy production. In embodiments, bioenergetic capacity includes a value for oxygen consumption (e.g., oxygen consumption rate (OCR)) and a value for proton flux (e.g., proton flux rate (PER)). In embodiments, the value for oxygen consumption (e.g., OCR) is a response to mitochondrial uncoupling. In embodiments, the value for proton flux (e.g., PER) is a response to ATPase inhibition. In embodiments, PER is glycolytic PER, which is mathematically expressed as CO 2 The contribution of is removed.

[0127] "Bioenergetic balance" as the term is used herein refers to the balance between aerobic energy production and glycolytic energy production. In an embodiment, bioenergetic balance describes the proportion of ATP produced by glycolysis of oxidative phosphorylation. In an embodiment, bioenergetic balance includes the relationship, e.g., ratio, between ATP produced by mitochondria and ATP produced by glycolysis, between ATP produced by mitochondria and total ATP production, between ATP produced by glycolysis and total ATP production, or any combination thereof.

[0128] "Bioenergetic work" as the term is used herein refers to the amount of ATP produced by a cell.

[0129] The term "cell sample" as used herein refers to a sample that includes cells or cell products or by-products. In embodiments, the cell sample includes a plurality of cells. In embodiments, the cells are disposed in a medium. The cell sample can be or include one or more of a cell, a tissue, a cell or tissue construct, a cellular organelle, an enzyme, and / or a conditioned medium.

[0130] "Cellular metabolic function" as the term is used herein refers to the ability of a living organism to carry out chemical reactions necessary to sustain life. In an embodiment, the cellular metabolic function of a cell sample can be monitored by measuring the OCR and ECAR.

[0131] As used herein, the term "extracellular acidification rate (ECAR)" refers to the measurement of proton extrusion in the extracellular medium over time. ECAR can be reported as the rate of change in pH units over the assay run time, e.g., millipH / min (mpH / min).

[0132] As used herein, "glycolysis" or "glycolytic activity" refers to the cellular metabolic function that converts glucose into lactate.

[0133] "Mitochondrial respiration" as the term is used herein refers to the oxygen-requiring metabolic reactions and processes that occur in mitochondria to convert energy stored in macronutrients into ATP.

[0134] The term "mitochondrial toxicity index" (also referred to as "mitotox index" or "MTI") as used herein refers to an index value derived from OCR measurements. The MTI is a parameter that provides information about both the type and magnitude of mitochondrial toxicity. Positive MTI values ​​(typically between 0 and 1) identify mitochondrial toxicity due to uncoupling, conversely negative MTI values ​​(typically between 0 and -1) identify mitochondrial toxicity due to inhibition.

[0135] As used herein, "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. The use of the term "and / or" in various places herein does not imply that the use of the term "or" is not interchangeable with the term "and / or," unless the context clearly indicates otherwise.

[0136] "Oxygen Consumption Rate (OCR)" as the term is used herein refers to a quantitative measurement of oxygen consumption by a sample over time. Thus, OCR can provide a measurement of cellular and mitochondrial respiration over time. OCR values ​​are the quantitative measurement of O2 consumption over the assay run time. 2 The rate of change of content can be reported, for example, in picomoles per minute (pmol / min).

[0137] In one embodiment, OCR includes scenarios where oxygen consumption is not determined in a completely sealed system, e.g., a system that allows oxygen back diffusion or substantial oxygen back diffusion into the sample, or where oxygen consumption is oxygen depletion in the sample corrected for oxygen back diffusion into the sample, or where oxygen consumption is oxygen depletion uncorrected for oxygen back diffusion into the sample, or where oxygen consumption is determined in a sealed system, e.g., a system that does not allow oxygen back diffusion or substantial oxygen back diffusion into the sample, or where oxygen consumption is equal to or substantially equal to oxygen depletion in the sample.

[0138] In one embodiment, oxygen consumption is determined directly or indirectly, e.g., within a test well or across a capillary, e.g., inferred from a measured oxygen gradient, or by measuring oxygen at preselected time points.

[0139] In one embodiment, oxygen consumption is measured by 2 It is reported in units other than the rate of change of content, such as sensor response per unit time (microseconds / minute, relative fluorescence units / minute, etc.).

[0140] "Primary cells" as the term is used herein refers to cells that are isolated or harvested directly from a subject, organ, or tissue. For example, primary cells may be isolated from blood obtained from a living subject. Primary cells may be isolated or harvested using enzymatic or mechanical methods. Once isolated or harvested, primary cells may be cultured in a medium containing essential nutrients and growth factors to support growth. Primary cells may be suspension cells that do not require attachment for growth (e.g., anchorage-independent cells) or adherent cells that require attachment for growth (e.g., anchorage-dependent cells).

[0141] As used herein, the term "proton efflux rate (PER)" refers to a quantitative measure of extracellular acidification that takes into account media buffering capacity and plate geometry. The PER value is the rate of H over the assay run time. + The rate of change of H can be reported, for example, in picomoles per minute (pmol / min). + is a quantifiable analyte that is proportional to the pH value.

[0142] A "sample" as the term is used herein refers to a biological sample obtained or derived from a source of interest. In embodiments, the source of interest includes an organism such as an animal or a human. The source of the sample may be blood or blood components, bodily fluids, solid tissues such as from fresh, frozen and / or preserved organs, tissues, biopsies, resections, smears, or aspirates, or cells from any stage of a subject's pregnancy or development. In embodiments, the source of the sample is blood or blood components. In embodiments, the sample is a primary sample, e.g., obtained directly from the source of interest by any suitable means. In embodiments, the sample is a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample).

[0143] "Sample carrier" as the term is used herein refers to a substrate on which a sample can be carried. In an embodiment, the sample carrier can include one or more wells. Exemplary sample carriers include, but are not limited to, microplates, microtiter plates, multi-well plates, single-well plates, microwell plates, microfluidic chips, microfluidic devices, dishes, slides, flasks, and tubes. Sample carriers can be used to hold a variety of types of samples, including, but not limited to, cells, tissues, small organisms, animal models, multicellular structures, and 3D samples. As used herein, at least one well of a sample carrier can mean at least 1, 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 192, 288, 384, or 1536 wells, or any number or more wells in between. As used herein, at least two wells of a sample carrier can mean at least 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 192, 288, 384, or 1536 wells, or any number or more wells in between.

[0144] [Bioenergy measurement] The two major pathways for producing energy, mitochondrial respiration and glycolysis, involve the intracellular consumption of oxygen and the efflux of protons, respectively. The devices and methods disclosed herein include sensors, e.g., label-free sensors, for detecting extracellular changes in test components and measuring the rates of cellular respiration, glycolysis, and ATP production. The apparatus described herein can be employed to determine extracellular, intracellular, and pericellular test components.

[0145] According to certain embodiments, disclosed herein is an analytical instrument, also referred to herein as an apparatus. The apparatus can include a stage adapted to support a sample carrier, also referred to herein as a sample carrier or sample carrier cartridge, (e.g., a multi-well plate). The apparatus can include a sensor adapted to sense a cellular constituent associated with a cell sample in a well of the sample carrier (e.g., a multi-well plate). The apparatus can include a dispensing system adapted to introduce a fluid into the well. The apparatus can include a plunger adapted to receive a barrier to create a reduced volume of medium in the well containing at least a portion of the cells, the barrier being adapted for insertion into the well by relative movement of the stage and the plunger.

[0146] In particular, the device may include a plurality of sensors, each sensor adapted to sense a cellular constituent of a corresponding well of a sample carrier (e.g., a multi-well plate). Thus, the device may include an array of sensors. The sensors may independently and selectively sense a cellular constituent of at least one well (e.g., each well). The dispensing system may include one or more injectors. The dispensing system may be configured to independently and selectively introduce a fluid or agent into at least one well (e.g., each well). The plunger may be adapted to independently and selectively insert into at least one well (e.g., each well).

[0147] The apparatus can include a motion actuator assembly, also referred to herein as an elevator mechanism, constructed and configured to position or orient one or more components along at least one coordinate axis. The motion actuator assembly can include one or more high torque motors configured to drive the components of the system.

[0148] The motion actuator assembly can include at least one axis actuator assembly. In some embodiments, the motion actuator assembly can include at least one x-axis actuator assembly configured to position the stage relative to the sensor. The x-axis actuator assembly can additionally or alternatively be configured to position the sensor relative to the stage. The x-axis actuator assembly can additionally or alternatively be configured to position the stage relative to the housing. The motion actuator assembly can include at least one z-axis actuator assembly configured to position the sensor and / or the dispensing system relative to the stage. The z-axis actuator assembly can additionally or alternatively be configured to position the stage relative to the sensor and / or the dispensing system. The motion actuator assembly can include at least one y-axis actuator assembly configured to position the stage relative to the sensor. The y-axis actuator assembly can additionally or alternatively be configured to position the sensor relative to the stage.

[0149] In use, the movement actuator assembly can be configured to align or substantially align the array of sensor units and / or injectors with corresponding wells of a sample carrier (e.g., a multi-well plate) positioned on the stage. In use, the movement actuator assembly can be configured to provide fluid communication between one or more components, for example between the sensor units or injectors of the dispensing system and samples in wells of the sample carrier (e.g., a multi-well plate).

[0150] In an exemplary embodiment, the one or more sensors can be adapted to sense changes in oxygen level and pH (proton concentration) of the cell medium associated with metabolic activity of the cell sample in a well of a sample carrier (e.g., a multi-well plate). The stage, sensor, and dispensing system can cooperate to simultaneously measure a basal oxygen consumption rate and a basal extracellular acidification rate of the cell sample using the sensor. The dispensing system can then be used to sequentially administer one or more agents to the cell sample. In an exemplary embodiment, the one or more agents can include a mixture of a mitochondrial ATP synthase inhibitor (Oligomycin A), a mitochondrial uncoupler BAM15, and / or a mitochondrial complex I and complex III inhibitor (rotenone and antimycin A, respectively). The sensor can measure the oxygen consumption rate and the extracellular acidification rate, optionally substantially simultaneously, after each dispensing of the one or more agents. Additional agents, such as modulator reagents, may optionally be dispensed prior to dispensing the described reagents, or the extracellular membrane ionophore monensin may be injected after injection of rotenone / antimycin A into the cells. The same measurements of oxygen consumption rate and extracellular acidification rate may be performed before and after each dispense.

[0151] Components of the apparatus are further described, for example, in U.S. Pat. No. 7,276,351, entitled "Method and device for measuring multiple physiological properties of cells," and U.S. Pat. No. 8,658,349, entitled "Cell analysis apparatus and method," each of which is incorporated herein by reference in its entirety for all purposes.

[0152] One or more of the following features may be included: The sensor may be configured to analyze the constituent without disturbing the cells. The well may include any feature, e.g., a step, a ring bump, a lip, that allows a corresponding sensor unit plunger from the array of sensor units to move to a defined position in the well to create a microchamber. The sensor unit plunger or the barrier may be adapted to agitate the medium prior to and / or after the analysis of the constituent. The sensor may be a photoluminescent based sensor. The sensor may be, for example, a fluorescent sensor, a luminescent sensor, an ISFET sensor, a surface plasmon resonance sensor, a sensor based on the principle of optical diffraction, a sensor based on the principle of Wood's anomaly, an acoustic sensor, or a microwave sensor. At least a portion of the well may be adapted to receive a sensor. The reduced volume of the medium achieved by the plunger may include the sensor and / or at least a portion of the barrier may include the sensor.

[0153] The instrument may include a light source, such as a fluorescent lamp, a light emitting diode (LED), or a laser, configured to excite the sensor of the sensor unit to generate a signal responsive to the target analyte or property being measured. In some embodiments, the light source may be configured to generate a reference signal. Fluctuations in intensity from the light source may be corrected in proportion to the drift by monitoring the reference signal generated by the light source. The light source may be positioned on a thermally conductive printed circuit assembly configured to minimize drift from the light source. In some embodiments, the thermally conductive printed circuit assembly may be formed of a material configured to minimize drift caused by thermally induced fluctuations from the light source by at least 20%, such as at least 15%, 10%, or 5%. In some embodiments, the thermally conductive printed circuit assembly reduces drift by approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%. FIG. 38 shows a correction factor reduced from a 10% drift correction to less than 1%. In some embodiments, the thermally conductive printed circuit assembly is formed from materials configured to substantially minimize drift such that a reference signal is not required, thereby reducing the cost of the system.

[0154] In certain embodiments, the one or more sensors include a CO 2 , O 2 , Ca ++ , H + The wells may be adapted to analyze (determine the presence or concentration of) extracellular components, such as cellular components, or extracellular metabolic products consumed or secreted. 2 The test components that are proportional to their content are, for example, CO 2 , O 2 The analyte that is proportional to the pH of the sample is, for example, Ca. ++ , H + More than one test component, for example at least one test component, can be measured to analyze the extracellular components.

[0155] The one or more sensors can be adapted to analyze a first extracellular constituent. In some embodiments, the one or more sensors can be adapted to analyze multiple extracellular constituents, such as more than one, more than two, more than three, more than four, or more constituents. Each sensor can analyze multiple constituents simultaneously. Each sensor can analyze multiple constituents individually, such as sequentially. The present disclosure generally relates to a method for analyzing a first target analyte, such as O 2 The sensor unit is described as being configured to analyze at least one analyte proportional to content, and at least one analyte proportional to a second target analyte, e.g., pH value, however, it should be understood that the sensor unit may be configured to analyze additional or alternative target analytes.

[0156] In certain embodiments, the sensor is an optical sensor. The optical sensor can be a fluorescence or phosphorescence based sensor. The sensor can alternatively utilize a solid-state sensor, a nanoparticle sensor, a microparticle sensor, and / or a magnetic sensor, etc. For example, a solid-state sensor can include one or more spots or films on a lid, a base, a protrusion, or a combination thereof, and a particle-based sensor can generally be in solution or suspension. Alternatively, in one aspect, a particle-based sensor can be loaded into a cell or coated on a surface. Nevertheless, such sensors can be optical, optical, or optical sensors. 2 , pH, temperature, CO 2 , or a combination thereof.

[0157] Further, in one aspect, the sensor may be an electrochemical or potentiometric sensor. Additionally or alternatively, electrodes may be included in the well to measure electrical properties including impedance. Regardless of the sensor selected, it should be understood that in one aspect, the well or chamber may also include one or more reference probes, in the form of any of the sensors discussed above, that generate a signal of known value for instrument calibration, as discussed above.

[0158] One exemplary sensor unit is an oxygen-sensitive photoluminescent dye. The photoluminescent dye can be selected from any oxygen-sensitive photoluminescent dye. The suitable dye can be selected based on the intended use of the probe. A non-exhaustive list of suitable oxygen-sensitive photoluminescent dyes includes, but is not limited to, ruthenium(II)-bipyridyl and ruthenium(II)-diphenylphenanthroline complexes, porphyrin-ketones such as platinum(II)-octaethylporphine-ketone, platinum(II)-porphyrins such as platinum(II)-tetrakis(pentafluorophenyl)porphine, palladium(II)-porphyrins such as palladium(II)-tetrakis(pentafluorophenyl)porphine, phosphorescent metal complexes of tetrabenzoporphyrins, chlorins, azaporphyrins, and long-decay luminescent complexes of iridium(III) or osmium(II).

[0159] Typically, in such an embodiment, the hydrophobic oxygen-sensitive photoluminescent dye can be combined with a suitable oxygen-permeable and hydrophobic carrier matrix. A suitable oxygen-permeable hydrophobic carrier matrix can be selected based on the intended biological sample to be tested and the properties of the selected dye. A non-exhaustive list of polymers suitable for use as an oxygen-permeable hydrophobic carrier matrix includes, among others, but is not limited to, polystyrene, polycarbonate, polysulfone, polyvinyl chloride and some copolymers. An alternative example is to dye oxygen-permeable microbeads with the oxygen-sensitive photoluminescent dye, mix the dyed beads with silicone or polyurethane, and apply the mixture as a polymer coating.

[0160] Regardless of the type of solid-state sensor selected, in one embodiment, by way of example only, the sensor may be embedded in a permeable medium, such as a permeable medium selected from hydrogel, silicone, and matrigel. In some embodiments, the sensor is attached to at least one of the protrusions by solidifying or removing the medium (such as by drying, curing, cooling, evaporation, or other techniques). The solid-state sensor may be applied by dipping or spotting a distal end of at least one of the protrusions into a mixture of a fluorescent indicator in the medium.

[0161] However, it should be understood that in certain embodiments, the sensor may be spotted or immersed in whole or in part on one or more of the protrusions. Moreover, it should be understood that in certain embodiments, the sensor may be removably connectable to the body of one or more of the protrusions of the assembly. Moreover, it should be understood that in certain embodiments, the sensor may be integrally formed with one or more of the protrusions. Integral formation of the sensor on one or more of the protrusions may be accomplished by one or more techniques, such as deposition, chemical coating, spin coating, immersion, robotic spotting, etc.

[0162] The dispensing system can include one or more injectors configured to independently and selectively introduce a fluid or agent into at least one well (e.g., each well). In some embodiments, the dispensing system can include an array of injectors, such as at least one injector positioned to correspond to at least one well (e.g., each well) of a well plate. In some embodiments, the dispensing system can include one or more movable injectors, each configured to introduce a fluid or agent into multiple wells of a well plate.

[0163] In certain embodiments, for example to actuate the movement of one or more injectors across multiple wells, the device can include an injector movement actuator assembly positioned to drive at least one injector. The injector movement actuator assembly can drive the one or more injectors in a preselected pattern across rows of wells, across columns of wells, or across any configuration of wells.

[0164] Thus, the device can have one, two, three, four, five, six, seven, eight, nine, or ten movable injectors positioned to be driven across a plurality of wells, rows of wells, or rows of wells. Alternatively, the dispensing system can have an array of one or more injectors, e.g., one, two, three, four, five, six, seven, eight, nine, or ten injectors, fixedly positioned to correspond to at least one well (e.g., each well). The device can have a well to injector ratio of 1:1 to 1:384, e.g., 1:1, 1:2, 1:3, 1:4, 1:8, 1:12, 1:24, 1:36, 1:48, 1:64, 1:72, 1:96, 1:192, or 1:384. The device can have a syringe to well ratio of 1:1 to 1:384, for example 1:1, 1:2, 1:3, 1:4, 1:8, 1:12, 1:24, 1:36, 1:48, 1:64, 1:72, 1:96, 1:192, or 1:384.

[0165] The assemblies and processes according to exemplary aspects of the present disclosure may be suitable for measuring components in any of a variety of types of samples, such as biological samples. In one aspect, for example, the systems and processes according to exemplary aspects of the present disclosure may be used to measure one or more components or parameters related to components in cellular material. The one or more components may be contained in the medium surrounding the cells or may be contained in the cells themselves. In some embodiments, the biological sample being tested may include cellular material derived from cells, such as organelles, mitochondria, cell extracts, cell products or by-products, or conditioned medium. The measurements may be completed in a label-free manner.

[0166] An exemplary analytical instrument is shown in Figures 1-4. As shown in Figures 1-4, the instrument or device 100 includes a housing 10 having an opening formed in a side wall of the housing 10. The opening may optionally be closable by a door 12. A stage 20 adapted to receive a sample carrier (e.g., a multi-well plate) 30 is provided within the housing 10. The stage 20 may be movable by an x-axis actuator assembly to be positioned within the housing 10 through the opening or outside the housing 10. The door 12 may be closed when the stage 20 is positioned within the housing 10 for testing. The housing may include one or more electronic ports 14 connectable to a computer and / or a power source.

[0167] The electronic port 14 may be compatible with one or more of USB, mini-USB, HDMI, DVI, dual DVI, mini-DVI, micro-DVI, display port, mini-display port, VGA, mini-VGA, RS-232, Ethernet / LAN, or any other electronic port capable of transmitting data. It should be noted that although the device shown in Figures 1-4 includes an electronic port 14, the device may be connectable to an external computer by any means known in the art, such as wireless fidelity network (WiFi), ultra-high frequency radio waves (also known as Bluetooth), or any other data transmission connection. In an embodiment, the device may be connectable to an external computer through the cloud.

[0168] An exemplary assembly 110 is shown in FIG. 5. The assembly 110 can be housed in the housing 10 shown in FIGS. 1-4. The assembly 110 includes components of a sensing system (e.g., fiber optics) 40 including an array of sensor units and a dispensing system 50 including an array of injectors arranged on a manifold. In some embodiments, the manifold includes holes, and pressurized air is forced through a sensor cartridge including a drug / compound / substance port that includes a substance corresponding to the manifold hole, which is "sealed" by a gasket. A force is applied to the manifold, allowing the substance to be delivered to the sample. One or more components of the manifold can be independently moveable on the z-axis as directed by a z-axis actuator assembly 54 of the motion actuator assembly. The temperature of the manifold and / or the cartridge can be controlled by a manifold temperature controller 52. The assembly 110 includes a stage 20 adapted to receive a sample carrier (e.g., a multi-well plate) 30 (on which the cartridge is illustrated). The temperature of the samples in the sample carrier (e.g., a multi-well plate) 30 is controlled by a sample temperature control element 22. The stage 20 is movable along the x-axis as oriented by an x-axis actuator assembly 24 of the motion actuator assembly. The motion actuator assembly also includes a y-axis actuator assembly 26 configured to move the stage 20 along the y-axis.

[0169] The device may include an automatic measurement system. The device may also include a computer or may be connectable to a computer, with the automatic measurement system in electrical communication with the computer. In certain embodiments, the device may also include a controller for performing the addition of one or more fluids or agents to one or more of the wells of the sample carrier (e.g., a microplate). The controller may operate the sensor to perform sensing of one or more components in one or more wells of the sample carrier (e.g., a microplate). The system may communicate with the controller and the sensor via a graphical user interface present on the computer. The graphical user interface may be configured to receive instructions for designing a multi-well experiment, i.e., a multi-well experiment, according to the methods disclosed herein, to instruct the controller to execute the multi-well experiment, and to receive data acquired by the sensor in response to execution of the multi-well experiment.

[0170] In certain embodiments, the graphical user interface can include a plurality of display areas, each area belonging to one of the wells. The graphical user interface can be configured to receive instructions written in the respective areas belonging to one of the wells for the design of a multi-well experiment, and to receive data acquired by the sensor in response to the execution of the multi-well experiment for display in the respective areas belonging to one of the wells. Thus, the method executable by the controller can be independently and selectively applied to one or more wells through instructions from the graphical user interface.

[0171] FIG. 6 shows an exemplary system including an analytical instrument (laboratory instrument) connectable to a cloud-based computing network and a computer through the cloud-based network. The analytical instrument includes a detector or sensor unit and other electronics such as a signal processing module and a motion actuator. The detector and electronics can be controlled by one or more controllers, such as a motion controller operably connected to the motion actuator assembly, and a control system operably connected to the sensing system and / or the dispensing system. Protocols for the components of the analytical instrument can be provided through a user interface accessible on the computing device or cloud-based computing network. The user interface can be provided on a web browser software platform and / or on a desktop software platform. It should be noted that the desktop software platform can be provided on a desktop computer, a laptop computer, and / or a tablet or other mobile device. The web browser software platform can provide cloud-based data processing, cloud-based data storage, and / or cloud-based connectivity between the computer and the analytical instrument. Other mechanisms for connecting to the cloud can also be used, such as desktop software or driver software. A data storage module, such as a local memory storage device, such as a server, an external drive, a portable drive, and / or a cloud-based memory storage device, may also be included in the system. The data storage module may store historical data, protocols, data processing algorithms, and / or controller executable instructions.

[0172] 7 and 8 are schematic diagrams of the system disclosed herein, with the electronic components shown in more detail. FIG. 7 is a diagram of an analytical instrument operably connected to a central control computer. The baseboard includes a microcontroller or system controller operably connected to temperature control elements for the manifold and tray (i.e., sample temperature control elements) and a dispensing system or injection unit. A further microcontroller, also referred to herein as a "motion controller", is shown operably connected to the controller and a motion actuator assembly including a z-motor for operating the z-axis actuator assembly and an x-motor for operating the x-axis and optionally y-axis actuator assemblies. The apparatus described herein can include stepper motors with higher torques that improve the accuracy of measurements over the life of the instrument and reduce the need to provide maintenance and / or replace motor components.

[0173] A proximity sensor is also provided as part of the motion actuator assembly configured to sense the relative positioning of the stage or sample carrier (e.g., a multi-well plate) and the sensor unit and other equipment components such as an injector of a dispensing system. The proximity sensor may be configured to generate a notification signal, and optionally pause a protocol, when a component is positioned within a predetermined distance from another component, e.g., when a sensor unit is positioned within a predetermined distance from a corresponding well of a sample carrier. Additionally or alternatively, the proximity sensor may be configured to generate a notification signal, and optionally pause a protocol, when an opening in a side wall of the housing is ajar and / or when external light is detected within the housing.

[0174] The system may also include a stall sensing module programmed to generate a notification signal, and optionally pause the protocol, e.g., stop motor movement, if a given protocol step is not completed within a given time interval. The stall sensing module may be configured to detect stalls through the use of an encoder. For example, the encoder may operate by looking for timing related delays in the movement of the encoder and flag a stall.

[0175] The diagram of FIG. 7 also illustrates a microcomputer operatively connected to a signal processing module including an amplifier and a microcontroller configured to receive and amplify signals from the sensor unit. 2 The system further includes a sensing unit for the test component and a pH test component. The signal processing module is further operably connected to the system controller and the central control computer. The system further includes a barcode scanner configured to scan a barcode encoding information operably transmittable to the central control computer.

[0176] FIG. 8 is a schematic diagram of the system showing a computer operably connected to a system control board or system controller and a barcode scanner. The barcode scanner is configured to decode and transmit information from the barcode to the computer. The system controller is operably connected to a tray heater or sample temperature control element configured to control the temperature of the consumable or sample in the sample carrier (e.g., a multi-well plate). The system controller is also operably connected to a light emission amplifier or a signal processing module. The signal processing module is operably connected to an optical fiber or a sensor unit. In some embodiments, the system controller is also operably connected to a manifold heater or manifold temperature control element configured to control the temperature of the injection manifold or the dispense system. Optionally, a separate system controller operably connected to the manifold heater or manifold temperature control element may be provided.

[0177] 9 shows an exemplary sensor unit 41 deployed in a well 31. The exemplary sensor unit 31 is a fluorescent sensor. A fluorophore may be disposed on a surface of the well 31, the fluorophore having a fluorescent property that depends on at least one of the presence and concentration of a component in the well 31. The sensor unit 41 may include a housing for receiving a waveguide for at least one of stimulating the fluorophore and receiving a fluorescent emission from the fluorophore.

[0178] The present disclosure provides methods, devices, and measurement systems for adding test compounds to wells and measuring well constituents with sensors. The methods can be performed as high throughput assays by adding one or more test compounds to one or more wells, respectively, or by adding the same or different test compounds to multiple wells of a sample carrier (e.g., a microplate). In certain embodiments, the test compounds are introduced while the sensor probe remains in equilibrium with, e.g., submerged within, the liquid contained within at least one well (e.g., each well). In such embodiments, equilibration times can be reduced because the sensor probe remains submerged during compound delivery. Thus, systems and methods are provided for storing and dispensing a single preselected test compound, or a preselected concentration of a compound per well.

[0179] In certain embodiments, the device and method store and deliver one or more test compounds or target agents per well. The test compounds can be delivered using a supply of compressed gas from a remote source to actuate the delivery of the compounds. In certain embodiments, both the sensor probe and the test compound delivery structure are incorporated into a single disposable cartridge. Also described is a pneumatic multiplexer that, when temporarily attached to the cartridge, allows a single actuator to initiate the delivery of test compounds from multiple ports using a supply of compressed gas from a remote source.

[0180] In one aspect, a cartridge is provided that is adapted to mate with a sample carrier (e.g., a multi-well plate) having a plurality of wells. The cartridge may include a substantially planar element having a plurality of regions corresponding to respective openings of a common number of wells in the sample carrier (e.g., a multi-well plate). At least one port may be formed in the cartridge in at least one region, the port adapted to deliver a test fluid, e.g., an aqueous solution of a candidate drug / compound / substance or other agent, to each well. The cartridge may also include at least one of a) a sensor or a portion thereof adapted to analyze constituents in the well, and b) an opening adapted to receive a sensor disposed in a sub-region of at least one region of the cartridge.

[0181] The components and features of the cartridge are further described, for example, in U.S. Pat. No. 9,170,255, entitled "Cell analysis apparatus and method," which is incorporated herein by reference in its entirety for all purposes.

[0182] The apparatus may include an elevator mechanism adapted to move the cartridge relative to a stage or plate to place a sensor in a well, typically multiple sensors in multiple wells simultaneously. A pressure source adapted to be fluidly engaged with the cartridge to deliver test fluid from a port in the cartridge to the well. The apparatus may also include a multiplexer disposed between the pressure source and the cartridge, the multiplexer adapted to be in fluid communication with a plurality of ports formed in the cartridge. The multiplexer may be in selective fluid communication with an exclusive set of ports formed in the cartridge. A controller may be included to control the elevator mechanism, the multiplexer, and / or the pressure source to enable delivery of test fluid from a given port or set of ports to a corresponding well or set of wells when an associated sensor is disposed in the well. The controller may be in communication with a computer or a graphical interface as described above.

[0183] In certain exemplary embodiments, the cartridge opening adapted to receive the sensor can include a sensor sleeve structure having a surface adjacent to a well of a sample carrier (e.g., a multi-well plate). Disposed on the surface can be a fluorophore having fluorescent properties that depend on at least one of the presence and concentration of a component in the well. The sensor sleeve can include an elongated housing for receiving a waveguide for at least one of stimulating the fluorophore and receiving fluorescent emission from the fluorophore.

[0184] The array of sensors corresponding to the array of wells can be integral to the cartridge, or can be a separate element that fits into and is positioned within an opening formed in the cartridge. The array of sensors can be fitted to a well plate.

[0185] A method of analyzing cells using the devices disclosed herein is provided. The method can be employed to measure cells disposed in a medium in a sample carrier (e.g., a multi-well plate). The method can include one or more of disposing at least a portion of a sensor in a medium in a well in the sample carrier (e.g., a multi-well plate), analyzing constituents for the cells in the medium in the well, delivering a test fluid to the well while the sensor remains disposed in the medium in the well, and further analyzing the constituents to determine any changes therein. In certain embodiments, one or more constituents can be analyzed substantially simultaneously. In particular, changes in the rate of one or more constituents can be measured over an assay time, for example to determine metabolic or other activity of the cell sample.

[0186] The analyzing step can include analyzing the respective components for the respective cells in the medium in each well. The respective components can be the same components. The delivering step can include delivering a respective test fluid or target agent to each well while the respective sensors remain disposed in the medium in each well. The respective test fluid or agent can include the same test fluid or agent.

[0187] The analyzing step can include analyzing respective constituents for respective cells in the medium in each well to determine any respective changes therein. The delivering and further analyzing steps can be repeated. Different test fluids or agents, or additional aliquots of the same test fluid or agent, can be delivered between measurements. The method can include substantially maintaining equilibrium between the sensor and the medium during the delivering step, or maintaining thermal equilibrium between the test fluid and the medium during the delivering step.

[0188] The method may include controlling the temperature and / or environment of the cell sample before, during, and / or after the analytical step. In certain embodiments, the method may include controlling the temperature and / or environment of the cell sample throughout the performance of the analytical method. Controlling the environment may include, for example, controlling the relative humidity (RH) and / or N 2 , O 2 , and / or CO 2 For example, in certain embodiments, controlling the environment may include controlling the composition of the environmental gas, such as the concentration of N 2 This can include creating a low oxygen environment by purging with a gas.

[0189] The method may further comprise imaging or scanning the sample during the analyzing step, during the delivering step, and / or following the analyzing and / or delivering steps.

[0190] The devices and methods disclosed herein can be used to analyze biological samples, also referred to herein as cell samples. In particular, the devices and methods disclosed herein can be used to analyze live cell samples. The sample can include or take the form of one or more of free cells, cell constructs, free tissues, tissue constructs, organelles, enzymes, cell products or by-products, and conditioned media. The cell sample can include mammalian cells or tissues. The cell sample can include non-mammalian cells or tissues. The sample can include animal cells or tissues. The sample can include insect cells or tissues. The sample can include plant cells or tissues, such as seeds, pods, or other plant material. The sample can include single-cell organisms, such as microorganisms. In certain exemplary embodiments, the sample can include whole plant or animal model tissues, such as zebrafish, C. elegans, Drosophila.

[0191] The biological material to be analyzed may include cellular material. The biological material may include living cells, including bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, or insect cells, etc. The cells may be animal cells, human cells, immune cells, or immortal cells.

[0192] Exemplary cells include human T cells (CD4+, PanCD3+, CD8+, PBMCs, e.g., naive, activated, effector and memory), mouse T cells (spleen-derived CD8 naive and activated), immortalized mouse myoblasts (e.g., C2C12), Jurkat cells, lung cancer cell models (A549, PC9, H1373), leukemia cancer cell models (THP-1), human hepatoma cells (e.g., HepG2), human epidermoid carcinoma cells (e.g., A431), and analysis of whole organisms such as zebrafish, C. elegans, and Drosophila. Certain embodiments of the devices and methods disclosed herein allow for analysis of live cells that require temperatures between 28° C. and 40° C. without the need to place the equipment in a temperature-controlled room.

[0193] The devices and methods disclosed herein can be employed to facilitate research in the areas of cancer, immunology, toxicology, drug / compound / agent discovery, and immunotherapy, among others.

[0194] In one aspect, the cell sample is obtained or derived from a subject, such as a human or non-human animal. In one aspect, the subject is a mouse, which in one aspect has or is at risk of having a disorder. Nevertheless, in one aspect, the cell sample may include primary cells, cells isolated or harvested directly from a tissue or organ of a living organism, cultured cells, and / or immortalized cells. For example, the cell sample may include primary cells, or cells isolated or harvested directly from a tissue or organ of a living organism and then cultured ex vivo. In one aspect, the cell sample includes modified cells, such as, for example, cells from a knockout mouse or a CRISPR KO library, genetically engineered for heterologous expression of a gene of interest and / or genetically engineered for inhibition of expression of a gene. Nevertheless, in one aspect, the cell sample includes stem cells or cells derived from stem cells. Nevertheless, regardless of the cells used, in one aspect, the cell sample includes a medium, such as a culture medium or an expansion medium, and the cells may be disposed in the medium. Furthermore, as will be appreciated, in one aspect, the cell sample includes a plurality of cells, such as a plurality of cells described herein.

[0195] The cells to be tested may include any suitable cell sample, including, but not limited to, cultured cells, primary cells, human cells, neurons, T cells, B cells, epithelial cells, muscle cells, stem cells, induced pluripotent stem cells, immortalized cells, pathogen-infected cells, bacterial cells, fungal cells, plant cells, archaeal cells, mammalian cells, avian cells, insect cells, reptilian cells, and amphibian cells. The cells to be tested may also include monolayer cells, two-dimensional cell samples, three-dimensional cell samples, such as tissue samples, cell spheroids, organoids, biopsy samples, cell scaffolds, and organs-on-a-chips. Examples of parameters that may be measured and relate to the above cell functions include carbon dioxide concentration, oxygen concentration or oxygen tension, calcium ions, and hydrogen ions. However, in one aspect, the parameter measured is oxygen concentration, such as oxygen consumption. Through these tests, an understanding of what drives the phenotype and function of the cells and / or an accurate picture of the cellular environment or microenvironment can be gained.

[0196] The assemblies and processes according to exemplary aspects of the present disclosure may be used to measure metabolic data of living cells or (micro)environmental conditions of any living cells. Cellular materials to be tested may include, for example, bacterial cells, fungal cells, yeast cells, prokaryotic cells, and eukaryotic cells, etc. Cells that may be tested include mammalian cells, including animal cells and human cells. Specific cells that may be tested include cancer cells, immune cells, immortal cells, primary cells, induced pluripotent stem cells, and cells infected with viral or bacterial pathogens, etc.

[0197] For example, in one embodiment, the assemblies and processes according to the exemplary embodiments of the present disclosure may be used to assist in immunotherapy. Immunotherapy is a type of treatment that strengthens a patient's immune system to fight cancer, infections, and other diseases. Immunotherapy processes may include adoptive cell-based therapy, such as the production of T cells, Natural Killer (NK) cells, monocytes, macrophages, and combinations thereof. During T cell therapy, T cells are removed, for example, from the patient's blood. The T cells are then sent to a bioreactor and expanded or cultured. In addition to this, the T cells may be altered to have specific proteins called receptors. The receptors on the T cells are designed to recognize and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in the bioreactor to achieve a certain cell density and then delivered into the patient's body to fight cancer or other diseases. T cell therapy may also be referred to as adoptive T cell therapy or T cell transfer therapy, one example of which is called chimeric antigen receptor (CAR) T cell therapy. The use of T cells for adoptive T cell therapy or T cell transfer therapy has boomed in recent years due to great success in fighting blood diseases. In some embodiments, aspects of the invention can be used to monitor the health of T cells used in adoptive T cell therapy or T cell transfer therapy. In some embodiments, aspects of the invention can be used to monitor T cell activation, T cell exhaustion, and T cell metabolism, including that of starting material and modified products, etc.

[0198] NK cells are a type of cytotoxic lymphocyte that can seek out and destroy infected cells in the body. NK cells can exhibit very fast immune response. Therefore, the use of NK cells in anti-cancer drug therapy has attracted great interest and popularity. However, there are only limited numbers of NK cells in mammalian blood, which requires that NK cells be expanded to a relatively high cell density in a bioreactor.

[0199] Culturing cells, such as T cells, NK cells, or other mammalian cells, typically requires a somewhat complicated process from seeding to use in a patient. The disclosed assemblies and processes can be used to monitor the metabolism of cells during any time during the culture process to ensure that the cells are healthy and / or have the desired metabolic phenotype, and that the medium in which the cells are growing contains optimized levels of nutrients. The systems and processes can be used, for example, to make adjustments to ensure metabolic fitness of the cells as they are growing.

[0200] In addition to immune cells, the metabolism of cancer cells can also be monitored to provide an understanding of which nutrients are nourishing the cancer cells. For example, the assemblies and processes according to exemplary embodiments of the present disclosure can reveal mechanisms or components that affect the metabolism of cancer cells to inhibit proliferation. The assemblies and processes according to exemplary embodiments of the present disclosure can also be used to determine the rate at which cancer cells can proliferate. The systems and processes of the present disclosure are also suitable for use in toxicology. For example, the processes and assemblies of the present disclosure can be used to detect mitochondrial liability of potential therapeutics. The risk of mitochondrial toxicity can, for example, be evaluated with high specificity and sensitivity. In this manner, the mechanism of action of some mitochondrial toxicants can be determined.

[0201] [Temperature control] The devices described herein include one or more temperature control elements designed to reduce temperature gradients between the outer (e.g., border) and inner wells of a sample carrier (e.g., a multiwell plate). Sample temperature control elements and manifold temperature control elements are described herein. The temperature control elements can be designed to control temperature independently of each other. The temperature control elements are generally formed of a temperature conductive material and are optionally positioned in close proximity to or in direct contact with one or more components, such as the sample carrier (e.g., a multiwell plate), the sensor unit, and / or the injector. For example, the sample temperature control element can be dimensioned to fit the sample carrier (e.g., a multiwell plate). The manifold temperature control element can be dimensioned to fit the sensor, the injector, and / or the cartridge, and can optionally be dimensioned to cover the sample carrier (e.g., a multiwell plate) when the cartridge is positioned to mate with the sample carrier (e.g., a multiwell plate), e.g., when the sensor unit and / or the injector are in fluid communication with the wells of the sample carrier (e.g., a multiwell plate). In some embodiments, a microenvironment is created that includes a manifold and heater, heated components surrounding the sensor cartridge, and a tray heater in direct contact with the sample carrier, allowing for temperature maintenance over extended periods of time. The manifold temperature control element can be configured to mate with the sample temperature control element and cover the sample carrier (e.g., a multi-well plate).

[0202] The design of the temperature control elements creates controlled temperature zones or microenvironments within the device. The controlled temperature zones typically include an array of wells in the sample carrier. In particular, the controlled temperature zones do not include the headspace of the housing, or a significant portion of the headspace, e.g., the temperature control does not extend to the entire interior chamber of the device, such that the temperature of components outside the controlled temperature zones is not substantially altered, e.g., increased or decreased, by actuation of the temperature control elements. In some embodiments, the volume of the controlled temperature zones does not exceed the volume of the sample carrier by more than 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x. In some embodiments, the volume of the controlled temperature zones does not exceed 10% of the volume of the housing, e.g., not exceeding 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0203] Surprisingly, it has been discovered that the design of the temperature control element allows the device to operate at lower temperatures than expected, e.g., temperatures below 8°C, as compared to a typical lower operating temperature limit of 12°C. The lower operating temperature limit may be limited by heat generated by components of the system, such as the motor or motor control components, power supplies, circuit boards, and light sources. The lower operating temperature allows the device to be used to test sample types that could not previously be tested using such devices, e.g., zebrafish, whole cell organisms, or non-mammalian cells. Thus, in some embodiments, the temperature control element can control the temperature of the sample in at least one well (e.g., each well) to be less than 12°C, e.g., less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C.

[0204] Creation of a controlled temperature zone or microenvironment generally enables the device to bring the temperature of the sample in at least one well (e.g., each well) of the sample carrier within a predetermined range of the target temperature within about 5 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, or 1 minute of activation of the temperature control element and / or introduction of the sample carrier into the controlled temperature zone.

[0205] Additionally, the design of the temperature control elements allows the instrument to achieve temperature uniformity and a wider range of operating temperatures than previous designs. The wider operating temperature range allows the instrument to be used with a wider variety of cell types, such as non-mammalian cells that may require lower or higher temperatures than previously achievable, improving viability during the assay. The greater operating temperature and better temperature control elements can improve the sensitivity of the sensing unit, for example, allowing the device to have a lower OCR detection limit than previous instruments. In some embodiments, the uniformity and / or accuracy of the measurements is improved.

[0206] The manifold temperature control elements can be configured to control the temperature of the target agent and / or sensor unit to within 3° C., e.g., within 2° C., within 1° C., within 0.6° C., within 0.5° C., within 0.4° C., within 0.3° C., within 0.2° C., or within 0.1° C., of another injector and / or sensor unit. In certain embodiments, the manifold temperature control elements can be configured to control the temperature of the target agent and / or sensor unit, and the sample temperature control elements are configured to control the temperature of the samples in the array of wells of the sample carrier to within 3° C., e.g., within 2° C., within 1° C., within 0.6° C., within 0.5° C., within 0.4° C., within 0.3° C., within 0.2° C., or within 0.1° C., of each other. Thus, the temperature control elements disclosed herein are generally capable of maintaining temperature uniformity between different samples in a well plate, e.g., between samples in the interior and at the boundary of a well plate, and / or between a cartridge component and a corresponding sample in the well plate.

[0207] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier to be within a predetermined range. Exemplary predetermined ranges include temperatures between 0°C and 70°C above ambient temperature, such as temperatures between 8°C and 20°C above ambient temperature, such as temperatures between 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 30°C, 40°C, 50°C, 60°C, or 70°C above ambient temperature. In some embodiments, the sample temperature control element is configured to control the temperature of a sample, such as two identical or substantially identical samples, in at least one well (e.g. each well) of the sample carrier, such that, for example, when the samples are analyzed under the same or substantially the same conditions, a sensor signal corresponding to the level, production or consumption of a target analyte does not differ by more than a predetermined amount between the two identical or substantially identical samples, such as not differing by more than 10% between the two identical or substantially identical samples, such as not differing by more than 5%, 3%, 1%, or 0.1%. In particular, the temperature control element may be configured to reduce or suppress variations in sensor readings, such as photoluminescence sensor readings, cell metabolism and other functions, and / or analyte concentrations that may occur as a result of temperature differences.

[0208] The design of the temperature control element reduces evaporation of the sample during the execution of the protocol. Evaporation can affect cell function if it is intense enough to change the concentration of the test component in the medium. The temperature uniformity achieved by the sample temperature control element and / or the manifold temperature control element exhibits reduced evaporation of the sample compared to conventional devices. In some embodiments, the temperature control element can be configured to control evaporation of the sample in the array of wells to less than 25%, e.g., less than 20%, less than 15%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Evaporation can be controlled by such percentages for assays of long duration, such as 6-hour assays, 8-hour assays, 10-hour assays, or longer. Additionally, the sample carrier (e.g., multi-well plate) can be designed to reduce evaporation during the cell culture and incubation process.

[0209] Surprisingly, the design of the temperature control element allows for low O 2 It has been found to provide detection limits of 0.01 pmol / min and improved accuracy of measurement. For example, the analytical instruments disclosed herein can have an OCR detection range of 2000 pmol / min to 0.01 pmol / min, e.g., 700 pmol / min to 0.01 pmol / min, e.g., 50 pmol / min to 0.01 pmol / min. In some embodiments, the analytical instruments can have improved OCR detection limits of less than 50 pmol / min, e.g., less than 40 pmol / min, less than 30 pmol / min, less than 20 pmol / min, less than 10 pmol / min, less than 5 pmol / min, less than 3 pmol / min, less than 1 pmol / min, less than 0.1 pmol / min, or less than 0.01 pmol / min.

[0210] In addition, the design of the temperature control element can reduce, limit, or inhibit differential (gradient) diffusion of gas in the sample carrier, cartridge, and / or the internal environment near the sample carrier or controlled temperature zone. The temperature control element can be configured to control, e.g., reduce, limit, or inhibit, diffusion of gas within the controlled temperature zone, cartridge, well plate, such that the composition of gas in the environment does not change significantly during the assay, e.g., does not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% during the assay.

[0211] [Environmental Control] The devices described herein may include or be associated with one or more environmental control modules designed to control the environment surrounding the sample carrier (e.g., a multi-well plate). The environmental control modules may be designed to control the environmental gas and / or relative humidity (RH) of the environment surrounding the sample. For example, the environmental control modules may control the N 2 Concentration, O 2 concentration, and CO 2 The environmental control module can be configured to control one or more of the concentrations of RH, CO, Hg, CO2, CO3, CO4, CO5, CO6, CO7, CO8, CO9, CO10, CO11, CO12, CO13, CO2, CO14, CO2, CO2, CO3, CO4, CO5, CO6, CO15, CO2, CO2, CO2, CO3, CO4, CO5, CO2, CO3, CO4, CO5, CO6, CO3, CO4, CO5 ...7, CO7, CO8, CO8, CO9, CO9, CO9, CO9, CO9, CO9, CO9, CO10, CO11, CO12, CO13, CO14, CO15, CO15, CO16, CO17, CO18, CO19, CO2, CO2, CO3, CO4, CO5, CO4, CO5, CO5, CO6, CO6, CO7, CO8, CO9, CO9, CO9, CO10, CO11, CO12, CO13, CO14, CO15, CO16, CO17, CO18, CO19, CO2, CO2, CO3, CO4, CO5, CO4, CO5, CO6, CO5, CO6, CO7, CO8, CO9, CO9, CO11, CO12, CO13, CO14, CO15, CO15, CO16, CO17, CO18, CO19, CO2, CO2, CO3, CO4, CO5, CO4, CO5, CO5, CO6, CO6, CO7, CO4, CO5, CO6, CO6, CO7, CO8,

[0212] The environmental control module contains a gas, e.g., N, 2 , O 2 , and CO 2The environmental control module may include one or more sources of gas, air, or liquid. The environmental control module may form a controlled environment zone that includes the array of wells of the sample carrier. The controlled environment zone may be open or closed to the surrounding environment. The environmental control module may include a pump or fan configured to direct gas to or remove gas from the well plate.

[0213] In certain embodiments, the environmental control module is integrated into the instrument. The environment can be created by moving a heated component to surround or cover or encase the heated sample carrier. The heated component can be made of a thermally conductive material, such as metal, aluminum, steel, etc. These thermally conductive materials can be anodized to reduce / eliminate electrical conductivity. The thermally conductive heated component can also block stray (ambient) light. In some embodiments, the container is substantially enclosed such that there is minimal air flow. The sealed container can house a well plate, for example a stage that holds the well plate. In some embodiments, the container can house a cartridge with a well plate. To create a controlled environmental zone, the sealed container can be fluidly connected to a gas source and appropriately purged with one or more selected gases.

[0214] In certain embodiments, an environmental control module is associated with the device. For example, in some embodiments, the device can be disposed in a gas-controlled incubator or hypoxic chamber. Thus, the device can be configured for use in a gas-controlled environment and can be formed of materials suitable for use in a gas-controlled environment, such as materials having low gas solubility.

[0215] The environmental control modules can be integrated with the system software and can be operably connected to, for example, the controller and / or the system processor. The software can be programmed to cycle the environmental control modules according to a selected protocol.

[0216] The environmental control module may be operatively connected to, for example, a controller and / or system processor, and may include a controller and / or system processor that may be operable to receive measurements of the cellular microenvironment (e.g., intracellular O 2 , pericellular O 2 or in close proximity to the cell sample. 2 The system can be integrated with system software that receives input from the environmental control modules (measurements) to allow the environmental control to provide the target cell microenvironment. The software can be programmed to cycle the environmental control modules to provide the target microenvironment according to a selected protocol.

[0217] [Signal processing module] High impedance transimpedance amplifiers are susceptible to parasitic current paths. Such parasitic current paths can be caused by flux residues from soldering and manufacturing or surface contamination from surface cleaners. Parasitic current paths can also be exacerbated by high humidity environments and the absorption of moisture in the dielectric materials used to insulate the conductive paths.

[0218] The device disclosed herein is designed to reduce parasitic current paths by including a signal processing module capable of operating at high relative humidity, for example, 75%, 85%, or even 95% relative humidity. It has been unexpectedly discovered that the performance of the signal processing module at high relative humidity allows assays and experiments to be performed on the instrument for longer durations. In some embodiments, performing an assay for a longer duration requires higher humidity and / or longer humidity time, which requires electronic circuitry in the device to be more robust for operation at higher humidity and / or longer humidity time. Thus, real-time cell data can be collected from cell samples without adversely affecting the sensitivity of the sensor unit, and assays can be performed for longer than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours.

[0219] The signal processing module is a processor operatively connected to the array of sensor units configured to receive and amplify signals from the sensor units. The signal processing module can receive and amplify multiple signals from the array of sensor units simultaneously or individually, e.g., sequentially. In some embodiments, the signal processing module can be capable of adjusting the amplification of the signal to acquire data at a faster or slower rate, e.g., reducing the amplification to increase the acquisition rate. In some embodiments, the signal processing module is configured to operate with reduced parasitic currents, e.g., interference, dark current, or noise, associated with detection and / or amplification of signals from the sensor units.

[0220] The signal processing module can be configured to detect the signal using time-based detection or intensity-based detection. Briefly, radiation emitted by the excited probe can be measured in intensity units and / or in the lifetime / time domain (including, for example, decay rate, phase shift, or anisotropy detection). Intensity-based detection can include detection and / or processing of ratiometric measurements. Briefly, the measurement can include a analyte-sensitive signal measurement and a analyte-insensitive or mostly analyte-insensitive reference measurement. A ratio between the references can be incorporated to facilitate a ratiometric evaluation of the analyte flux or concentration.

[0221] In some embodiments, the signal processing module includes a printed circuit assembly formed of an insulating material having a high dielectric constant. In some embodiments, the signal processing module can include one or more photosensitive components, such as semiconductor diodes, photomultiplier tubes, avalanche photodiodes, CMOS sensors, CCDs, etc. In some embodiments, the one or more photosensitive components can be connected to a transimpedance amplifier. In some embodiments, the signal processing module includes a printed circuit assembly having a transimpedance amplifier including a grounded guard trace. In some embodiments, the signal processing module includes a printed circuit assembly having an integrator design. In some embodiments, the signal processing module includes a printed circuit assembly having an op-amp design. In some embodiments, the signal processing module includes a printed circuit assembly formed of surface mount components, and is substantially free of, e.g., secondary hand soldered high gain components. In some embodiments, the signal processing module includes a printed circuit assembly including a thermally conductive excitation source, and optionally, the thermally conductive excitation source is in thermal communication, e.g., thermal contact, with a heat sink. The thermally conductive excitation source can be any excitation source that changes intensity with respect to temperature, such as a laser diode or a light emitting diode (LED). In some embodiments, the signal processing module includes a printed circuit assembly having an integrator design. In some embodiments, the signal processing module includes a printed circuit assembly having an operational amplifier design.

[0222] Surprisingly, it has been discovered that the design of the thermal conductive excitation source significantly reduces thermal drift, such that typically less reference correction is required, which in turn reduces correction errors and thereby improves the accuracy of the measurements (FIG. 38). The data shown in the graph of FIG. 38 shows the reduction in thermal drift after including the thermal conductive excitation source. In some embodiments, the improved design of the thermal conductive excitation source can reduce (or eliminate) the need to include a reference signal detector, reducing the complexity of the optical fiber routing and the cost of the device while achieving similar and / or improved performance. Thus, in some embodiments, the design of the signal processing module eliminates the need for a reference signal detector and / or a light source configured to generate a reference signal. The device can be without a reference signal detector.

[0223] The components and features of the signal processing module are further described in "Section 5: High Impedance Sensors" by Kester et al. (Appendix), which is incorporated herein by reference in its entirety for all purposes.

[0224] [Optical module] The apparatus may further include an optical module positioned to image or scan a sample in a sample carrier (e.g., a multi-well plate). The optical module may be positioned within the housing. The optical module may be operably connected to the controller. The optical module may be controlled or operated via a graphical user interface. Furthermore, images or scans acquired by the optical module may be viewed and / or recorded, optionally in real time, via the graphical user interface. Thus, in some embodiments, the optical module is operably connected to a computer, the computer configured to display and / or record images or scans of the sample in real time.

[0225] Cell-based assays, especially live cell assays, are becoming more and more common in the field of life science research. Sample carriers (e.g. microplates) are increasingly used as vessels for the investigation of cell proliferation processes by qualitative and quantitative means. In many cases, work with cells is carried out by researchers utilizing multiple specialized instruments.

[0226] Reading photoluminescence, e.g., fluorescence and / or phosphorescence, with an instrument having a light beam diameter large enough to obtain a representative measurement of fluorescence throughout the well, or a beam diameter to perform an area scan and mapping of the signal across the well, can be accomplished with dedicated conventional fluorescence readers or multi-detector readers. Most instruments provide for plate incubation, fluid injection, and also allow the option of gas control (CO2 and / or O2) similar to tissue culture incubators.

[0227] With wide-field imaging modalities, more information can be obtained from cells than just the fluorescent signal level of the well. Laboratory microscopes are commonly used, using bright field and phase contrast for unstained cells, and fluorescent imaging for stained cells. Some instruments allow for incubation chambers and environmental control. For clearer imaging or sectioning of 3D cell clusters such as spheroids, confocal microscopy is used as a third instrument choice.

[0228] Typically, these instruments are purchased from various vendors, and the user may be forced to physically transfer, for example, sample carrier (e.g., microplate) vessels from one instrument to another as needed, and may be forced to track the entire sample analysis process and collate and combine data from several instruments to obtain a complete comprehensive analysis of the cell sample. Without robotics, it may be nearly impossible to properly perform lengthy and complex experiments or assays. The use of robotics further increases both the cost and complexity of the analysis. The combination of non-imaging analysis modalities (fluorescence, absorbance, chemiluminescence), wide-field fluorescence imaging at the cell level, confocal fluorescence imaging, environmental control, and reagent injection in a single instrument may provide a complete comprehensive analysis approach, relieving the user from tedious sample carrier (e.g., microplate) handling, sample carrier (e.g., microplate) tracking, and data transfer. Described herein is an approach for a composite system that can store, collate, and analyze data acquired from individual instruments.

[0229] [Multimodal measurements in a cloud-based system] In certain embodiments, the devices and methods disclosed herein can be used to perform a complete analysis of a cell sample by qualitatively and quantitatively measuring various parameters of the same cell sample. The methods include measuring the metabolic function, bioenergetic balance, bioenergetic capacity, and bioenergetic work of cells, e.g., by measuring O using a sensing subsystem. 2 , CO 2The method can include visually observing characteristics of the sample, such as cell proliferation, cell health, cell microenvironment, morphological changes, ultrastructural changes, and marker expression, using an optical module with an automated cell imaging reader, such as Cytation® 5 or Cytation® 7, as disclosed in U.S. Pat. No. 10,072,982, which is incorporated by reference in its entirety for all purposes. The method may be any of the methods described in U.S. Pat. Nos. 10,551,371, 10,539,523, 10,215,748, 10,067,121, 9,709,548, 9,612,234, 8,263,375, 8,041,515, 8,026,080, 7,470,533, 7,468,255, 7,560,269, 7,732,127, or U.S. Pat. This can include detecting attachment, ultrastructural changes, augmentation, morphological changes, and cell-cell interactions by impedance measurements using sensing systems or devices such as those described in Application Publication No. 2018 / 0246019 and International Application Publication No. 2021 / 202264, each of which is incorporated herein by reference in its entirety for all purposes.

[0230] Cell-substrate impedance monitoring generally allows for continuous real-time monitoring of cells. Cell-substrate impedance monitoring can be used to assess interactions between cells and electrodes, where changes in cell attachment, growth, morphology, and motility on the electrode result in detectable changes. To this end, cell-substrate impedance monitoring is a useful tool that can be employed to assess cell proliferation and cell lysis. In combination with real-time cell analysis by impedance, as described in U.S. Patent Application Publication No. 2021 / 0301245 (which is incorporated herein by reference in its entirety for all purposes), the bright field and fluorescence detection optical modules of xCELLigence® eSight are exemplary optical modules that provide live cell imaging during impedance measurements.

[0231] It will be appreciated that successive analyses may be performed by further instruments performing different measurements of the same sample, such as mass spectrometry, spectroscopy, phosphorescence lifetime imaging microscopy (PLIM) including two-photon excitation imaging and / or fluorescence lifetime imaging microscopy (FLIM).

[0232] [Device combination] In certain embodiments, cells can be analyzed sequentially by performing successive measurements of the same cell sample. Samples can be analyzed in any order, 2 , CO 2The bioenergetic work of cells, such as pH, can be measured. The data can be stored in a cloud-based storage and can be optionally analyzed on a cloud-based data processing visualization system. Samples from the same cell sample, different samples, or the same cell line can be analyzed using electrochemical measurements, such as impedance measurements. The data can be stored in a cloud-based system. Samples from the same cell sample, different samples, or the same cell line can be visually observed for cell growth and morphology. The data can be stored in a cloud-based system. Data obtained from independent measurements can be correlated with the corresponding sample / measurement, for example by labeling the samples with a barcode or other digital identification system. The data can be collected and collated in a cloud-based storage and can be optionally processed in a cloud-based data processing visualization system. The collated data from analyzing the same cell sample can be queried for patterns and information.

[0233] Each of the measurements can be performed within the equipment described herein, or a combination of equipment, each operably connected to a data storage and processing system, e.g., a cloud-based system or computer.

[0234] In certain embodiments, samples can be analyzed in parallel by taking one or more aliquots of the original cell sample or samples from the same cell line to generate multiple substantially identical cell samples for each measurement to be made, for example to generate three or more corresponding substantially identical samples. Each sample can be analyzed in a separate instrument as described above. The samples can be analyzed simultaneously or substantially simultaneously. Data can be collected and collated in a cloud-based storage system as described above. The collated data can be queried for patterns and information as described above.

[0235] In certain embodiments, the sample or an aliquot of the sample is 2 , CO 2 The cells can be analyzed to measure the bioenergetic work of the cells, for example by measuring parameters such as pH, or other metabolic related parameters, as well as being visually observed for cell growth and morphology, e.g., simultaneously or substantially simultaneously. In some embodiments, the sample or an aliquot of the sample is subjected to O 2 , CO 2 The cells may be analyzed to measure the bioenergetic work of the cells, for example by measuring parameters such as temperature, pH, or other metabolic related parameters, and may be simultaneously, i.e., simultaneously or substantially simultaneously, analyzed for electrochemical measurements, e.g., impedance measurements. In some embodiments, the sample or an aliquot of the sample may be visually observed for cell growth and morphology, and may be simultaneously, e.g., simultaneously or substantially simultaneously, analyzed for electrochemical measurements, e.g., impedance measurements.

[0236] It should be noted that although the present disclosure generally refers to measuring metabolism, similar methods can also be used to measure or detect cellular microenvironment features, such as environmental conditions experienced by a sample. The conditions can be manipulated toward desired microenvironmental conditions, possibly via environmental control. The conditions can be manipulated to relate to cellular responses. As an exemplary embodiment, impedance, specific imaged cellular parameters, cellular oxygenation, fluorometric parameters (e.g., cellular metabolism) that vary as a function of oxygen or pH can be controlled to realize models to depict the effects of tumor microenvironmental conditions on cellular function. As a further example, such characteristics can be controlled to analyze the beating rate and / or metabolism of cardiomyocytes in response to reduced oxygen and / or nutrient availability, where the beating rate is controlled pharmacologically or using electrical pacing via a device.

[0237] The embodiments described herein overcome the above-mentioned drawbacks, as well as other drawbacks not described above, and an embodiment is not required to overcome the above-mentioned drawbacks, and an example embodiment may not overcome any of the above-mentioned problems.

[0238] According to one aspect of the exemplary embodiment, there is provided a device for analyzing one or more samples, the device including a support for a receptacle for holding the samples, an imaging subsystem for imaging the samples, and an analysis subsystem for analyzing the samples.

[0239] According to one aspect of an exemplary embodiment, there is provided a method of sample analysis comprising selecting at least one subsystem from among a plurality of subsystems of a sample analysis device for inspecting one or more samples, the plurality of subsystems including an imaging subsystem for imaging the one or more samples and an analytical subsystem for analyzing the one or more samples, and controlling the at least one selected subsystem to perform an inspection on the one or more samples, the inspection including an imaging operation of the imaging subsystem for imaging the one or more samples and an analytical operation of the analytical subsystem for analyzing the one or more samples.

[0240] According to one aspect of an exemplary embodiment, there is provided a non-transitory computer readable medium having a program embodied thereon which, when executed by a computer, causes the computer to perform a method for inspecting a sample, the method including: selecting at least one subsystem from among a plurality of subsystems of a sample analysis device to inspect one or more samples, the plurality of subsystems including an imaging subsystem to image the one or more samples and an analytical subsystem to analyze the one or more samples; and controlling the at least one selected subsystem to perform an inspection on the one or more samples, the inspection including an imaging operation of the imaging subsystem to image the one or more samples and an analytical operation of the analytical subsystem to analyze the one or more samples.

[0241] According to one aspect of the exemplary embodiment, a device for analyzing a sample is provided. The device may include a receptacle support configured to support a sample carrier (e.g., a microplate) including a sample carrier (e.g., a microplate) well configured to hold a sample, also referred to herein as a sample carrier, e.g., a multi-well plate, a plate, or a sample carrier. In one embodiment, imaging of the sample is performed using an automated cell imaging reader, such as Cytation® 5, Cytation® 7, or other instrument, as disclosed in U.S. Pat. No. 10,072,982, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, imaging of the sample is performed using a confocal imaging device comprising a receptacle support configured to support a sample carrier (e.g. a microplate) including a sample carrier (e.g. a microplate) well configured to hold a sample, an objective lens configured to image the sample, a laser point scanning confocal system configured to image the sample through the objective lens, and a spinning disk and / or wide field imaging system configured to image the sample through the objective lens, wherein at least a portion of both the laser point scanning confocal system and the spinning disk and / or wide field imaging system are movably mounted such that the laser point scanning confocal system and the spinning disk and / or wide field imaging system are configured to be selectively aligned with the objective lens to image the sample.

[0242] It will be appreciated that the cell sample may be observed using any type of imaging modality that allows for visual inspection of the cells.

[0243] In certain embodiments, the cell sample can be observed using phosphorescence lifetime imaging microscopy (PLIM), including two-photon excitation imaging, and / or fluorescence lifetime imaging microscopy (FLIM).

[0244] In certain embodiments, an imaging modality known as confocal imaging may be suitable for imaging cell samples, e.g. 3D cellular structures such as spheroids. In confocal imaging, a point or a portion of a sample may be illuminated at a time. Light may be passed through a small aperture, e.g. a pinhole, positioned in an optically conjugate plane. Point illumination significantly eliminates out-of-focus and background light, thereby improving the optical resolution and contrast of the image. The complete image, constructed or stitched point-by-point via the scanning function, is very sharp and has well-defined features. The scanning function may be performed using a spinning disk, also known as a scan disk or Nipkow disc.

[0245] Confocal imaging is a particularly suitable imaging modality for use with spheroids: it allows spheroids to be sectioned layer by layer and 3D models to be created in silico for both accurate cell counting and 3D image manipulation to view the spheroids from various angles.

[0246] Figures 13A and 13B are comparative images of spheroids. Figure 13A shows a spheroid photographed at twenty times (20X) magnification using wide-field imaging. Figure 13B shows a spheroid photographed at twenty times (20X) magnification using confocal imaging. The size of the spheroid can be assessed using the image in Figure 13A, but individual cells and spheroid structure only become visible with confocal imaging in Figure 13B.

[0247] The resolution advantage resulting from confocal imaging in FIG. 13B comes at the expense of reduced light intensity caused by the confocal aperture, so longer exposure times are often required compared to wide-field imaging in FIG. 13A.

[0248] The addition of confocal fluorescence imaging to an instrument that also includes non-imaging analytical modalities (fluorescence, absorbance, chemiluminescence, etc.) and wide-field fluorescence imaging at the cellular level, combined with a controlled live-cell environment, may provide modern researchers with the single most versatile instrument for analyzing sample carrier (e.g. microplate) based assay formats, including those aimed at studying 3D cell spheroids.

[0249] In one example, wide-field imaging is performed for faster screening, while O captured from the sample is 2 , CO 2 , there may be a workflow in which confocal imaging is performed for publishing images related to pH measurements.

[0250] Widefield imaging can be performed for HCS type assays, where widefield imaging has faster throughput and the resulting image analysis remains statistically robust. Confocal imaging can then be employed to capture representative wells of "hits" compared to "controls" for publication or presentation purposes.

[0251] In one example, there may be a workflow where widefield imaging is performed for a quick initial screening of spheroids based on size, then confocal imaging is used for a deeper assessment of the size of each "hit" well based on nuclear count, which is more accurate when confocal imaging is used.

[0252] Typically, wide-field imaging cannot "see" deep enough into the 3D spheroid to reliably count individual nuclei, but wide-field can still determine "hits" based on the overall spheroid size. Once "hit" wells are identified using wide-field imaging, the identified wells may then be imaged with confocal imaging to provide improved image analysis to count all nuclei in the spheroid, which could not be done using wide-field imaging alone.

[0253] In one example, there can be a proliferation assay (3D Endothelial Cell Spheroid Assay) to determine candidate wound healing drugs / compounds / substances. Primary drug / compound / substance screening can be performed in a sample carrier (e.g., a microplate) where small endothelial spheroids are treated with a library of unknown compounds to determine which compounds induce increased cell proliferation / growth. Compounds that cause further proliferation can be candidates for further wound healing studies.

[0254] In the analysis workflow, a plate reader can be used to rapidly screen sample carriers (e.g., microplates) using GFP fluorescence intensity to determine wells with increased size spheroids. Wells that meet a threshold GFP intensity (the threshold is statistically determined during assay development) are considered "hits" and selected for further imaging. A control well is always further imaged as a reference well for comparison with the hit wells. Confocal imaging of the 3D spheroids can be performed to acquire a two-channel z-stack image set (Hoescht33342 nuclear marker and GFP marker) of the entire spheroid sample. In image processing and analysis of the maximum projection of the Z-stack, the cell count of the spheroid is determined to quantify the size of the spheroid. A visual inspection of the distribution of the nuclear mask in the image is performed to determine the presence or absence of cell death within the spheroid. The results from the image analysis of the hit wells are then compared to the control to determine the percentage increase relative to the control.

[0255] In an exemplary workflow, a 3D tumoroid cytotoxicity immune response assay (3D tumoroid assay from surgical specimens to determine immune and cytotoxicity treatment response) is performed. This assay involves culturing tumoroids obtained from animal models or surgical specimens from patients. Since these tumoroids are from animals / patients, the immune cell response from the tumors in vitro can be evaluated, allowing the analysis of tumor response to various treatments. This assay can use heterogeneous multicellular tumor models to evaluate the efficacy of novel treatments in a sample carrier (e.g., microplate)-based format.

[0256] For example, tumoroids can be stained for nuclei counting (e.g., blue) and for immune cell markers (e.g., red). A sample carrier (e.g., microplate) reader can be used to evaluate wells with high cytotoxicity, indicated as a low blue signal, and wells with high immune response, indicated as a high red signal. Wells that meet one or both threshold criteria for cytotoxicity or immune response (thresholds are statistically determined during assay development) are considered "hits" and selected for further imaging. Control wells are always also further imaged for comparison with the hit wells. Confocal imaging of 3D tumoroids is performed to acquire a two-channel z-stack image set (Hoescht33342 nuclear market and CY5 marker) of the entire tumoroid sample. Image processing and analysis are performed on the maximum projection of the z-stack, and cell counting of the tumoroids is performed to quantify the cell count. For immune response, the count of red positive cells is determined. Results from image analysis of the hit wells are compared to the control to determine the percentage of cytotoxicity or immune response relative to the control.

[0257] Some of the above examples take advantage of the ability of a single instrument to perform the assay as a "hit picking". An initial rapid read identifies samples of particular interest using a rapid read method, which may typically be a fluorescent non-imaging read or a fluorescent or brightfield wide-field imaging read performed at low magnification. Once wells of interest, called hits, are identified, a second, more time-consuming modality is deployed to determine results of particular interest. This process is particularly important when the final result is high-resolution confocal imaging, where large data stores are required and collecting vast amounts of information for only a few samples of interest provides significant savings in data store space. This process also saves processing time during data acquisition and data viewing, since most samples are not "hits" and are discarded during the initial assay steps. A single unified device for performing various heterogeneous processing steps may streamline the analysis.

[0258] Other applications of the capabilities of a single instrument with multiple functions to study spheroids are possible. Spheroids are typically grown in round-bottom wells. Often, for the final imaging step, the spheroids are transferred to flat-bottom plates to prevent the round well bottom from acting like a lens during imaging, thereby unnecessarily inducing optical aberrations and adversely affecting the resulting image quality. High-quality microscope objectives are not designed for such "round-well" bottom lenses in the optical path. After transfer to another well, dish, or plate for best image quality, the exact location of the spheroid in the well is no longer known. In a preferred embodiment, wide-field imaging with low magnification but large field of view may be performed to image the well to locate the spheroid (area of ​​interest) and position the well to align the location of the found spheroid (area of ​​interest) with the optical axis, then a high magnification objective with a smaller field of view is used to image the spheroid in a confocal modality, performing a Z-stack by collecting multiple images while the objective moves along the focus axis of the objective perpendicular to the well bottom. The spheroid (area of ​​interest) can be identified by using the non-imaging analysis modality of the instrument by performing a fluorescent reading area scan and selecting the area of ​​maximum fluorescent signal far imaging.

[0259] FIG. 14 is a block diagram illustrating a multi-detection system according to an embodiment.

[0260] 14, the multi-detection system includes a controller 1000, a fluid injection subsystem 1100, an imaging subsystem including a wide-field imaging component 1200 and a confocal imaging component 1500, a non-imaging analysis subsystem 1300, an imaging illumination subsystem 1600 for wide-field imaging, a housing 1900, a sample carrier (microplate) 300, a transport (e.g., a microplate transport) 310 for the sample carrier, an incubation chamber 320 for incubating the samples in the wells 200, an environmental control subsystem 2000, and a confocal imaging subsystem. The multi-detection system may also include an external subsystem 2100.

[0261] Samples are deposited in wells 200 (e.g., microwells) of a sample carrier (e.g., microplate) 300. The sample carrier (e.g., microplate) 300 is transported in and out of a measurement incubation chamber 320 by a sample carrier (e.g., microplate) transport 310. When positioned to be exposed to the external environment of the multi-detection system, the sample carrier (e.g., microplate) 300 can be accessible outside of the incubation chamber 320 and / or housing 1900 for access by a technician or a robotic arm. Once the sample carrier (e.g., microplate) 300 is positioned within the chamber, a variety of supported imaging and non-imaging analysis modalities can be performed.

[0262] The sample carrier (e.g., microplate) transport 310 is part of a sample carrier (e.g., microplate) transfer subsystem for position manipulation of the sample carrier (e.g., microplate) 300 and may include any suitable combination of belts, platforms, sample carrier (e.g., microplate) holders, motors, and positioning software running under hardware control for position manipulation. When the sample carrier (e.g., microplate) 300 is placed in the incubation chamber 320, the entire sample carrier (e.g., microplate) 300 remains incubated. The incubation system and incubation chamber 320 are described in detail below.

[0263] The non-imaging analytical subsystem 1300 may be based on illumination via flash bulbs, dual excitation and dual emission monochromators, photomultiplier tubes (PMTs), and silicon detectors. The non-imaging analytical subsystem 1300 supports absorbance, fluorescence, and chemiluminescence analytical modalities for detection of corresponding properties of the samples in the wells 200. The non-imaging analytical subsystem 1300 may be implemented as a filter-based subsystem or as a hybrid of any or all of the above.

[0264] The imaging subsystem includes wide-field imaging components 1200 and confocal imaging components 1500, such as objectives, lenses, LEDs, filter cubes, spinning disks, cameras and other components. The imaging illumination subsystem 1600 includes illumination components for wide-field imaging and can provide illumination for bright-field, color bright-field, and phase-contrast imaging modalities.

[0265] The external subsystem 2100 may be an external confocal illumination subsystem for confocal imaging that may be modularly connected to and disconnected from an imaging subsystem within the housing 1900 via optical fibers to add flexibility to the physical arrangement of the external subsystem 2100 relative to the instrument. Alternatively, the confocal imaging illumination subsystem may be arranged to be integrated within the housing 1900.

[0266] The fluid injection subsystem 1100 injects reagents into the wells 200 as required by the assay. The fluid injection subsystem 1100 can include any combination of pumps, reservoirs, lines or tubing, pipettes and tips, and software running under hardware control to deliver, and if necessary, aspirate fluids to and from the wells.

[0267] The environmental control subsystem 2000, shown disposed externally to the housing 1900, can include a gas control module that provides control of atmospheric conditions inside the housing 1900. Other control modules can include modules for control of temperature, humidity, and other conditions that can be controlled within the housing 1900 under the control of the environmental control subsystem 2000. The environmental control subsystem can include any combination of pumps, reservoirs, lines or tubes, fans, and heating and cooling elements, etc., to control all conditions within the housing 1900. The housing 1900 contains most of the subsystems and defines a physical space in which a gaseous atmosphere conducive to living cells can be effectively maintained and controlled by the environmental control subsystem 2000.

[0268] The controller 1000 can control all operations of the multi-detection system. The controller 1000 can communicate with each of the various subsystems in the multi-detection subsystem via wires or wirelessly. The controller 1000 can include any combination of hardware (e.g., CPU, memory, cables, connectors, etc.) and software for execution by the hardware to control the operation of the multi-detection system.

[0269] FIG. 15 is a block diagram illustrating a multi-detection system according to an embodiment.

[0270] The multi-detector system allows for several imaging modalities. In addition to the confocal imaging modality, wide-field imaging in fluorescence, bright field, and phase contrast can be performed. The optical elements of both the confocal and wide-field imaging systems are shown in FIG.

[0271] The sample carrier (e.g., microplate) 300 can be disposed on a transport 310 for the sample carrier (e.g., microplate transport) that positions the well 200 of interest in line with the imaging optical axis of the objective 1230. The objective can be selected from several objectives of various magnifications disposed on the objective turret 1232. The relative position of the imaging illumination subsystem 1600 is shown in FIG. 15, which can be used for brightfield, color brightfield, and phase contrast imaging of the sample. Many optical elements are shared between the widefield and confocal systems, and a more detailed description of such parts is provided below in FIGS. 16 and 17, where some elements of FIG. 15 have been omitted for clarity.

[0272] FIG. 16 is a block diagram illustrating a multi-detection system according to an embodiment.

[0273] Confocal imaging deployed as shown in Figure 16. The widefield imaging subsystem elements (e.g., LED cube 1201 and filter cube 1210) are automatically removed from the optical path to the sample and the system shown in Figure 15 is transformed into the confocal optical system shown in Figure 16 for understanding the confocal optical path.

[0274] A spinning disk confocal system is deployed as an exemplary embodiment of a confocal imaging system. The system is based on the use of a spinning disk (FIG. 18), an optical path. The disk is arranged in an intermediate image plane conjugate with the sample and the detection plane. Thus, the disk is in both the excitation and emission optical paths. The disk is typically about 2 mm thick in one exemplary embodiment and is made of glass or quartz. The disk can be coated to be opaque or to have a given transparency or opacity, except for transparent areas left as a pattern of pinholes or slits. Ideally, the disk surface is made not to reflect incident light. The sample to be imaged is illuminated by the excitation light transmitted through the pinholes. Only radiation emitted by the sample, originating from these illuminated spots on the sample, reaches the detector through the pinholes in the disk. The pinholes or slits are numerous but spaced apart from each other so that they act optically independently. Energy from neighboring pinholes ideally does not affect the sample spot illuminated by a given pinhole. The disk spot pattern is typically arranged in several spirals, as shown in Figure 18. The disk is controlled to spin continuously, thus scanning the sample. As the disk rotates, the sample is illuminated one spot at a time, and the complete sample image is detected on the detector for reconstruction as a complete image of the sample.

[0275] Returning to FIG. 16, the confocal light source 1540 can be any light source suitable for confocal microscopy. For example, the confocal light source 1540 can be a solid-state light source, such as a light emitting diode (LED) or a solid-state or semiconductor-based laser (laser diode). In an exemplary embodiment, the output tip of the optical fiber can be the light (radiation) source. Radiation is taken as an embodiment because the excitation spectrum can be outside of the 380 nm to 630 nm range commonly referred to as light. However, the term "light source" is more commonly used in imaging, and the term light is used interchangeably herein with radiation. The input tip of the fiber may be illuminated, i.e., illuminated, from a light source module external to the instrument, providing flexibility in selecting the best light source matching the sample imaging needs. The fiber also provides flexibility in branching inputs from multiple external light sources. The output tip of the fiber is imaged by a condenser 1522 onto or near the intermediate sample image plane where the spinning disk 1504 is located. Light from the fiber may be sent through an excitation filter 1531 and then reflected off a dichroic mirror 1533 and focused by a tube lens 1520 onto the spinning disk 1504. Here and throughout this specification, the term "lens" may refer to a single lens or a group of lenses depending on the embodiment and function as will be understood by those skilled in the art. As discussed, the disk has a spiral pattern of slit holes. A field lens 1519 minimizes light loss and directs the light leaving the disk to be collected by a tube lens 1250. The tube lens 1250 directs the excitation radiation via mirror 1220 to the objective lens 1230. The objective lens 1230 illuminates a small spot on the sample near the bottom of the well. The sample components are stained with a dye that corresponds to the excitation wavelength. These components are excited by the incident radiation and typically emit radiation having a longer wavelength. This emitted light is directed to a detector as follows:

[0276] Light emitted by the sample is collimated by objective lens 1230, reflected by mirror 1220, and collected by tube lens 1250 and field lens 1519 onto spinning disk 1504. An intermediate image of the sample in the emitted light is formed on the surface of spinning disk 1504. Tube lens 1520 and lens 1521 invert the image to form a sample image at detector 1560, which is typically a pixelated digital camera such as a charge coupled device (CCD) camera or a complimentary metal-oxide semi-conductor (CMOS) camera. The sample image is captured by the camera, can be stored in the memory of the multi-detection system or an external computing system, may be enhanced and analyzed for various properties, and / or may be presented to a user on a visual display.

[0277] A confocal cube 1530 (e.g., a confocal excitation / dichroic mirror / emission cube) is shown between the tube lens 1520 and the lens 1521, which is a configuration for fluorescence microscopy. The filters and dichroics can be thin film coatings on glass. The excitation filter 1531 forms a bandpass for excitation, the emission filter 1532 forms a bandpass for emission, and the dichroic mirror 1533 separates the excitation and emission to fully use the available energy and suppresses the amount of excitation light reflected from multiple optical surfaces that reach the detector as the excitation light travels toward the sample, including the disk surface. The lens 1521 (e.g., emission filter) provides the majority of the excitation light suppression, but the dichroic mirror 1533 also plays a role in suppression. An alternative configuration of the described cube could be several filter wheels carrying excitation filters, emission filters, and dichroics. In the illustrated embodiment, the cube is one way of arranging the described elements, allowing very easy replacement by the user when imaging needs change. Some filter cubes (e.g., confocal cube 1530) may be placed on motorized sliders and identified by settings in software executed by the user, or may be electronically or optically labeled with a code that is automatically read via a barcode or some other automatically available method.

[0278] The surface of the spinning disk is imaged onto the detector along with the sample. Therefore, dust particles that adhere to the disk surface may appear as artifacts in the image, such as bright streaks of light due to the rotation of the disk. Small particles can easily adhere to the disk surface with enough force to resist the centrifugal force. The spinning disk 1504 and the disk drive motor 1509 are part of the disk module 1553. The disks in the module are typically assembled in a clean environment, such as a clean room, and are sealed from the surrounding environment to prevent dust particles from accumulating on the disk. Windows 1551 and 1550 in the module allow light to pass through but do not allow dust to pass through. Ideally, these dust-proof windows should be located as far away as possible from the intermediate image plane so that dust that may accumulate on the window glass does not result in artifacts in the image. The disks are fully contained within the disk modules 1502 and 1553. Therefore, users should not open the modules to avoid introducing dust particles to the disks.

[0279] 16 shows two disk modules 1553 and 1502 mounted on a multi-detection instrument. The disks can be moved to position one disk or the other into the optical path. Alternatively, both disks can be moved out of the optical path and out of the space 1501 disposed along the optical axis. This allows wide-field imaging modalities such as fluorescence imaging, bright-field imaging, or phase contrast imaging to be performed.

[0280] A major advantage of allowing the user both confocal and wide-field imaging options in the same instrument is that images of various imaging modalities can be overlaid, for example the same image in a wide-field image and a confocal imaging modality. Alternatively, a bright-field image can be used to locate the area of ​​interest and then confocal imaging. For this configuration to properly acquire images, the magnification of both modalities should match exactly, otherwise the images will not be overlaid properly. The light in the section between the tube lens 1520 and the tube lens 1250 is not parallel. In the confocal modality, there are several flat windows in the light path in this section, such as the confocal disk and dust-proof windows. In the wide-field modality, there is no need for these windows. However, to match the optical path length of the non-parallel optical path, glass 1505 is added in the space 1501 between the confocal disks where the wide-field imaging takes place. This ensures that the sample remains in focus with respect to the fixed objective lens position even when the image modality is changed. This ensures that the magnification is consistent between the confocal and wide-field imaging modes. The thickness of glass 1505 should match the sum of the flat windows of the disk used in confocal imaging (window 1551, spinning disk 1504, and window 1550). Glass 1505 should be placed as far as possible from the intermediate image plane so that dust that may accumulate on the glass does not result in artifacts in the image.

[0281] The size of the pinhole on the confocal disk is ideally selected based on the parameters of the imaging objective 1230. In an embodiment, the size of the image of the disk pinhole created on the sample can be matched to the distance between the first two minima of the Airy diffraction pattern of the objective. The formula for the disk pinhole size given in "Introduction to Spinning disk microscopy" by Zeiss is: Diameter of the pinhole in the disk = 1.2 * objective lens magnification * emission wavelength / objective lens numerical aperture

[0282] Both the numerical aperture (NA) and magnification of the objective are part of this equation. If the pinhole is too small, too much light will be lost and it will take a long time to capture an image. If the pinhole is too large, the confocal effect may be reduced or lost entirely. Most commercially available spinning disk microscopes employ non-interchangeable spinning disks with pinholes in the range of 50um to 70um. This works well enough as a compromise with the high magnification objectives typically deployed in confocal microscopy. However, it is preferable that a disk with an appropriate pinhole can be matched to the objective being used.

[0283] Some spinning disks do not have a spiral pattern of round holes, but instead employ a slit aperture. The slit aperture can provide a brighter illumination of the sample and a stronger luminescence signal, while the pinhole aperture can provide a better axial resolution. Therefore, for some imaging applications, including biological fluorescence applications, the slit can be preferred to allow for shorter image acquisition times, which is another reason to change the disk even with a fixed objective lens.

[0284] Multiple discs may be presented to the imaging device and selection among the discs may be performed by the user or automatically by a multi-detection system.

[0285] 16 shows an example of two disk modules 1502, 1553 used in a multi-detection instrument. All disk modules may be configured to be interchangeable by the user. Modules may be identified by settings in user-controlled software to allow automatic configuration by the multi-detection system, or may be electronically or optically labeled with a code that is automatically read via a bar code or any other available method.

[0286] A further advantage of the modular disk module is that the user can clean windows 1551 and 1550 when the disk module is removed from the device and both windows are easily accessible, which can provide dust protection.

[0287] The identification of the modules allows for automated software setup and automatic resetting and calibration of the axial position of the modules in the optical path. In a spinning disk confocal imager, the plane of the disk surface, the detector sensing element plane, and the sample plane should be conjugate to each other. This means that when tracing the emitted light from the sample, the image of the sample plane coincides with the disk plane, and the images of the disk plane and the sample plane coincide with the detector plane. The sensitive tip plane of the detector 1560 is fixed by the camera design. The objective lens 1230 can be moved along the focal axis to sharpen the sample image on the detector. The disk should then ideally be disposed in an intermediate plane conjugate to both the detector plane and the intermediate sample image plane, so that all three planes are conjugate. In the proposed embodiment, the disk module design keeps the axial position of the disk very close to the ideal conjugate position, but the final position of the disk surface may be automatically adjusted by observing the disk pattern on the detector and sharply focusing this pattern on the detector. Several image-based focusing methods are available and well known in the industry. Once the optimal disk surface location is found, this location may be stored in software and memory and associated with the disk module. If a disk module is removed and reinstalled, the correct disk location may be automatically restored by the software. If a new disk module is introduced, the system will instead run a disk focusing routine and select the optimal axial location for the new disk module. The user may thus be relieved from having to keep track of which disk modules are deployed in the device and their various positioning.

[0288] Alternatively, if only a few disk modules are expected to be used, the user may configure the disk modules via a settings screen in the calibration portion of the user interface of the software included in the multi-detection system.

[0289] The two concepts of user-interchangeable disk modules and automated axial disk positioning are best worked in tandem, but can also be implemented separately. If automated axial disk positioning is not available, the disk modules can be configured to be interchangeable with some criterion on the module that ensures disk position and proper placement in the instrument. The concept of an easily interchangeable disk module that does not need to be opened by the user and therefore not exposed to the environment is still applicable and may benefit users who want the flexibility of multiple disks to best suit their deployed imaging objectives and samples.

[0290] Even if disk modules are limited to one or two in the device, automatic axial adjustment may be used to alleviate the need to tightly control the location of the detector image sensor sensitive surface on the detector 1560 (such as a camera), allowing maximum user flexibility in camera selection and allowing camera upgrades in multi-detector systems. If the sensor surface is moved after a camera change, the disk surface may be automatically repositioned to be conjugate with the sensor surface via an image-based autofocus routine.

[0291] FIG. 17 is a block diagram illustrating a multi-detection system according to an embodiment.

[0292] In Fig. 17, wide-field imaging is shown deployed in an exemplary embodiment. As explained above, the optical part (with elements labeled 15xx) allows both confocal imaging (with spinning disks 1504 or 1503 in the optical path) and wide-field imaging (through the space between disks 1501). However, for wide-field modalities that researchers may want to deploy in a single universal instrument, there may be drawbacks to using this optical system and the confocal light source 1540 and confocal cube 1530. For confocal imaging, the excitation radiation should be directed onto the disk through multiple optical elements (e.g. dichroic mirror 1533, tube lens 1520, window 1551) positioned prior to the disk surface. After the disk, the excitation radiation is directed to the sample through more optical elements (e.g. window 1550, field lens 1519, tube lens 1250, mirror 1220, objective lens 1230). For confocal imaging, this approach is not an option. However, on any surface encountered, some of the excitation light is reflected. A suitable design then relies on careful ray tracing to ensure that the reflected light is kept as far away from the detector as possible, and an emission filter 1532 to suppress unwanted reflected light. The optical elements preceding the disk surface, such as the tube lens 1520 and window 1551, and the spinning disk 1504 surface, are exposed to very strong levels of excitation radiation that are partially reflected. Any dust particles may also be excited to fluoresce. Despite the designer's best intentions, some of the light will enter the detector, reducing the signal-to-noise ratio. Thus, a non-fluorescent sample that would otherwise appear very dark on the image may not appear very dark. This may be due to a noticeable background signal from reflected light, the effect of which tends to be uniform across the entire image. For wide-field microscopy using the confocal partial excitation element described above in FIG. 16, significant compromises in image quality and system capabilities may be involved.

[0293] In an exemplary embodiment, an alternative subsystem that can be used for wide-field fluorescence imaging is included in the same instrument. The confocal cube 1530 of the confocal subsystem is positioned out of the path, and the spinning disk module is positioned in the space 1501 for wide-field imaging. This transforms the configuration of FIG. 15 into that of FIG. 17. The dedicated wide-field sub-elements are the LED cube 1201 and the wide-field excitation / emission / dichroic imaging filter cube 1210. The excitation filter 1211, dichroic mirror 1212, and emission filter 1213 are mounted in a filter cube that will typically be matched with the LED cube 1201 for best signal-to-noise performance. Several of these cube pairs corresponding to the particular chemistry to be investigated may be included on the slider.

[0294] This design has several advantages.

[0295] First, the LED excitation optics are much closer to the sample, so the excitation light encounters fewer optical surfaces on its way to the sample, thus significantly reducing reflections from these surfaces that can reach the detector, improving the signal-to-noise ratio in the image.

[0296] Second, a wide range of commercially available LEDs are used in the LED cube 1201 which may not be powerful enough to be used in the confocal optical path, but which can provide sufficient excitation when placed close to the sample as shown in FIG.

[0297] Third, and of particular importance when samples need to be excited in the UV range, some objectives are rated as UV objectives, transmit ultraviolet light, and exhibit very low fluorescence when excited by UV. However, there is no guarantee that the commonly available optics for the rest of the optical path, such as tube lenses, will not fluoresce when illuminated by UV light. If a wide-field image of a sample stained with the common DAPI nuclear stain is required, a common approach in the confocal optical path is to use a wavelength of about 400 nm, thus avoiding strongly exciting the optics in addition to the sample. However, shifting the excitation from 360 nm, the ideal wavelength for DAPI stain excitation, towards 400 nm significantly reduces the light emitted. Researchers will need to put a higher concentration of dye in the sample or increase the gain of the detector, thus reducing the signal-to-noise ratio of the imaging. Ideally, excitation of the DAPI stained sample would be at 360 nm, but the UV excitation light would not pass through any optics that could fluoresce. The LED cube 1201 and filter cube 1210, in an exemplary embodiment, allow for just such an optimal option. The UV excitation light is incident only on the objective lens 1230, which may be selected not to fluoresce. The emitted light returns to the detector through multiple optical elements common to the confocal and widefield paths, but because the emitted light is in the visible spectrum, the optical elements it encounters typically do not fluoresce at the levels found in UV light.

[0298] 17 shows the relative locations of an imaging illumination subsystem 1600 for wide-field imaging in non-fluorescence modalities. This system may be a brightfield, color brightfield with three color LEDs switchable one at a time, or a phase contrast illumination system with a ring aperture that can be matched to a phase contrast objective.

[0299] Additional embodiments and components of the imaging system are further described in PCT Publication No. 2022120047, "Universal multi-detection system for microplates with confocal imaging," which is incorporated herein by reference in its entirety for all purposes. Such components include, for example, laser point scanning confocal (LSC) modalities, laser point scanning confocal (LSC) systems, spinning disk confocal systems, and wide-field capabilities in a single device.

[0300] Components of wide-field imaging systems are further described, for example, in U.S. Pat. No. 10,072,982, entitled "Universal multidetection system for microplates," which is incorporated herein by reference in its entirety for all purposes.

[0301] Configurations according to embodiments of the present disclosure may also incorporate or include a laser point scanning confocal system, a spinning disk confocal system, and wide field capabilities in a single device, as described below, although embodiments of the present disclosure may include any combination of the above systems and capabilities.

[0302] FIG. 19 illustrates a confocal disk imaging module according to an embodiment.

[0303] The disk drive motor 1509, a DC brushless motor in an exemplary embodiment capable of high speed rotation of several thousand RPM at a constant speed, is mounted to the housing base 1800. The spinning disk 1504 is secured on the motor shaft by hub pieces 1820 and 1830. A cover 1810 is mounted to the housing base 1800 to complete a dust-free environment for the disk. The disk is not accessible to the user. The optical windows 1550 and 1551 keep the interior of the module dust-free while allowing light to pass through. It is advantageous from an imaging standpoint to keep both windows as far away from the disk surface as possible within the overall space constraints to avoid dust on the windows affecting the image. The disk modules may be identified via barcode labels, simple binary code labels, or some other machine-readable means, so that the multi-detection system may automatically identify which disk modules are present and available at any given time.

[0304] With reference to FIG. 18, the disk speed and the exposure time of the confocal image need to be closely correlated. As seen in FIG. 10, the disk is equipped with multiple spirals, and as the disk rotates, the sample is swept by a pattern of pinholes. There is a minimum angle of disk rotation that is required to sequentially but completely illuminate the sample once. For most commercially available disks, and for the disk of the exemplary embodiment, this angle is 30 degrees. If the exposure time is not a multiple of the time to move the disk 30 degrees, some artifacts such as stripes will be visible in the image. This is a well-known problem in the industry. In the exemplary embodiment, the disk rotation speed is set to 2400 rpm and the exposure time is set to a multiple of one full rotation (e.g., 25 msec, 50 msec, 75 msec, etc.). This approach results in a good compromise between image quality and the shortest time to capture an image. This approach of using full revolution time exposure increments also resulted in minimizing image artifacts caused by potential non-concentricity between the spiral pattern of the disk and the axis of rotation of the disk.

[0305] FIG. 20 shows a disc changing mechanism and a disc focusing mechanism according to an embodiment.

[0306] 20, a disc changing mechanism and a disc focusing mechanism may be implemented in an exemplary embodiment. However, the configuration of the disc changing mechanism and the disc focusing mechanism is not limited thereto.

[0307] A base 1701 supports all elements of the mechanism. A linear track rail 1705, such as part of an IKO or HTK guide system, is attached to the base 1701. A linear track carriage 1706 supports a bracket 1710. The bracket 1710 is translated by a motor 1715 via a timing belt 1717 in a direction perpendicular to the optical axis. This movement allows either the disk module 1502 or the disk module 1553 or the space 1501 to be positioned in alignment with the imaging optical axis. Other mechanical implementations are possible, but the main advantage of the timing belt is the speed of exchange achievable with this particular method. Axis homing sensors and / or possible encodings are not shown for clarity.

[0308] Bracket 1710 also carries linear rail 1720 and motor 1725. In the exemplary embodiment, the motor shaft is in the form of a lead screw. The motor, via a lead nut 1727, translates support 1730 attached to linear carriage 1721 along the optical axis to provide axial focusing for the confocal disk. Axial homing sensors and / or possible encoding are not shown for clarity.

[0309] The disk module may be attached directly to the support 1730 and accessed by the user. This attachment may be via fasteners or via magnets for easy removal. Alternatively, the disk module may be attached to guides 1732, which may be secured to the support 1730 with a slip fit for easy removal from the device by the user.

[0310] As will be appreciated by those skilled in the art, other mechanisms may be deployed to accomplish the functions of accessing, positioning, and focusing the disk modules.

[0311] FIG. 21 is a diagram of a non-imaging analysis subsystem according to an embodiment.

[0312] Referring to FIG. 21, a non-imaging analysis subsystem 1300 of a multi-detection system is provided.

[0313] The analytical modalities of the non-imaging analytical subsystem 1300 can be absorbance, fluorescence from above and below, and chemiluminescence. The Xe flash bulb 13001 emits radiation in the range of 200 nm to 1000 nm. Two stages 13002 and 13003 of a fluorescence excitation / absorption dual monochromator select a narrow bandpass of the radiation. The radiation is guided towards the sample by fiber optic cables into either the absorbance channel via fiber 13030, the top fluorescence via 13005, or the bottom fluorescence via 13033. Since only one fiber is active at a time, there is no optical crosstalk between the various analytical modes. The absorbance is measured by a silicon detector 13060 via lenses 13040 and 13050.

[0314] Top fluorescence excitation and emission pickup is via lens 13020, which can move up and down to accommodate various sample carriers (e.g. microplates) and fluid levels. Bottom fluorescence is similarly achieved with lens 13055. Both top and bottom emission are guided by fiber optic cables to the first stage of the emission dual monochromators 13010 and 13011 and then to photomultiplier tube 13012. A chemiluminescence fiber 13021 may be connected directly to the photomultiplier tube to provide measurements for very low light levels via bypassing the monochromator.

[0315] Fluid injection subsystem 1100 provides the researcher with the ability to inject reagents via fluid lines 1112 and 1111 and rapidly measure the results of the injection via the analytical subsystem, further expanding the range of tests that can be performed on the device.

[0316] FIG. 22 illustrates an injection subsystem according to an embodiment.

[0317] With reference to Fig. 22, an optional injection subsystem is provided. Injection subsystem 1100 may be disposed on top of the multi-detection instrument as shown in Fig. 23, with fluid lines 1112 and 1111 fed through septum accesses at the top of the housing. Reagents are delivered to the microwells by pumps in fluid injection subsystem 1100 via fluid lines 1111 and 1112, which may be PTFE lines, as shown in Fig. 22, and delivered to the wells via injection needles 1102 and 1101.

[0318] Referring to FIG. 21, environmental control can be deployed in a multi-detection system.

[0319] As shown in FIG. 21, a transport 310 for sample carriers (e.g. microplate transport) supports a sample carrier (e.g. microplate) 300 and is located in an incubation chamber 320. This ensures that the sample carrier (e.g. microplate) 300 is maintained at the desired temperature at all positions of the sample carrier (e.g. microplate transport) 310 in the incubation chamber 320. The incubation chamber 320 may be constructed of a material suitable for maintaining a constant temperature, such as a continuous aluminum sheet, while still providing access to the optical elements through a small opening. The incubation chamber 320 is typically insulated. The design of such chambers will be known to those skilled in the art from many multi-detection instruments. A typical controlled temperature range can be from room temperature up to 65C.

[0320] FIG. 23 is a diagram illustrating a multi-detection system according to an embodiment.

[0321] For live cells, the temperature is typically 37C, but in addition, control of the gas around the sample is required. This control is achieved by filling the entire housing 1910 of the instrument in FIG. 23 with the appropriate gas mixture. This design avoids attempting to contain the gas controlled environment only in the measurement chamber or an isolation partition. The purpose of this design is to allow the atmosphere within the housing 1910 to equalize. Therefore, the design of the housing 1910 is made as airtight as possible by avoiding gaps in the housing and using soft gasket materials around the user access doors.

[0322] 24A-24C are diagrams illustrating a gas control subsystem according to an embodiment.

[0323] 24A-24C, an environmental control subsystem 2000 (e.g., a gas control subsystem) can be located external to the instrument. The environmental control subsystem 2000 allows a user to set the CO2 and / or O2 concentration levels in the chamber to higher CO2 and lower O2 than normal atmosphere. Gas sampling lines connect the environmental control subsystem 2000 to the interior of the instrument housing. Based on the composition of the gas sampled or extracted from the instrument through the sampling lines, a control system can adjust the flow rate of CO2 or N2 gas supplied to the instrument, for example, with the inlet gas dispersed using a small fan. This allows for the placement of all gas sensors and valves external to the main instrument, preserving the complexity and reliability of gas control in an external gas controller.

[0324] The combination of an incubation chamber around the XY transport movement zone and gas control of the atmosphere inside the housing and therefore around the sample carrier (eg, microplate) provides the user with the ability to perform long term live cell experiments.

[0325] Referring to FIG. 23, an external view of the entire instrument implemented in an exemplary embodiment and the elements subject to user interaction with the instrument is shown. A transport for sample carriers (e.g. microplate transport) 310 presents itself to the user (illustrated on the right), and a microplate 300 is placed on the transport for sample carriers (e.g. microplate transport) 310, for example by the user or a robotic arm, and then positioned in the multi-detection system. Access to the confocal cube 1530, the widefield LED cube 1201, and the widefield filter cube 1210, the confocal disk module and the objective lens 1230 is via the front of the instrument, via a door 1905. This makes it easy for the user to access most user-replaceable elements at once.

[0326] According to certain embodiments, an objective of the present disclosure (eg, objective 1230 or objective 2210) may be a fluid immersion objective.

[0327] One way to improve optical performance in microscopy is to use a fluid immersion objective. In optical microscopy, a fluid immersion objective is a specially designed objective lens used to increase the resolution of a microscope. According to an embodiment of the present disclosure, the optical system is an inverted microscope, meaning that the objective lens is located below the sample and views the sample from below. In the inverted microscope configuration of the present disclosure, when performing fluid immersion, a drop of fluid (e.g., water or other fluid) is placed on the objective lens and held in place by the surface tension of the fluid. The objective lens is then brought close to the sample, and the droplet is sandwiched between the sample and the objective lens. In this way, light passing to and from the sample to the objective lens does not pass through air. The refractive index of the fluid, which is higher than air, results in an increased numerical aperture. This increases the resolution and increases the signal level. According to an embodiment, the objective lens can be brought close to the sample, and then a drop of fluid is placed on the objective lens.

[0328] In addition to water immersion objectives, the objectives of the present disclosure can also be provided with other types of fluids to increase the numerical aperture. Some examples of fluids include, for example, oil and glycerol. In embodiments of the present disclosure, the fluid can be water, oil, glycerol, or any other type of fluid that can increase the refractive index.

[0329] With reference to FIGS. 27A and 27B, an immersion objective according to an embodiment of the present disclosure will be described below. According to an embodiment, the objective 1330 can include a sleeve 1332 that fits over the objective 1330. The sleeve 1332 can be configured to provide a fluid path into and out of the sleeve 1332. In addition, the sleeve 1332 helps to hold the droplet 33 of fluid in place. According to an embodiment, the sleeve 1332 has a port for pumping fluid in and a port for pumping fluid out. According to an embodiment, the inlet and outlet ports can be the same port 31, as shown in FIGS. 27A and 27B. With reference to FIG. 27B, an excess portion 34 of the droplet of liquid can exit the sleeve 1332 through the port 31. In an exemplary embodiment, the sleeve 1332 can be formed of, for example, anodized aluminum, plastic, or other materials.

[0330] According to an embodiment, referring to FIG. 28, a fluid pump system can be provided. The fluid pump system can include a first pump 1336, a second pump 1337, a first reservoir 1338 (source reservoir), and a second reservoir 1339 (waste reservoir), and fluid can be pumped from the first reservoir 1338 to the head of the objective lens 1330 by the first pump 1336. As shown in FIG. 28, the first pump 1336 can be a syringe pump. The fluid is then removed from the objective lens 1330 via the second pump 1337, which pumps the fluid to the second reservoir 1339. The second pump 1337 can be referred to as a waste pump or can be a syringe pump, as shown in FIG. 28. The first pump 1336 and the second pump 1337 can be other types of pumps that achieve the same or similar functions. The sleeve 1332 can fit over the objective lens 1330 and direct fluid to the top of the objective lens 1330, helping to hold the droplet of fluid in place. The sleeve 1332 can also have a waste port that can be configured to allow fluid to be removed from the sleeve 1332. The objective lens 1330 can be a specially designed objective lens optimized for fluid (e.g., water) immersion applications. In FIG. 28, the first reservoir 1338 and the second reservoir 1339 are shown as separate source and waste reservoirs, respectively. However, according to an embodiment, instead of two separate reservoirs, a single reservoir can be provided and fluid can be reused. In addition to this, the pump can be multi-purpose. For example, the BioTek C10 has a fluid dispensing module that can be used to dispense reagents to samples. This same dispense module may be configured to have additional purposes (including those of first pump 1336 and / or second pump 1337) to reduce costs.

[0331] 28, the objective lens 1330 can be attached to the objective lens turret 1232 by an objective lens coupling 1334. A description of the objective lens coupling 1334 is provided below with reference to FIG.

[0332] As shown in FIG. 29, the objective lens coupling portion 1334 may include a kinematic connection 1334A and a magnet 1334B configured to couple the objective lens 1330 and the objective lens turret 1232 together. For example, the objective lens 1330 may include at least one of a convex portion or a concave portion as a first portion of the kinematic connection 1334A, and the objective lens turret 1232 may include at least one of the other of a convex portion or a concave portion as a second portion of the kinematic connection 1334A corresponding to the first portion. The magnet 1334B may be included in one or more of the objective lens 1330 and the objective lens turret 1232. According to an embodiment, both the objective lens 1330 and the objective lens turret 1232 may include a magnet 1334B corresponding to each other and configured to connect to each other via a magnetic force. In other embodiments, only one of the objective lens 1330 and the objective lens turret 1232 may be provided with a magnet 1334B, and the magnet 1334B may be configured to connect to a magnetic material (e.g., a metal) provided on the other of the objective lens 1330 and the objective lens turret 1232.

[0333] According to a comparative embodiment, the objective lens can be screwed into the objective lens turret. However, the use of a sleeve and tube with the objective lens can make it difficult to screw the objective lens into the objective lens turret, at least in some embodiments. According to an embodiment of the present disclosure, the use of an objective lens coupling 1334 including a kinematic connection 1334A and a magnet 1334B allows the objective lens with the sleeve and tube to be easily installed.

[0334] 30A-33C, the objective lens 1330 and the sleeve 1332 may have various configurations. According to an embodiment, the sleeve 1332 may also be referred to as a cap.

[0335] FIG. 29 is a diagram showing an objective lens coupling portion according to an embodiment, FIG. 30A is a perspective view showing an immersion objective lens according to a first embodiment, FIG. 30B is a top view showing an immersion objective lens according to a first embodiment, FIG. 30C is a first cross-sectional view taken along line AA in FIG. 30B, showing an immersion objective lens according to a first embodiment with a spherical liquid provided thereon, FIG. 30D is a second cross-sectional view taken along line AA in FIG. 30B, showing an immersion objective lens according to a first embodiment with a sample carrier (e.g., a microplate) provided thereon, FIG. 31A is a top view showing an immersion objective lens according to a second embodiment, FIG. 31B is a first cross-sectional view taken along line BB in FIG. 31A, showing an immersion objective lens according to a second embodiment with a spherical liquid provided thereon, and FIG. 31C is a second cross-sectional view taken along line BB in FIG. 31A, showing a sample carrier (e.g., a microplate) provided thereon. FIG. 32A is a top view of an immersion objective lens according to a third embodiment, FIG. 32B is a first cross-sectional view taken along line CC in FIG. 32A showing an immersion objective lens according to the third embodiment with a spherical liquid provided thereon, FIG. 32C is a second cross-sectional view taken along line CC in FIG. 32A showing an immersion objective lens according to the third embodiment with a sample carrier (e.g., a microplate) provided thereon, FIG. 33A is a top view of an immersion objective lens according to a fourth embodiment, FIG. 33B is a first cross-sectional view taken along line DD in FIG. 33A showing an immersion objective lens according to the fourth embodiment with a spherical liquid provided thereon, and FIG. 33C is a second cross-sectional view taken along line DD in FIG. 33A showing an immersion objective lens according to the fourth embodiment with a sample carrier (e.g., a microplate) provided thereon.

[0336] In the following description of Figures 30A to 33C, the same or similar features are given the same or similar reference numerals, and for the purpose of clarity, the repeated description of the same or similar features may be omitted.

[0337] 30A-30D, a top surface 10A of the sleeve 1332A can be flush with a lens top surface 11A of the objective lens 1330A, and the sleeve 1332A can be configured to clamp to the objective lens 1330A.

[0338] The sleeve 1332A may include, for example, an upper portion 50A, a middle portion 60A, and a lower portion 70A. According to embodiments, the upper portion 50A, the middle portion 60A, and the lower portion 70A may be provided separately or integrally with each other to form a single body or multiple bodies. According to embodiments, two of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be provided integrally to form a single body, and the other one of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be provided as a separate body configured to be attached to the other two. According to embodiments, the upper portion 50A, the middle portion 60A, and / or the lower portion 70A may be subdivided into separate bodies and / or may comprise additional bodies. According to embodiments, any number of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be formed of aluminum.

[0339] According to an embodiment, any number of the top portion 50A, middle portion 60A, and bottom portion 70A may be formed to exhibit substantial rotational symmetry about a central axis of the objective lens 1330A, which may be, for example, an optical axis of the objective lens 1330A.

[0340] The middle part 60A may be provided above the lower part 70A. The middle part 60A may include an inlet port 62 and an outlet port 63. Fluid may be pumped into the sleeve 1332A via the inlet port 62 and out of the sleeve 1332A via the outlet port 63 by a fluid pump system (see, for example, FIG. 28). The inlet port 62 and the outlet port 63 may be provided on both sides of the sleeve 1332A, away from each other. However, the positions of the inlet port 62 and the outlet port 63 are not limited to such a configuration and may be variously changed. According to an embodiment, the inlet port 62 and the outlet port 63 may be constituted by a single port.

[0341] The intermediate portion 60A may further include a tapered portion 64A that follows the contour of the objective lens 1330A. For example, the tapered portion 64A may extend upward and radially inward from an outer portion of the intermediate portion 60A. The tapered portion 64A may be formed to exhibit substantial rotational symmetry about a central axis of the objective lens 1330A. According to an embodiment, the tapered portion 64A may have a shape other than tapered, so long as the shape follows the contour of the objective lens 1330A. The shape of the tapered portion 64A (e.g., an inverted "V" shape that follows the contour of the objective lens 1330A) allows the liquid droplet 90 to have a desired shape on the objective lens 1330A for immersion. According to an embodiment, the tapered portion 64A may alternatively be referred to as a protruding portion.

[0342] According to an embodiment, the inlet port 62 may include a passage extending through and to the inside of the tapered portion 64A, such as configured to supply liquid for the liquid droplet 90 to the space between the objective lens 1330A and the tapered portion 64A.

[0343] The upper portion 50A may include a body. For example, the body may include a side wall 52A extending upward from the middle portion 60A and a top wall 53A extending radially inward from the side wall 52A. The side wall 52A and the top wall 53A may extend substantially at 90 degrees from each other. However, the angle is not limited thereto and may be varied in various ways according to the embodiment. The body including the side wall 52A and the top wall 53A may be formed to exhibit substantially rotational symmetry around the central axis of the objective lens 1330A.

[0344] The groove 84 may be formed by and between the top portion 50A and the middle portion 60A. For example, the groove 84 may be defined by an inner surface of the top wall 52, an inner surface of the side wall 53, and an outer surface of the tapered portion 64A. According to an embodiment, the groove 84 may be formed to exhibit substantial rotational symmetry about a central axis of the objective lens 1330A. The groove 84 may be configured to receive and contain an excess amount of liquid. According to an embodiment, the groove 84 may be in communication with the outlet port 63 such that an excess amount of liquid in the groove 84 may exit the sleeve 1332A via a passage of the outlet port 63 in communication with the groove 84.

[0345] 30C and 30D, at least an upper surface of the upper wall 53A may form an upper surface 10A of the sleeve 1332A that is flush with the upper surface 11A of the objective lens 1330A. According to an embodiment, the upper surface of the tapered portion 64 may also be flush with the upper surface 11A of the objective lens 1330A.

[0346] According to an embodiment, one or more O-rings 32 can be provided between the sleeve 1332A and the objective lens 1330A. For example, the O-ring 32 can be provided between the middle portion 60A and the objective lens 1330A. The O-ring 32 can be configured to seal a bottom side of a space in which liquid is received between the objective lens 1330A and the tapered portion 64A.

[0347] 30D, a sample carrier (e.g., microplate) 80 holding a sample in at least one well 82 may be provided directly above the sleeve 1332A and objective lens 1330A. A droplet 90 of liquid on the lens of the objective lens may contact a bottom surface of the sample carrier (e.g., microplate) 80 at a location directly below the well 82. The sample carrier (e.g., microplate) 80 may correspond, for example, to sample carrier (e.g., microplate) 300 described in this disclosure, or other sample carriers (e.g., microplates).

[0348] Referring to Figures 31A to 31C, the top surface 10B of the sleeve 1332B can be above the lens top surface 11B of the objective lens 1330B, and the sleeve 1332B can be configured to clamp to the objective lens 1330B.

[0349] The sleeve 1332B can include, for example, an upper portion 50B, a middle portion 60B, and a lower portion 70B.

[0350] The intermediate part 60B can include a tapered portion 64B, and the upper part 50B can include a body including a side wall 52B and a top wall 53B. At least an upper surface of the top wall 53B can constitute the top surface 10B of the sleeve 1332B above the lens top surface 11B of the objective lens 1330B. According to an embodiment, the top surface of the tapered portion 64B can also be above the lens top surface 11B of the objective lens 1330B and be flush with the top surface of the top wall 53B.

[0351] 32A-32C, a top surface 10C of the sleeve 1332C can be below a lens top surface 11C of the objective lens 1330C, and the sleeve 1332C can be configured to clamp to the objective lens 1330C.

[0352] The sleeve 1332C can include, for example, an upper portion 50C, a middle portion 60C, and a lower portion 70C.

[0353] The intermediate part 60C can include a tapered portion 64C, and the upper part 50C can include a body including a side wall 52C and a top wall 53C. At least an upper surface of the top wall 53C can form the top surface 10C of the sleeve 1332C below the lens top surface 11C of the objective lens 1330C. According to an embodiment, the top surface of the tapered portion 64C can also be below the lens top surface 11C of the objective lens 1330C and be flush with the top surface of the top wall 53C.

[0354] 33A-33C, a top surface 10D of the sleeve 1332D can be flush with a lens top surface 11D of the objective lens 1330D, and the sleeve 1332D can be configured to screw onto the objective lens 1330D.

[0355] According to an embodiment, the inner surface of the sleeve 1332D and the outer surface of the objective lens 1330D may include correspondingly engaging threads such that the sleeve 1332D and the objective lens 1330D can be attached to and detached from each other by rotational movement of at least one of the sleeve 1332D and the objective lens 1330D.

[0356] The sleeve 1332D can include, for example, a first portion 60D and a second portion 50D.

[0357] The first part 60D may include a tapered portion 64D and the second part 50D may include a body including a side wall 52C and a top wall 53C. At least an upper surface of the top wall 53D may constitute a top surface 10D of the sleeve 1332D that is flush with a top surface 11D of the objective lens 1330D. According to an embodiment, the top surface of the tapered portion 64D may also be flush with the top surface 11D of the objective lens 1330D.

[0358] According to an embodiment, the interior surface of the first portion 60D may include threads.

[0359] According to an embodiment, the upper surface 10D of the sleeve 1332D can be above or below the upper surface 11D of the objective lens 1330D. For example, the upper surface of the upper wall 53D can be above or below the upper surface 11D of the objective lens 1330D, and the upper surface of the tapered portion 64D can be flush with the upper surface of the upper wall 53D.

[0360] According to the embodiments of the present disclosure, various embodiments of confocal microscopy can be provided alternatively or additionally. For example, a laser point scanning confocal system can be provided. Laser point scanning confocal microscopy can include focusing a single point of laser light through a small aperture (pinhole) and sequentially scanning the sample point by point in a zigzag pattern. The sample fluoresces and the light is sent back through the optical system. This light can then be read point by point by a detector, which can be a photomultiplier tube (PMT), but may also be detected using other light measuring sensors. The signal from the sensor can be recorded point by point, with each point constituting a single pixel in the image. Laser point scanning systems have advantages and disadvantages compared to spinning disk confocal systems. Laser point scanning systems are typically slower than spinning disk confocal and therefore often not suitable for high throughput applications or live cell imaging. Laser point scanning confocal systems, on the other hand, penetrate deeper into the sample and provide better axial and lateral resolution. In recent years, improvements have been made to laser point scanning systems to increase their speed, approaching that of spinning disks while still providing improved depth penetration. The speed of laser point scanning confocal systems is limited by the scan speed of the motor that drives the scanning mirror of the system.

[0361] According to an embodiment, the confocal subsystem of the present disclosure can include both laser point scanning confocal and spinning disk confocal. The spinning disk confocal system can be used for biological sample imaging and high throughput applications, while the laser point scanning confocal system can be used to penetrate deeper into the sample with improved resolution. Similar to the way wide-field imaging or other measurement modalities are used to provide "hits", the embodiments of the present disclosure can implement spinning disk confocal to rapidly scan the 3D sample and identify the location of several points of interest. The laser point scanning system can then be used to capture a more detailed image of the area of ​​interest. Both the laser point scanning confocal system and the spinning disk system are commercially available as two separate instruments. However, using two separate instruments in such a manner has several problems. For one, the cost of both the spinning disk and the laser confocal microscopes can make it impractical to implement a workflow as described above. In addition to this, there is also the technical problem of relocating the area of ​​interest to the other microscope. By implementing both the laser point scanning confocal system and the spinning disk system on the same instrument, the "hits" can be found and the area of ​​interest can be scanned by switching the optics without moving the stage. Finally, studying live cells also poses the problem that the sample changes over time. Moving the sample to another instrument takes too much time compared to the rate at which the biological features change. Moving the sample to another instrument can result in the area of ​​interest that was "hit" changing and no longer being of interest.

[0362] Another advantage of having both laser point scanning confocal and spinning disk confocal in the same instrument is that the laser point scanning confocal system can be utilized to target and photobleach specific areas of the sample, rather than for imaging. The laser point scanning confocal system, and the specific control it provides over the XY scanning mirror, allows very small specific regions of the sample to be targeted with the laser. This can be a single spot, or a block defined by a zigzag scan. Then, once photobleaching has occurred, the instrument can quickly switch to spinning disk confocal and monitor Fluorescence Recovery after PhotoBleaching (FRAP). Some specific applications include (a) analyzing molecular diffusion within cells (e.g., examining the diffusion of F-actin in primary dendritic cells after an area of ​​interest has been photobleached), (b) quantifying biological membrane fluidity (e.g., membrane fluidity in C. elegans), and (c) analyzing protein binding (e.g., monitoring the dynamic binding of chromatin proteins in vivo).

[0363] According to embodiments of the present disclosure, the pinpoint precision of a laser point-scanning confocal system combined with the imaging speed of a spinning disk system solves an unmet market need in FRAP assays.

[0364] 30A and 30B, a configuration according to an embodiment of the present disclosure that includes a laser point scanning confocal system, a spinning disk confocal system, and wide field capabilities in a single instrument is described below, however, an embodiment of the present disclosure may include any combination of the above systems and capabilities.

[0365] FIG. 30A shows the device set to laser point scanning confocal (LSC) modality. FIG. 30B shows the indication set to wide field or spinning disk confocal modality. According to an embodiment, a mechanism for switching between the LSC system and the wide field or spinning disk confocal system can be provided. As shown in FIG. 30A and FIG. 30B, the elements in block 2220 are movable, allowing switching between laser point scanning optics and spinning disk / confocal. For example, block 2220 can be a multiple disk module that can be moved to select between disks (and therefore modalities) as described in this disclosure.

[0366] With reference to FIG. 30A, an embodiment of the present disclosure can include a laser point scanning confocal system. Light, typically from a laser light source, enters such a system at an optical input device 2201. The optical input device 2201 can be, for example, a fiber-coupled input or a directly coupled laser without fiber. The light is then collimated as it passes through a lens 2202. The light then hits a long pass dichroic 2203, which is designed to reflect the input light and allow higher wavelengths of emitted light to pass through. It is typical for a light source to have multiple input wavelengths.

[0367] An embodiment of the present disclosure can support an automated means of switching the longpass dichroic 2203 to correspond to the input wavelength. The light is then reflected off the scan mirror 2204. The scan mirror 2204 can be controlled using two-axis motors 2205 and 2206. In some embodiments, both motors are Galvo type motors, while in other embodiments, one motor is driven by a galvo and the other is a resonant scanner. A resonant scanner is much faster than a galvo motor, but offers less control over positioning. Both types of motors are known to those skilled in the art. According to an embodiment, the scan mirror 2204 can be configured as multiple (e.g., two) separate scan mirrors. For example, the multiple separate scan mirrors can include a first mirror configured for x-scanning and a second mirror configured for y-scanning, and the positioning of each of the separate scan mirrors can be controlled, for example, by a respective motor.

[0368] After the light is reflected from the scan mirror 2204, it travels through a focus lens 2207 and then through a tube lens 2208. The light then travels to a reflecting mirror 2209, an objective lens 2210, and finally to the sample 2211, where the spot illuminated on the sample can be made smaller. The light then travels back through the laser point scanning system and to the long pass dichroic 2203, assuming the sample is fluorescent. If the emitted light is within the passband of the long pass dichroic 2203, it will pass to the focus lens 2213 and through the pinhole 2214. The pinhole 2214 can be a single size pinhole or can be variable in size. The size variation can be achieved by having multiple pinholes on a selector wheel or a variable aperture. The light then travels through a lens 2215 and then to the dichroic 2216.

[0369] The configuration shown in Figure 30A includes a dual PMT 2218 setup, allowing for measurement of multiple emission wavelengths simultaneously. This configuration can be expanded to additional numbers of PMTs 2218. This may also be a single PMT 2218 configuration where the emission wavelength is selected via a version of the dichroic 2216 and EM filter 2219 that includes a switching mechanism. The switching mechanism may be a cube and slider or multiple wheels, all of which will be understood by those skilled in the art.

[0370] In a laser point scanning system, the location of the optical input device 2201 may require precise alignment with the pinhole 2214. This makes the implementation, installation, and maintenance of the laser point scanning system difficult. After shipment or maintenance, an adjustment is typically required to realign the pinhole 2214 with the fiber location. A solution to this problem is that both the optical input device 2201 (e.g., optical fiber input) and the pinhole 2214 are on motorized shafts, allowing the instrument (e.g., its controller) to automatically align the optical input device 2201 with the pinhole 2214 by controlling the corresponding motors. Such an embodiment may provide benefits after shipment, after maintenance, or during thermal changes of the instrument. In addition to this, the location of the fiber input may be smaller than the pinhole size so that there is some margin in the design. With automated alignment, the pinhole size may be reduced, thus improving the confocality of the system, thereby improving the resolution and sample penetration.

[0371] FIG. 25 is a functional block diagram illustrating control of device modalities according to an embodiment.

[0372] The operation of the modalities may be controlled by a central control unit (e.g., a processor, CPU, microprocessor, etc.) According to an embodiment, the central control unit may also be referred to as a controller (e.g., controller 1000).

[0373] The central control unit 900 can be connected to communicate with and control elements of embodiments of the present disclosure, for example, the central control unit 900 can be connected to communicate with and control elements of the sample environment 90A, the sample selection and positioning elements 90B, the monochromator module elements 90C, the imager module 90D, the external light source module 932, and the injection module 934.

[0374] The controlled sample environment elements 90A can provide temperature control 902 and gas control 904 as described above.

[0375] Sample selection and positioning 90B can be controlled through the use of motors to position the sample in any X-direction 906 and Y-direction 908.

[0376] The controlled monochromator module elements 90C may include monochromator excitation 910, monochromator emission 912, monochromator PMT 916, fiber optic selection 918, and a light source such as a flash lamp 914.

[0377] The elements 90D of the imager module under control may include an objective lens selector 930, an image capture device such as a camera 920, a focus drive 924 for the objective lens, an LED and filter cube selector 922 for wide field imaging, a confocal cube selector 928, as well as a spinning disk module and control 926 (e.g., selection and focusing), and a laser scanning confocal module control 927.

[0378] FIG. 26 is a flow diagram of a method for controlling a multi-detection system in accordance with an exemplary embodiment.

[0379] Control of the device can be coordinated through the use of a controller, e.g., as discussed above with respect to Figure 25 and / or Figures 34A-34B. Input to the device (step S1805) can be accomplished through a local user interface of the device, such as a touchpad or graphical display, or through communication with the device through a wired or wireless connection, such as over a network.

[0380] In the case of input to the device, input may be performed through the use of a user interface or graphical user interface displayed on a computer or other terminal running a control application.

[0381] The inputs may be user inputs such as settings and parameters for implementing control of the device.

[0382] In response to receiving input, control of the instrument can be effected through various elements of the instrument, e.g., as discussed above with respect to Figure 25 and / or Figures 34A-34B. For example, in response to receiving user input, the instrument can be controlled to perform a gas control procedure for a gas module (step S1810), a sample positioning control procedure for controlling the positioning of the sample (step S1820), a monochromator control procedure for controlling the operation of the monochromator (step S1830), an imager control procedure for controlling the imager (step S1840), and outputting the results of the control of the elements of the instrument (step S1850).

[0383] Although the controls are presented as shown in Figure 26, the elements can be individually controlled in any order and control of all elements is not required. Thus, multiple modalities of the device can be controlled within a single assay.

[0384] The control method shown in Figure 26, and other functions described herein that may be performed by the controller, may be implemented through the execution of a processing unit (e.g., CPU) that controls the elements of the device by executing one or more control programs. The programs may be stored in memory (i.e., RAM, ROM, Flash, etc.) or other computer readable medium (i.e., CD-ROM, disk, etc.). The programs may be executed locally by the device or by a controlling device such as a computer that sends commands to be executed by the device.

[0385] Referring to FIG. 35, an embodiment of the present disclosure can include a display, and the controller can be further configured to cause the display to display a user interface. FIG. 35 shows an example of a user interface when the instrument has a combination of various optical modes. Element 2300 is an image of a sample. Element 2301 is a drop-down menu for selecting magnification. Element 2302 is a selection box for enabling / disabling water immersion. If selected and the objective is configured for water immersion, the controller can automatically pump water into the objective and automatically remove the water when imaging is completed or the checkbox of element 2302 is deselected. Element 2303 is a drop-down list for EM wavelength selection. Although FIG. 35 shows that a selection between four different EM wavelengths can be provided, any number of EM wavelength selections can be provided. Element 2304 is a drop-down list for EX wavelength selection. Although FIG. 35 shows that a selection between four different EX wavelengths can be provided, any number of EX wavelength selections can be provided. Element 2305 is a drop-down menu that allows to select between various modes of instruction. Fig. 35 shows the selection between modalities, the system including spinning disk, laser scanning, and wide field modalities. According to an embodiment, the modalities listed in element 2305 may depend on the modalities present in the system. The system may for example have any combination of the above mentioned modalities (and / or additional modalities) or may have only a single modality. If only a single modality is provided, element 2305 may not be provided. According to an embodiment, elements 2301, 2302, 2303, 2304 and 2305 are not limited to being drop-down menus and selection boxes, but may present options for selection in any manner known to those skilled in the art.

[0386] According to an embodiment, the interface may include a display element that allows a user to select multiple modalities to be automatically performed in sequence. For example, the controller may be configured to control a sequence to be automatically performed based on one or more inputs from the user on the interface. The sequence may include any order of modality operations, including those orders of modality operations described in this disclosure. For example, an operation using a spinning disk or wide field imaging system may be performed followed by an operation using a laser point scanning confocal system.

[0387] The components and features of the optical module are further described in U.S. Pat. No. 7,782,454, entitled "Universal multidetection system for microplates," which is incorporated herein by reference in its entirety for all purposes.

[0388] For example, according to one aspect, an optical module is provided that includes a first optical device that transmits a narrow wavelength band of light, the first optical device including a first filter and a first monochromator that provide an alternative path for the narrow wavelength band of light. The optical module can also include a light source that generates light as broadband excitation light, the first optical device transmitting the narrow wavelength band of the broadband excitation light and blocking other wavelength bands of the broadband excitation light through the first filter or the first monochromator, a second optical device that directs the narrow wavelength band of the broadband excitation light to a sample and receives emitted light from the sample, a third optical device that transmits the narrow wavelength band of the emission, and a detector that converts the narrow wavelength band of the emission into an electrical signal, where the third optical device includes the second filter and the second monochromator that provide an alternative path for the narrow wavelength band of the emission.

[0389] [Transportation Module] According to certain embodiments, the analytical instrument further comprises a transfer module configured to transfer optical signals from the array of sensor units to the signal processing module, for example the transfer module can transfer one or more of the excitation optical signal, the reference optical signal, and the emission optical signal.

[0390] The transfer module can be formed of multiplexed fiber optic material. Figures 36 and 37 show several views of an exemplary transfer module 60, including a side view (Figure 37) and a cross-sectional view (Figure 36) of the transfer module 60. In an embodiment, the transfer module 60 can include an array of fiber optic bundles, each fiber optic bundle communicating with a corresponding sensor unit of the array of sensor units. The fiber optic bundles can be positioned and configured to directly interface with one or more sensor units. Each fiber optic bundle can be formed of an array of fiber optic cables housed within a fiber and / or plastic probe housing, such as a metal fiber probe housing or a plastic housing, as shown in the cross-sectional view of Figure 36.

[0391] In certain embodiments, the transfer module can take the form of a homogenized fiber optic waveguide that optically connects the sensor units to the transfer module, e.g., optically connects each sensor unit to a corresponding fiber optic bundle of the transfer module. The homogenized fiber optic waveguide can be configured to distribute light uniformly to one or more sensor units. The homogenizer can improve mechanical and optical shuffling. EXAMPLES

[0392] The embodiments can be further understood with reference to the following examples, which are intended to serve as illustrations and not as limitations.

[0393] Example 1: Exemplary Protocol Cells are seeded into assay wells of a multi-well sample carrier (e.g., a microplate) at 50%-90% confluency. To maximize sensitivity, the suspension cells are allowed to attach to the bottom of the wells. For this exemplary protocol, a 96-well plate constructed and configured to fit the device is used. However, the sample carrier (e.g., a multi-well plate) can have any number of wells compatible with the device, such as 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384, or any other number of wells. The temperature of the cell suspension is controlled.

[0394] The apparatus lowers a sensor probe into the assay well. The sensor is positioned at a height of 200 microns from the bottom of the well, forming an approximately 2 microliter transient microchamber (also referred to herein as a "measurement chamber"). As the oxygen and pH levels change, the changes are measured by the sensor. Measurements are typically taken for a predetermined period of time, typically between 1 and 5 minutes, for example 3 minutes. The rate of change is automatically calculated by the computing device. At the end of this measurement period, the sensor probe is raised, allowing the extracellular medium to return to baseline conditions.

[0395] The sensor cartridge also contains ports (4 per well) that allow injection of modulators (target analytes) into the cell wells during the assay. When specified by the instrument's protocol, provided, for example, via the graphical user interface, the controller instructs the dispensing system to inject test compounds into the assay wells and perform a gentle mixing step to ensure dispersion of the compound throughout the assay medium. All wells are processed simultaneously in this manner. Subsequent measurement cycles, additional injections specified by the protocol, and percentage calculations are performed automatically.

[0396] The exemplary protocol was run using THP-1 cells (human monocytes derived from a patient with acute monocytic leukemia) for testing purposes. OCR and ECAR data were measured and reported using the analytical instrument described herein. This testing was also performed on a comparative analytical instrument, with conventional temperature control, signal processing, and motion actuator motor components. The results are presented in the graphs of Figures 10A-D.

[0397] The graph in Figure 10A shows OCR measurements over assay time measured using an analytical instrument disclosed herein. The graph in Figure 10B shows OCR measurements over assay time measured using a comparative analytical instrument. The graph in Figure 10C shows ECAR measurements over assay time measured using an analytical instrument disclosed herein. The graph in Figure 10D shows ECAR measurements over assay time measured using a comparative analytical instrument.

[0398] An exemplary protocol was also carried out using A549 cells (human lung cancer cells) and administration of 5 mM Metformin as a targeting agent. OCR data was measured using the analytical instruments described herein and comparative analytical instruments. The results are shown in the graphs of Figure 11A and Figure 11B.

[0399] The graph in Figure 11A shows OCR measurements over assay time measured using an analytical instrument as disclosed herein, and the graph in Figure 11B shows OCR measurements over assay time measured using a comparative analytical instrument.

[0400] Thus, an analytical instrument having a temperature control element, signal processing module, and motion actuator assembly motor described herein demonstrated significant improvements in lower limit OCR detection accuracy and readability over a comparative analytical instrument while simultaneously detecting ECAR. Without wishing to be bound by theory, it is believed that improving temperature uniformity across a sample within a controlled temperature zone can improve both the performance of the analytical instrument in sensing target analytes and cell biological characteristics.

[0401] Example 2: Evaporation test protocol using water samples Six assays were performed in the analytical instrument disclosed herein using known volumes of water in a sample carrier (e.g., a multi-well plate) for six hours with a modified protocol configured to perform four measurements per hour. Evaporation from the water samples was measured using a plate reader. A standard curve was generated by measuring the absorbance of known volumes of water. Absorbance measurements of the test plate were collected immediately after each assay. The standard curve was used to calculate the volume of water in each well of the test plate to assess the amount of water volume lost by evaporation during the six-hour assay. Results were calculated as a percentage of the total volume lost. The average evaporation for each assay is presented in the table of FIG. 12.

[0402] The maximum average percentage of water volume lost due to evaporation during the 6 hour assay was 10.04%, as shown in the table in Figure 12. Thus, the volume of sample fluid lost due to evaporation was small.

[0403] Without wishing to be bound by theory, it is believed that improving temperature uniformity across a sample within controlled temperature zones can reduce evaporation of the sample fluid and improve both the performance of the analytical instrument in sensing target analyte components and the biological characteristics of cells.

[0404] Example 3: Direct identification of mitochondrial toxicity using a novel index derived from mitochondrial oxygen consumption rate. Mitochondrial toxicity (Mito Tox) is a common problem in therapeutic development, contributing to the attrition of drug / compound / substance candidates and post-marketing drug / compound / substance withdrawal (Wallace, KB, 2008, "Mitochondrial off targets of drug therapy", Trends Pharmacol. Sci. 29, 361-366). Among the methods used to assess drug / compound / substance-induced mitochondrial toxicity in drug / compound / substance discovery and preclinical safety, direct measurement of mitochondrial oxygen consumption using Agilent's Seahorse XF technology is well reported as a specific and sensitive marker / indicator (Yvonne Will and James Dykens (2014), "Mitochondrial toxicity assessment in industry - a decade of technology development and insight", Expert Opinion on Drug Metabolism & Toxicology, 10:8, 1061-1067, DOI: 10.1517 / 17425255.2014.939628) (Tilmant Ka,*, Gerets Ha, De Ron Pa, Hanon Ea, Bento-Pereira Ca,b,1, Atienzar FA, "In vitro screening of cell bioenergetics to assess mitochondrial dysfunction in drug development", Toxicology in Vitro 52 (2018) 374-383).

[0405] Thus, disclosed herein is a standardized XF solution that allows for the assessment of compounds that exhibit mitochondrial toxicity. As described herein, the XF Pro analyzer has several novel design features that provide improved sensitivity, precision, and consistency. Here, we utilize these improvements to detect drug / compound / substance-induced mitochondrial dysfunction using OCR measurements.

[0406] The Agilent Seahorse XF Mito Tox assay workflow involves sequential injections of oligomycin and FCCP, but includes a separate control group that receives rotenone / antimycin A prior to the assay. Compounds to be assessed for mitochondrial toxicity are provided to cells at specified times prior to the assay.

[0407] Based on the response of test compounds in basal oligomycin and / or FCCP OCR compared to appropriate controls, the XF Mito Tox assay can distinguish three different types of mitochondrial toxicity: direct / indirect inhibition of the ETC or other mitochondrial processes, uncoupling of the ETC from OxPhos, and (potential) specific inhibition of the OxPhos machinery (CV, ANT, PiT).

[0408] A significant improvement is provided through the extraction of a novel parameter, the Mito Tox Index (MTI), derived from the oxygen consumption rate (OCR) measured by the analytical instrument disclosed herein (Agilent's Seahorse XF Analyzer).

[0409] This approach to derive easily interpretable mitochondrial toxicity metrics is surprisingly made possible by the increased sensing accuracy achieved through the implementation of the instruments and methods disclosed herein, providing an easy and robust method to screen and validate toxicity in vitro. This workflow allows the reduction of complex respirometric responses to the mitochondrial toxicity index (MTI) metric and provides two types of MTIs that score inhibitor and uncoupler effects on the electron transport chain (ETC) on a negative and positive scale, respectively. The inhibitor MTI is designed to calculate the relative inhibitory effect on maximum OCR against the effect of the rotenone / antimycin A mixture, which is an ETC inhibitor control. In contrast, the uncoupler MTI calculates the relative increase in minimum OCR measured after oligomycin injection against the effect of FCCP in the vehicle control group. Since ATP synthase inhibitors do not affect maximum OCR, potential ATP synthase inhibitors can be identified among compounds that did not show significant scores in either MTI by monitoring basal OCR specific inhibition. The ability to derive defined metrics allows for additional functionality, such as convenient generation of dose-response relationships or convenient threshold setting for "hit" identification.

[0410] [Define the Mitochondrial Toxicity Index (MTI)] To distinguish between the three modes of mitochondrial toxicity described above and to quantify the magnitude of toxicity, Mito Tox Index (MTI) values ​​were derived, taking advantage of the improved measurement accuracy enabled by the device disclosed herein. Mitotoxicity due to inhibition, defined and detected as a decrease in the FCCP OCR of the test compound compared to the maximum FCCP OCR of the vehicle group, results in a negative MTI value (typically 0 to -1), as shown and described in Figures 39A to 39C.

[0411] Figure 39A shows a scenario in which a test compound results in a decrease in FCCP-induced OCR compared to the vehicle (negative) control (MTI=0), where the compound is classified as an inhibitor with a negative MTI value (e.g., MTI=-0.8). Note that the OCR of rotenone / AA serves as a positive (+) control for inhibition (MTI=-1). Figures 39B and 39C provide a summary of the measurements and groups used to control for inhibition.

[0412] Mitotoxicity due to uncoupling, where uncoupling is defined and detected as an increase in the oligo-OCR of the test compound compared to the minimal oligo-OCR of the vehicle group, results in a positive MTI value (typically 0-1) and is shown and illustrated in Figures 40A-C.

[0413] Figure 40A shows a scenario where a test compound results in an increase in oligo-induced OCR compared to the vehicle (negative) control (MTI=0), where the compound is then classified as an uncoupler with a positive MTI value (e.g., MTI=0.6). Note that the OCR of the vehicle FCCP serves as a positive (+) control for uncoupling (MTI=1). Figures 40B and 40C provide a summary of the measurements and groups used to control for uncoupling.

[0414] In summary, MTI is the value of the fraction of the effect of the test compound compared to the respective control for either uncoupling and / or inhibition. The MTI of the uncoupler is calculated as a positive index and is defined as the fraction of uncoupling induced by the test compound compared to the maximum uncoupling (OCR of FCCP in the vehicle group, the positive control). Note that the oligo OCR of the vehicle group serves as a negative control for uncoupling. Conversely, the MTI of the inhibitor is calculated as a negative index and is defined as the fraction of inhibition induced by the test compound compared to the maximum inhibition (OCR of FCCP in the rotenone / AA group, the positive control). Note that in this case, the OCR of FCCP in the vehicle group serves as a negative control. Upon transformation, both the MTI of the uncoupler and the inhibitor can be generated for each well. An exemplary MTI detection graph is shown in FIG. 41.

[0415] A specific example of mito tox by reducing mito function is the direct inhibition of ATP synthase (CV) or other components of the OxPhos machinery (e.g. ANT, Pi transporter). This type of inhibition often shows a decrease in basal OCR, while oligo OCR and FCCP OCR are significantly less affected (Figures 42A-D). If treatment with a test compound results in a decrease in basal OCR but not a significant decrease in maximum / FCCP-induced OCR compared to the vehicle (negative) control (MTI=0), the compound is classified as an OPI.

[0416] XF Mito Tox Assay Performance Metrics The Z - value (Z - factor) is used as a measure of assay quality or assay performance (Zhang). The Z’ - value typically ranges from 0 to 1.0 and can be interpreted as follows. A Z’ = 1.0 is considered ideal assay performance. When 0.5 < Z’ < 1.0, it is considered an excellent assay, meaning that the likelihood of reporting false positives or false negatives is significantly reduced. When 0.0 < Z’ < 0.5, this is considered minimal assay performance, and the likelihood of reporting false positives or false negatives increases. When Z’ is less than 0, there is too much overlap between the positive and negative controls for the assay to be useful.

[0417] Therefore, the Z - value can be used as a measure of the quality or ability of a screening assay (note that Z’ is not the same as the z - score). In screening activities, typically, there are comparisons of a large number of single measurements of unknown samples against well - established positive and negative control samples. The purpose of the assay is to determine which, if any, of the single measurements are significantly different from the controls. For this purpose, the distributions of the measurements from the positive control, negative control, and other single measurements must be considered to determine the probability that each measurement occurred by chance. Furthermore, these distributions cannot be determined a priori, and the performance must be evaluated after the assay to indicate / predict that the assay is useful in a screening (or user - defined) setting. The larger the Z’ - value, the lower the likelihood that the assay will report false positives and / or false negatives.

[0418] In the XF Mito Tox assay, Z’ - values (z’ Factor) are provided for both decoupling and inhibition, each having its own positive and negative controls, thus enabling the evaluation of assay performance. The Z’ - value is calculated as follows: Z’ = 1 - [3(mean of positive control + mean of negative control) / (standard deviation of positive control - standard deviation of negative control)]

[0419] Surprisingly, the improved precision of the instrument described herein allows for a simplified Mito Tox Metric (MTI) capable of achieving excellent Z' values ​​(>0.5) even in the absence of cell normalization (wherein data is corrected to account for variability in cell expansion across sample carriers (e.g., microplates)) (Figure 43).

[0420] [Example of use] This capability means that the XF Mito Tox assay can be run as a compound screen (e.g., up to 80 individual compounds at a single dose per plate) or used to perform dose-response assays (e.g., 8 compounds per plate, 10 concentrations / compound). When used with the respective software tools, the resulting dynamic OCR data is automatically converted into MTI values ​​for each test compound.

[0421] The modes of Mito Tox detected and measured using the XF Mito Tox assay were calculated. Drugs / compounds / substances that affect transport, TCA, FAO, OCR of the ETC (drugs / compounds / substances resulting in a decrease in FCCP induction) are classified as inhibitors. Drugs / compounds / substances that act as protonophores uncoupling ETC from OxPhos resulting in an increase in oligo OCR are classified as uncouplers. Drugs / compounds / substances that cause inhibition of OxPhos machinery (ATP synthase, ANT, Pi transporter) resulting in a decrease in basal OCR only are classified as "OPI".

[0422] Depending on the context / purpose of the mito tox study, the test compounds can be further subjected to dose response assays, including dose response curves and IC50 / EC50 values. Figure 42 shows the kinetic dose response OCR data for three compounds, which were then converted to MTI values ​​for each dose and plotted against compound concentration (Figure 42). IC50 (or EC50) values ​​were calculated for each sample.

[0423] [Experimental Method] All cell lines were maintained according to the manufacturer's recommendations. HepG2 cells were cultured at 2.0 × 10 per well. 4 Cells were seeded onto XF Pro Moat cell culture sample carriers (e.g., microplates) at a density of 1000× and cultured in DMEM low glucose (Gibco 11885) supplemented with 2 mM Glutamax and 10% serum. All cells were incubated at 37°C, 5% CO 2 The next day, cells were washed twice with Mito Tox assay medium (XF DMEM pH 7.4 + 10 mM XF glucose, 1 mM XF pyruvate, 2 mM XF glutamine) and incubated at 37°C, CO 2 The plate was incubated for 60 minutes without addition. Pretreatment solution was added during cell washing. The cell plate was then transferred to the XF Pro analyzer for assay performance using sequential injections of oligomycin (1.5 μM), FCCP (1.5 μM), rotenone / antimycin A (0.5 μM each) (final concentrations). Cells were then counted using a Cytation5 instrument, where assessed.

[0424] All XF assays were performed as described in the XF Mito Tox kit user guide, including compound dilution and sensor cartridge preparation. Agilent's Seahorse Analytics is a web-based software platform that provides an easy and streamlined data analysis workflow for the XF Mito Tox assay. Seahorse Analytics was used to calculate the Mito Tox Index (MTI value) and / or IC50 / EC50 values, which are key parameters of the XF Mito Tox assay. Instructions for performing data analysis using the Seahorse Analytics user guide.

[0425] Example 4: The disclosed analyzer exhibits improved measurement accuracy over a comparative analytical instrument. THP-1 cells were cultured in RPMI cell culture medium (supplemented with 10% FBS, 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate) at 37°C and 5% CO 2 The cell density was 10 6 The cell density was kept lower than 100 cells / mL, and the culture medium was renewed every 48–72 h.

[0426] The cell suspension was transferred to a centrifuge tube and the cells were centrifuged at 1000 x g for 10 min. The cells were resuspended in assay medium consisting of RPMI supplemented with 1 mM HEPES buffer (wherein sodium bicarbonate was replaced with an osmotically equivalent concentration of NaCl) pH 7.4, 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate. The resuspended cells were diluted to 2 x 10 in separate tubes. 4 Cells / well, 5 x 10 4 Cells / well, 1 x 10 5 Cells / well, 1.5 x 10 5 Cells / well, 2 x 10 5 Cells / well, 3 x 10 5 cells / well, and 4 x 10 5 The cells were diluted to a concentration of 1×10 cells / well. Six replicate wells for each concentration were seeded into each of two 96-well plates that were pre-coated with poly-D-lysine and pre-warmed overnight at 37° C. As shown in FIG. 44, the final concentration in each well was 1×10 3 Cells / well, 2.5 x 10 3 Cells / well, 5 x 10 3 Cells / well, 7.5 x 10 3 Cells / well, 1 x 10 4 Cells / well, 1.5 x 10 4 cells / well, or 2 x 10 4 50 μL of resuspended cells were added to each well so that there were 16 cells / well. The 96-well plate was centrifuged at 200×g for 1 min and assay medium was added so that each well had a final volume of 180 μL. The 96-well plate was incubated at 4°C for 1 h in a non-CO2 hood. 2 The mixture was incubated in an incubator at 37°C for 30 minutes.

[0427] One of the 96-well plates was placed in an analytical instrument described herein. The other plate was placed in a comparative analytical instrument having conventional temperature control, signal processing, and motion actuator motor components. Each instrument was programmed with command instructions. In this case, the instrument was programmed to perform three measurements, inject 20 μL of solution into each well from port A from a cartridge placed above the cell sample in the well, perform three measurements, inject 22 μL of solution into each well from port B from a cartridge placed above the cell sample in the well, and perform the final three measurements. The instrument was programmed to perform measurements every 6 minutes, with each 6 minute interval including a 3 minute mixing step and a 3 minute measurement step.

[0428] Oligomycin solution was prepared at 15 μM in assay medium. A mixed solution of 5 μM rotenone and 5 μM antimycin A was prepared in assay medium. The ports of the pre-hydrated cartridge for each well were loaded with 15 μM oligomycin solution (port A) and 5 μM rotenone + 5 μM antimycin A solution (port B). The hydrated assay cartridge containing the indicated reagents was loaded into the instrument and the experiment was run according to the instrument's protocol. The instrument measured OCR as described in the exemplary protocol of Example 1. Basal OCR was calculated as the average of six replicate wells in each plate of the three measurements.

[0429] This experiment was performed three times, and the results from each run are shown separately in Figures 45A-C. The basal OCR from all runs is summarized in Figures 46A and 46B, and the standard deviation across the runs is shown in Figure 47. Collectively, these data demonstrated improved measurement performance of the instruments described herein versus the comparative analytical instruments at low oxygen consumption rates (OCR). Specifically, data collected on the instruments described herein resulted in reduced incidence of negative rates at low concentrations or after rotenone + antimycin A injection, lower standard deviations, reduced inter- and intra-plate variability, and more consistent measurements at low OCR. This was demonstrated using a combination of titrated seeding densities and injection of a mitochondrial inhibitor compound. These improvements allowed for more confident interpretation of cell data due to better resolution and reproducibility between assay groups.

[0430] [Incorporated by reference] All publications, patents and accession numbers mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be a part of this specification by reference.

[0431] [Equivalent] While specific embodiments of the present invention have been described, the above specification is illustrative and not restrictive. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification and the appended claims. The full scope of the present invention should be determined with reference to the claims, along with their full scope of equivalents, and such variations, along with the specification.

[0432] [appendix] [Section 5] [High impedance sensor] Walt Kester, Scott Wurcer, Chuck Kitchin Many common sensors have output impedances higher than a few MΩ, and the associated signal conditioning circuitry must be carefully designed to meet the challenges of low bias current, low noise, and high gain. Most of this section is devoted to an analysis of photodiode preamplifiers. This application points out many of the problems associated with signal conditioning circuits for high impedance sensors and provides practical solutions that can be applied to almost all such sensors. Other examples of high impedance sensors that are discussed are piezoelectric sensors, charge output sensors, and charge coupled devices (CCDs).

[0433] [Table 1]

[0434] [Photodiode preamplifier design] Photodiodes generate a small electrical current that is proportional to the level of illumination, and have many applications, from precision light meters to high-speed fiber optic receivers.

[0435] An equivalent circuit for a photodiode is shown in Figure 5.3. One standard way to specify the sensitivity of a photodiode is to express the short-circuit photocurrent (I SC The most commonly used light source is an incandescent tungsten lamp operating at a color temperature of 2850 K. At an illuminance of 100 fc (foot-candle) (approximately the light level on an overcast day), a small area (1 mm 2 For diodes with a capacitance of 1000 Ω or less (less than 100 Ω), the short circuit current is typically in the picoamp to hundreds of microamp range.

[0436] [Table 2]

[0437] [Table 3]

[0438] The short-circuit current is very linear over light intensities of 60x to 90x and is therefore often used as a measure of absolute light level. The open-circuit forward voltage drop across a photodiode varies logarithmically with light level, but because of its large temperature coefficient, the diode voltage is rarely used as an accurate measure of light intensity.

[0439] Shunt resistance R SH is typically on the order of 1000 MΩ at room temperature, decreasing by a factor of two for every 10°C increase in temperature. J is a function of the junction area and the diode bias voltage. A value of 50 pF at zero bias is typical for small area diodes.

[0440] Photodiodes can be operated with either zero bias (photovoltaic mode, left) or reverse bias (photoconductive mode, right), as shown in Figure 5.4. The most linear operation is obtained in the photovoltaic mode, but higher switching speeds can be achieved at the expense of linearity if the diode is operated in the photoconductive mode. Under these reverse bias conditions, a small amount of current, called dark current, flows even when there is no illumination. In the photovoltaic mode, there is no dark current. In the photovoltaic mode, the noise of the diode is primarily thermal noise generated by the shunt resistor. In the photoconductive mode, shot noise due to conduction is an additional noise source. Photodiodes are usually optimized during the design process for use in either the photovoltaic or photoconductive mode, but not both. Figure 5.5 shows the photosensitivity of a small photodiode (silicon detector, part number SD-020-12-001), and the specifications for the diode are summarized in Figure 5.6. This diode was chosen for the following design example.

[0441] [Table 4]

[0442] [Table 5]

[0443] [Table 6]

[0444] One convenient way to convert the photodiode current into a usable voltage is to use an op-amp as a current-to-voltage converter, as shown in Figure 5.7. The diode bias is kept at zero volts by the virtual ground of the op-amp, and the short-circuit current is converted into a voltage. At maximum sensitivity, the amplifier must be able to detect a diode current of 30 pA. This means that the feedback resistor must be very large and the amplifier bias current must be very small. For example, 1000 MΩ would give a corresponding voltage of 30 mV for this amount of current. Larger resistor values ​​are not practical, so 1000 MΩ is used for the most sensitive range. This gives an output voltage range of 10 mV for a diode current of 10 pA and 10 V for a diode current of 10 nA. This gives a range of 60 dB. For higher values ​​of light intensity, the gain of the circuit must be reduced by using a smaller feedback resistor. At this maximum sensitivity range, it is possible to easily distinguish between the light intensity of a clear moonless night (0.001fc) and the light intensity of a full moon (0.1fc)!

[0445] [Table 7]

[0446] Note that we chose to get as much gain as possible from one stage rather than cascading two stages. This is to maximize the signal-to-noise ratio (SNR). Halving the value of the feedback resistor reduces the signal level by a factor of two, but the noise due to the feedback resistor (√(4kTR·bandwidth)) only drops by √2. This reduces the SNR by 3dB, assuming a constant closed-loop bandwidth. Later in the analysis, we will see that the resistor is one of the largest contributors to the overall output noise.

[0447] To accurately measure photodiode currents in the tens of picoamp range, the op amp bias current should be no more than a few picoamps. This narrows the options considerably. The industry standard OP07 is a bipolar op amp with ultra-low offset voltage (10μV), but its bias current is 4nA (4000pA!). Even super-beta bipolar op amps with bias current compensation (such as the OP97) have bias currents on the order of 100pA at room temperature, but these currents do not double for every 10°C increase as FETs do, so they may be suitable for very high temperature applications. FET-input electrometer-grade op amps are the choice for photodiode preamps because they only need to operate over a limited temperature range. Figure 5.8 summarizes the performance of several common "electrometer-grade" FET-input op amps. These devices are fabricated on a BiFET process and use P-channel JFETs as the input stage (see Figure 5.9). The remainder of the op-amp circuit is designed using bipolar devices. The BiFET op-amps are laser trimmed at the wafer level to minimize offset voltage and offset voltage drift. Offset voltage drift is minimized by first trimming the input stage so that the currents in the two JFETs that make up the differential pair are equal. A second trim of the JFET source resistance minimizes the input offset voltage. The AD795 was chosen for the photodiode preamplifier and its key specifications are summarized in Figure 5.10.

[0448] [Table 8]

[0449] [Table 9]

[0450] [Table 10]

[0451] Diode currents are measured in picoamps, so careful attention must be paid to potential leakage paths in practical circuits. Two parallel conductor stripes running 1 inch parallel, 0.05 inches apart, on a well-cleaned high-quality epoxy-glass PC board will have a typical leakage current of approximately 10 at +125°C. 11 ohms leakage resistance. If 15 volts is applied between them, a current of 150 μA will result.

[0452] The significant leakage paths for the photodiode circuit are circled by dotted lines in Figure 5.11. The feedback resistors should be ceramic or thin film on glass with glass insulation. The compensation capacitor across the feedback resistor should have a polypropylene or polystyrene dielectric. All connections to the summing junction should be kept short. If cables are used to connect the photodiode to the preamplifier, they should be kept as short as possible and have Teflon insulation.

[0453] Guarding techniques can be used to reduce parasitic leakage currents by isolating the amplifier's input from large voltage gradients across the PC board. Physically, the guard is a low-impedance conductor that surrounds the input line and is raised to the voltage of the line. It acts to buffer the leakage by shunting it away from sensitive nodes.

[0454] [Table 11]

[0455] The technique for guarding depends on the mode of operation, i.e., inverting or non-inverting. Figure 5.12 shows a PC board layout for guarding the inputs of an AD795 op amp in a DIP ("N") package. Note that the pin spacing allows traces to pass between the pins of this package. In inverting mode, the guard trace surrounds the inverting input (pin 2) and runs parallel to the input trace. In follower mode, the guard voltage is the feedback voltage to pin 2, the inverting input. In either mode, the guard traces should be located on both sides of the PC board if possible and connected together.

[0456] When using the guard technique with SOIC surface mount ("R") packages, things get a little more complicated because the pin spacing does not allow for PC board traces between the pins. Figure 5.13 shows the preferred method. In the SOIC "R" package, pins 1, 5 and 8 are "no connect" pins and can be used to route signal traces as shown. In the follower case, the guard traces are S It must be routed around the pins.

[0457] For extremely low bias current applications (such as using the AD549 with its 100 fA input bias current), all connections to the op amp inputs should be made to virgin Teflon standoff insulators. ("Virgin" Teflon is a solid piece of new Teflon material, machined to shape, not welded together from powder or grain.) If mechanical and manufacturing considerations permit, the inverting input pin of the op amp should be soldered directly to the Teflon standoff (see Figure 5.14) rather than through a hole in the PC board. The PC board itself should be carefully cleaned and sealed against moisture and dust using a high-quality compatible coating material.

[0458] [Table 12]

[0459] [Table 13]

[0460] [Table 14]

[0461] In addition to minimizing leakage currents, the entire circuit should be well shielded with a grounded metal shield to prevent pickup of stray signals.

[0462] [Preamplifier offset voltage and drift analysis] The offset voltage and bias current model for a photodiode preamplifier is shown in Figure 5.15. There are two important considerations in this circuit. First, the shunt resistance of the diode (R1) is a function of temperature and is halved for every 10°C increase in temperature. At room temperature (+25°C) R1 = 1000MΩ, at +70°C it drops to 43MΩ. This has a dramatic effect on the DC noise gain of the circuit and therefore the output offset voltage. In this example, at +25°C the DC noise gain is 2, but at +70°C it increases to 24.

[0463] The second drawback to this circuit is that the input bias current doubles for every 10°C increase in temperature. The bias current is I B This results in an output offset error equal to R2. At +70°C, the bias current increases from its room temperature value of 1 pA to 24 pA. Usually, the addition of a resistor (R3) with value R1||R2 between the non-inverting input of the op amp and ground can provide a first order cancellation of this effect. However, this method is ineffective because R1 varies with temperature. In addition, the bias current develops a voltage across the R3 cancellation resistor, which is applied to the photodiode, causing the diode response to become nonlinear.

[0464] The total referred to output (RTO) offset voltage error is summarized in Figure 5.16. Note that at +70°C the total error is 33.24mV. This error is acceptable for the design under consideration. The main source of error at high temperatures is of course the bias current. Operating the amplifier at reduced supply voltages, minimizing the output drive requirements, and heat sinking are some ways to reduce this source of error. The addition of an external offset nulling circuit can minimize the error due to the initial input offset voltage.

[0465] [Table 15]

[0466] [Table 16]

[0467] [Thermoelectric voltage as a source of input offset voltage] Thermoelectric potentials are generated by electrical connections made between different metals at different temperatures. For example, electrical contact of a copper PC board to a kovar input pin of a TO-99 IC package can generate an offset voltage of 40 μV / °C when the two metals are at different temperatures. Ordinary lead-tin solder generates a thermoelectric voltage of 1 μV / °C to 3 μV / °C when used with copper. Special cadmium-tin solders are available that reduce this to 0.3 μV / °C (Reference 8, p. 127). The solution to this problem is to ensure that the connections to the inverting and non-inverting input pins of the IC are made with the same material, and to arrange the thermal layout of the PC board so that these two pins remain at the same temperature. In cases where a Teflon standoff is used as the isolated connection point for the inverting input (as in the case of a photodiode preamplifier), it is advisable that the connection to the non-inverting input be made in a similar manner to minimize possible thermoelectric effects.

[0468] [Preamp AC design, bandwidth, and stability] A key to preamp AC design is understanding the noise gain of the circuit as a function of frequency. Plotting gain versus frequency on a log-log scale makes the analysis relatively simple (see Figure 5.17). This type of plot is also called a Bode plot. Noise gain is the gain seen by a small voltage source in series with the opamp input terminals. It is also the same as the non-inverting signal gain (gain from "A" to the output). In a photodiode preamp, the signal current from the photodiode passes through the C2 / R2 network. It is important to distinguish between signal gain and noise gain, since it is the noise gain characteristic that determines stability, regardless of where the actual signal is applied.

[0469] The stability of the system is determined by the net slope of the noise gain and the open-loop gain where they intersect. For unconditional stability, the noise gain curve must intersect the open-loop response with a net slope of less than 12 dB / octave (20 dB per decade). The dotted line shows the noise gain intersecting the open-loop gain with a net slope of 12 dB / octave, indicating an unstable condition. This condition occurs in our photodiode circuit if there is no feedback capacitor (i.e., C2=0).

[0470] [Table 17]

[0471] The general formula for determining the breakpoints and gain values ​​in the Bode plot is also shown in Figure 5.17. The zeros in the noise-gain transfer function are expressed as 1 / 2πτ 1 occurs at a frequency of τ 1 =R1||R2(C1+C2). The poles of the transfer function are 1 / 2πτ 2 This occurs at a corner frequency of τ 2 =R2C2, which is also equal to the signal bandwidth for a signal applied at point "B". At low frequencies, the noise gain is 1+R2 / R1. At high frequencies, it is 1+C1 / C2. Plotting this curve on a log-log graph is a simple matter of connecting the breakpoints with a straight line with a 45 degree slope. The point where the noise gain intersects with the open loop gain of the op amp is called the closed loop bandwidth. Note that the signal bandwidth for a signal applied at point "B" is fairly small, 1 / 2πR2C2.

[0472] Figure 5.18 shows the noise gain plot for the photodiode preamplifier using actual circuit values. The choice of C2 dictates the actual signal bandwidth and phase margin. In this example, a signal bandwidth of 16Hz was chosen. Note that a smaller value of C2 would result in a higher signal bandwidth and a corresponding reduction in phase margin. It is also interesting to note that even though the signal bandwidth is only 16Hz, the closed loop bandwidth is 167kHz. This will have important implications for the output noise voltage analysis that follows.

[0473] [Table 18]

[0474] It is important to note that temperature changes do not significantly affect the stability of the circuit. Changes in R1 (the shunt resistor of the photodiode) only affect the low frequency noise gain and the frequency at which the zero in the noise gain response occurs. The high frequency noise gain is dominated by the C1 / C2 ratio.

[0475] [Noise analysis of photodiode preamplifiers] To begin our analysis, consider the input voltage and current noise spectral densities of the AD795, shown in Figure 5.19. The AD795's performance is truly outstanding for a JFET-input op amp, with a 0.1Hz to 10Hz noise of 2.5µV pp and a 1 / f corner frequency of 12Hz, which compares favorably with all but the best bipolar op amps. As can be seen, the current noise is much lower than that of bipolar op amps, making it an ideal choice for high impedance applications.

[0476] A complete noise model for an op amp is shown in Figure 5.20. The model includes reactive elements C1 and C2. Each individual output noise contribution is calculated by integrating the square of the spectral density over the appropriate frequency bandwidth and then taking the square root: V 1RMS output noise due to = √(∫V 1 (f) 2 df)

[0477] In most cases, this integration can be done by inspection of the individual spectral density graphs superimposed on a graph of the noise gain. The total output noise is then obtained by combining the individual components in a root-sum-of-squares manner. The table below the diagram in Figure 5.20 shows how the individual sources are reflected in the output and the corresponding bandwidth for integration. To convert the single-pole bandwidth to an equivalent noise bandwidth, a factor of 1.57(π / 2) is required. The spectral density of resistive Johnson noise is given by: V R =√(4kTR) where k is the Boltzmann constant (1.38×10 -23 The Johnson noise is expressed as: (J / K), where T is the absolute temperature in K. A simple way to calculate this is to recall that the noise spectral density of a 1 kΩ resistor is 4 nV / √Hz at +25°C. The Johnson noise of another resistor value can be found by multiplying by the square root of the ratio of the resistance value to 1000 Ω. Johnson noise is broadband and its spectral density is constant with frequency.

[0478] [Table 19]

[0479] [Table 20]

[0480] [Input voltage noise] To obtain the spectral density plot of the output voltage noise due to the input voltage noise, the spectral density plot of the input voltage noise is multiplied by the noise gain plot. This is easily accomplished using a log-log scale Bode plot. The total RMS output voltage noise due to the input voltage noise is then obtained by integrating the square of the output voltage noise spectral density plot and then taking the square root. In most cases, this integral can be approximated. Usually, a lower frequency limit of 0.01 Hz in the 1 / f domain is used. If the bandwidth of the integration for the input voltage noise is greater than a few hundred Hz, the spectral density of the input voltage noise can be assumed to be constant. Usually, the value of the input voltage noise spectral density at 1 kHz will provide sufficient accuracy.

[0481] It is important to note that the input voltage noise contribution must be integrated over the entire closed-loop bandwidth of the circuit (closed-loop bandwidth f cl (is the frequency where the noise gain intersects with the op amp's open-loop response.) This is also true for the other noise contributors that are reflected to the output by noise gain (i.e., the noninverting input current noise and the noninverting input resistor noise).

[0482] The inverting input noise current flows through the feedback network, resulting in a noise voltage contribution at the output. The input noise current is approximately constant with frequency, therefore integration is accomplished by multiplying the noise current spectral density (measured at 1 kHz) by the noise bandwidth, which is 1.57 times the signal bandwidth (1 / 2πR2C2). The factor of 1.57 (π / 2) occurs when the single-pole 3 dB bandwidth is converted to an equivalent noise bandwidth.

[0483] [Johnson noise due to feedforward resistor R1] The noise current generated by the feedforward resistor R1 also flows through the feedback network, resulting in a contribution at the output. The noise bandwidth for integration is also 1.57 times the signal bandwidth.

[0484] [Non-inverting input current noise] Non-inverting input current noise I N+ generates a voltage noise across R3 that is reflected to the output by the noise gain of the circuit. The bandwidth for integration is therefore the closed-loop bandwidth of the circuit. However, if R3=0, or if R3 is bypassed with a large capacitor, which is usually desirable when operating an op amp in inverting mode, there is no contribution at the output.

[0485] [Johnson noise due to resistors at the non-inverting input] The Johnson voltage noise due to R3 is also reflected to the output by the noise gain of the circuit. If R3 is well bypassed, it does not contribute significantly to the output noise.

[0486] [Photodiode circuit noise performance summary] Figure 5.21 shows the output noise spectral density for each of the factors at +25°C. The non-inverting input of the op amp is grounded, so I N+ Note that there is no contribution from R or R3.

[0487] [Table 21]

[0488] [Noise reduction using output filtering] From the above analysis, the largest contributor to the output noise voltage at +25°C is the op-amp's input voltage noise reflected to the output by the noise gain. This contribution is large mainly because the noise gain, where integration takes place, extends to a bandwidth of 167 kHz (the intersection of the noise gain curve with the op-amp's open-loop response). When the op-amp output is filtered by a single-pole filter (shown in Figure 5.22) with a cutoff frequency of 20 Hz (R = 80 MΩ, C = 0.1 μF), this contribution is reduced to less than 1 μV rms. Note that simply increasing the feedback capacitor C2 does not achieve the same result. Increasing C2 reduces the high-frequency noise gain, but the integration bandwidth is proportionally higher. Also, a larger value of C2 may reduce the signal bandwidth to an unacceptable level. The addition of a simple filter reduces the output noise to 28.5 μV rms, roughly 75% of its pre-reduction value. After inserting the filter, the resistive noise and current noise become the largest contributors to the output noise.

[0489] [Table 22]

[0490] [Circuit performance summary] A diagram of the final optimized design of the photodiode circuit is shown in Figure 5.22. Performance characteristics are summarized in Figure 5.23. The total output voltage drift from 0°C to +70°C is 33mV. This corresponds to a diode current of 33pA or approximately 0.001 footcandles (the level of illumination on a clear moonless night). The offset nulling circuit shown at the non-inverting input can be used to null out the room temperature offset. Note that this method is superior to using the offset nulling pin because using the offset nulling pin increases the offset voltage TC by approximately 3μV / °C for every millivolt nulled. In addition, the AD795SOIC package does not have an offset nulling pin.

[0491] The input sensitivity based on a total output voltage noise of 44μV is obtained by dividing the output voltage noise by the value of feedback resistor R2. This results in a minimum detectable diode current of 44fA. If a 12-bit ADC is used to digitize the 10V full-scale output, the least significant bit (LSB) weight is 2.5mV. The output noise level is much smaller than this.

[0492] [Table 23]

[0493] [Photodiode circuit trade-offs] There are many tradeoffs that can be made with the basic photodiode circuit design described so far. More signal bandwidth can be achieved at the expense of a larger output noise level. Reducing the feedback capacitor C2 to 1pF increases the signal bandwidth to approximately 160Hz. Further reduction of C2 is not practical since the parasitic capacitance is probably on the order of 1pF to 2pF. Also, a small amount of feedback capacitance is required to maintain stability.

[0494] If the circuit is operated at higher levels of illumination (greater than approximately 0.3fc), the value of the feedback resistor may be reduced, resulting in a further increase in the bandwidth of the circuit and less resistor noise. If gain ranging is used to measure higher light levels, careful attention must be paid to the design and layout of the additional switching network to minimize leakage paths.

[0495] [Compensation of high-speed photodiode I / V converter] A classical I / V converter is shown in Figure 5.24. Note that assuming R1>>R2, this is the same as for a photodiode preamplifier. The total input capacitance C1 is the sum of the diode capacitance and the opamp input capacitance. This is a classical second order system and the following guidelines may be applied to determine the appropriate compensation:

[0496] [Table 24]

[0497] The net input capacitance C1 is expressed as a function of frequency f in the noise gain transfer function as shown in the Bode plot. 1 Form a zero at f 1 =1 / (2πR2C1) Here, we ignore the effect of the compensation capacitor C2, which is small relative to C1 and is added to the circuit without affecting the zero frequency f 1 Note that we are assuming that the approximation does not significantly affect . In most cases, this approximation will be close enough, taking into account other variables in the circuit.

[0498] If left uncompensated, the crossover frequency f 2 The phase shift in f causes instability and oscillations. Adding a feedback capacitor C2 reduces the 2 Introducing a pole at stabilizes the circuit and provides a phase margin of approximately 45 degrees. f 2 =1 / (2πR2C2) f 2 is the unity gain bandwidth frequency f of the op amp and u Since it is the geometric mean of f 2 =√(f 1 f u ) These equations can be combined and solved for C2: C2 = √(C1 / (2πR2·f u )) This value of C2 results in a phase margin of about 45 degrees. Increasing the capacitor by a factor of two increases the phase margin to about 65 degrees.

[0499] In practice, the optimum value of C2 should be determined experimentally by varying it slightly to optimize the output pulse response.

[0500] [Selecting an Op Amp for a Wideband Photodiode I / V Converter] The op-amp in the high-speed photodiode I / V converter should be of wideband FET input type to minimize the effects of input bias current and allow low values ​​of photocurrent to be detected. In addition, it should have a bandwidth of 3 dB, f 2 The formula for f u , rearranged with R2 and C1, we get f 2 =√(f u / (2πR2C1)) Here, C1 is the capacitance of the diode, C D and the op amp's input capacitance C IN In high speed applications, the diode capacitance will be much smaller than that of the low frequency preamplifier designs discussed above, possibly down to a few pF.

[0501] By examining this formula, f 2 To maximize the gain, a FET-input op amp should have a high unity gain bandwidth product, f u and low input capacitance C IN In fact, when evaluating various op amps for this application, it is clear that the C IN f for u The ratio of is a preferred figure of merit.

[0502] Figure 5.25 compares several FET-input op amps suitable for photodiode preamplifiers. Studies have shown that the AD823 op amp has the highest ratio of unity gain-bandwidth product to input capacitance, along with a relatively low input bias current. For these reasons, it was chosen for the wideband photodiode preamplifier design.

[0503] [Table 25]

[0504] [High-speed photodiode preamplifier design] The HP5082-4204 PIN photodiode will be used as an example for discussion. Its characteristics are shown in Figure 5.26. It is typical of many commercially available PIN photodiodes. As with most high speed photodiode applications, the diode is operated in reverse bias or photoconductive mode. This greatly reduces the junction capacitance of the diode, but causes the diode to conduct a small amount of dark current even when it is not illuminated (a circuit that compensates for this dark current error is shown later in this section).

[0505] This photodiode is linear with respect to illumination up to an output current of approximately 50 μA to 100 μA. The dynamic range is limited by the total circuit noise and the dark current of the diode (assuming no dark current compensation).

[0506] [Table 26]

[0507] Using the circuit shown in Figure 5.27, assume we want a full-scale output of 10V for a diode current of 100µA. This dictates the value of feedback resistor R2 to be 10V / 100µA = 100kΩ.

[0508] Diode capacitance C D= 4 pF and the input capacitance C of the AD823 IN = 1.8pF, the value of C1 is C1 = C D +C IN = 5.8pF. C1 = 5.8pF, R2 = 100kΩ, f u Solving the above equation using =16MHz we find: f 1 =274kHz C2 = 0.76 pF f 2 =2.1MHz

[0509] Note that in the final design (Figure 5.27), the 100 kΩ resistor is replaced with three 33.2 kΩ film resistors to minimize stray capacitance. The feedback capacitor C2 is a variable 1.5 pF ceramic and is adjusted for best bandwidth / pulse response in the final circuit. The bandwidth of the overall circuit is approximately 2 MHz.

[0510] The full-scale output voltage of the preamplifier for a diode current of 100 μA is 10 V, and the error (RTO) due to the photodiode dark current of 600 pA is 60 mV. The dark current error can be cancelled by using a second photodiode of the same type at the non-inverting input of the op amp, as shown in Figure 5.27.

[0511] [Table 27]

[0512] [Noise Analysis of High-Speed ​​Photodiode Preamplifiers] As with most noise analyses, only the major contributors need to be identified: noise sources are combined in an RSS fashion so that any single noise source that is at least three or four times larger than any of the others will dominate.

[0513] In the case of a wideband photodiode preamplifier, the dominant source of output noise is the op amp's input voltage noise, V Nand resistor noise V due to R2 N,R2 (see Figure 5.28). The input current noise of the FET input op amp is negligible. The shot noise of the photodiode (induced by the reverse bias) is negligible due to the filtering effect of the shunt capacitance C1. The resistor noise is easily calculated since a 1 kΩ resistor will generate about 4 nV / √Hz and therefore a 100 kΩ resistor will generate 40 nV / √Hz. The bandwidth for integration is the signal bandwidth, i.e. 2.1 MHz, and the total output rms noise is: V N,R2 RTO noise = 40√(1.57 2.1 10 6 )=73μVrms

[0514] The factor of 1.57 converts the approximate single-pole bandwidth of 2.1 MHz into an equivalent noise bandwidth.

[0515] The output noise due to input voltage noise is obtained by multiplying the noise gain by the voltage noise and integrating the whole function over frequency. Doing this exactly can be tedious, but some reasonable approximations can be made that greatly simplify the calculations. Obviously, for wideband circuits, the low frequency 1 / f noise can be neglected. The main source of output noise is the 1 / f noise. 1 and f u This is due to high frequency noise gain peaking occurring between . If we simply assume that the output noise is constant over the entire range of frequencies and use the maximum value for the AC noise gain [1+(C1 / C2)], we get: V N RTO noise ≒ V N (1+C1 / C2)√(1.57f 2 )=250μVrms

[0516] The total rms noise associated with the output is then the RSS value of the two components: Total RTO noise = √((73) 2 +(250) 2 )=260μVrms

[0517] The total output dynamic range can be calculated by dividing the full-scale output signal (10V) by the total output rms noise of 260μVrms and converting to dB to be approximately 92dB.

[0518] [Table 28]

[0519] [High impedance charge output sensor] High impedance transducers, such as piezoelectric sensors, hydrophones, and some accelerometers, require an amplifier to convert the movement of charge into a change in voltage. Due to the high DC output impedance of these devices, a suitable buffer is required. The basic circuit for an inverting charge-sensitive amplifier is shown in Figure 5.29. There are basically two types of charge transducers: capacitive and charge-dissipating. In a capacitive transducer, the voltage across a capacitor (V C ) is kept constant. The change in capacitance ΔC is the change in charge ΔQ = ΔCV C This charge is transferred to a voltage ΔV OUT =-ΔQ / C2=-ΔCV C / C2 is transmitted to the op amp output.

[0520] [Table 29]

[0521] A charge-emission transducer produces an output charge ΔQ, and its output capacitance remains constant. This charge normally produces an open-circuit output voltage at the output of the transducer equal to ΔQ / C. However, since the voltage across the transducer is held constant by the virtual ground of the op-amp (R1 is usually small), charge is transferred to capacitor C2, producing an output voltage ΔV OUT =-ΔQ / C2.

[0522] In practical applications, the charge amplifier responds only to AC inputs. The upper cutoff frequency is f 2 = 1 / 2πR2C2, the lower limit is f 1 = 1 / 2πR1C1.

[0523] [Circuit configuration of low noise charge amplifier] Figure 5.30 shows two methods of buffering and amplifying the output of a charge output transducer. Both require the use of an amplifier with very high input impedance, such as the AD745. The AD745 offers both low voltage and low current noise. This combination makes the device particularly suitable for applications requiring very high charge sensitivity, such as capacitive accelerometers and hydrophones.

[0524] [Table 30]

[0525] The first circuit in Figure 5.30 (left) uses an op amp in inverting mode. Amplification relies on the principle of conservation of charge at the amplifier's inverting input. Capacitor C S The charge on the capacitor C F and hence ΔQ / C F The input voltage noise of the amplifier is the AC noise gain of the circuit, 1 + C S / C F The signal is amplified by and appears at the output.

[0526] The second circuit (right) shown in Figure 5.30 is simply a high impedance follower with gain. Here the noise gain (1+R2 / R1) is the same as the gain from the transducer to the output. In both circuits, the resistor R B is needed as a return for the DC bias current.

[0527] To maximize DC performance over temperature, the source resistance at each input of the amplifier should be balanced. This is shown in Figure 5.30 by the resistor RB For best noise performance, the source capacitance is also represented by the capacitor C B The input capacitance should be balanced with the gate-source capacitance of the amplifier. In general, it is good practice to balance the source impedance (both resistive and reactive) as seen by the input of a precision low noise BiFET amplifier such as the AD743 / AD745. Balancing the resistive components will reduce the effect of bias current errors and therefore optimize DC performance over temperature. Balancing the input capacitance will minimize AC response errors due to the nonlinear common-mode input capacitance of the amplifier and also optimize noise performance as shown in Figure 5.30. In any FET input amplifier, the current noise of the internal bias circuitry can be coupled to the input via the gate-source capacitance (20 pF for the AD743 and AD745) and appear as excess input voltage noise. This noise component is correlated at the input so source impedance matching will tend to cancel its effect. Figure 5.30 shows the required external components for both inverting and noninverting configurations. A C greater than 300 pF is required. B For values ​​of , there is a decreasing effect on noise, and C B may simply be a large-capacity Mylar bypass capacitor of 0.01 μF or more.

[0528] [40 dB gain piezoelectric transducer amplifier operates at reduced supply voltage for low bias current] Figure 5.31 shows the piezoelectric transducer amplifier connected in voltage output mode. Reducing the power supply to +5V reduces the effect of the bias current in two ways: first, by lowering the total power dissipation, and second, by reducing the basic gate-junction leakage current. The addition of a clip-on heat sink such as the Aavid #5801 will further limit the internal junction temperature rise.

[0529] Without AC coupling capacitor C1, the amplifier will operate over the temperature range of 0°C to +85°C. If the optional AC coupling capacitor C1 is used, the circuit will operate over the entire temperature range of -55°C to +125°C, but DC information will be lost.

[0530] [Table 31]

[0531] [Hydrophone] Interfacing the output of highly capacitive transducers, such as hydrophones, some accelerometers, and condenser microphones, to the outside world presents many design challenges. Previously, designers had to use expensive hybrid amplifiers made of discrete low noise JFETs in front of traditional op amps to achieve the low levels of voltage and current noise required by these applications. Now, with the AD743 and AD745, designers can achieve nearly the same level of performance as the hybrid approach with a monolithic solution.

[0532] In sonar applications, piezoelectric ceramic cylinders are commonly used as the active element of hydrophones. A typical cylinder has a nominal capacitance of about 6000 pF and a series resistance of 10 Ω. The output impedance is typically 10 8 Ω or 100MΩ.

[0533] Since the hydrophone signals of interest are AC in nature with a wide dynamic range, noise is of primary concern among sonar system designers. The noise floor of the hydrophone and the hydrophone preamplifier combine to limit the sensitivity of the system and therefore the overall usefulness of the hydrophone. Typical hydrophone bandwidths are in the range of 1 kHz to 10 kHz. The AD743 and AD745 op amps have low noise figures of 2.9 nV / √Hz and 10 10It has a high input impedance of 30 Ω (i.e. 10 GΩ), making it ideal for use as a hydrophone amplifier.

[0534] The AD743 and AD745 are paired amplifiers with different levels of internal compensation. The AD743 is internally compensated for unity gain stability. The AD745 is stable for noise gains of 5 or greater and has a fairly high bandwidth and slew rate. This makes the AD745 particularly useful as a high gain preamplifier providing both high gain and wide bandwidth. The AD743 and AD745 also operate with extremely low levels of distortion, less than 0.0003% and less than 0.0002% (at 1 kHz), respectively.

[0535] [Op Amp Performance: JFET vs. Bipolar] The AD743 and AD745 op amps are the first monolithic JFET devices that offer low input voltage noise comparable to bipolar op amps, without the high input bias currents typically associated with bipolar op amps. Figure 5.32 shows the input voltage noise versus input source resistance for the bias current compensated OP27 and JFET input AD745 op amps. Note that the noise levels for the AD743 and AD745 are identical. From this figure, it is clear that at high source impedances, the low current noise of the AD745 results in lower overall noise than high performance bipolar op amps. It is also important to note that for the AD745, this noise reduction extends to low source impedances. As shown in Figure 5.32, at high source impedances, the low DC current error of the AD745 also reduces errors due to offset and drift.

[0536] [Table 32]

[0537] [pH probe buffer amplifier] A typical pH probe is 10 6Ω~10 9 To isolate the 20 Ohm source resistance from external circuitry, a buffer amplifier is required. Such an amplifier is shown in Figure 5.33. The low input current of the AD795 allows voltage errors caused by bias currents and electrode resistance to be minimized. The use of guarding, shielding, high insulation resistance standoffs, and other such standard picoamp methods used to minimize leakage are all required to maintain the accuracy of this circuit.

[0538] The slope of the transfer function of the pH probe is 50 mV per pH unit at room temperature with a temperature coefficient of approximately +3500 ppm / °C. The buffer shown in Figure 5.33 provides a gain of 20, resulting in an output voltage equal to 1 volt / pH unit. Temperature compensation is provided by resistor RT, a special temperature-compensated resistor (1 kΩ, 1%, +3500 ppm / °C, #PT146) available from Precision Resistor Co., Inc. (Reference 18).

[0539] [Table 33]

[0540] [CCD / CIS image processing] Charge-coupled devices (CCDs) and contact image sensors (CIS) are widely used in consumer imaging systems such as scanners and digital cameras. A general block diagram of an imaging system is shown in Figure 5.34. The imaging sensor (CCD, CMOS, or CIS) is exposed to an image or photograph, much like film is exposed in a camera. After exposure, the output of the sensor goes through some analog signal processing and then is digitized by an ADC. Most of the actual image processing is performed using high-speed digital signal processors. At this point, the image may be manipulated in the digital domain to perform functions such as contrast or color enhancement / correction.

[0541] The building blocks of a CCD are the individual light-sensing elements called pixels (see Figure 5.35). A single pixel consists of a light-sensitive element such as a photodiode or photocapacitor that outputs a charge (electrons) proportional to the light (photons) it is exposed to. The charge is accumulated during the exposure or integration time, after which it is transferred to the CCD shift register and sent to the output of the device. The amount of charge accumulated will depend on the light level, the integration time and the quantum efficiency of the light-sensitive element. A small amount of charge will accumulate even when no light is present; this is called the dark signal or dark current and must be compensated for during signal processing.

[0542] The pixels can be arranged in a linear or area configuration, as shown in Figure 5.36. A clock signal transfers the charge from the pixels into an analog shift register, and then a further clock is applied to move the individual pixel charges to the output stage of the CCD. Scanners commonly use a linear configuration, while digital cameras use an area configuration. Analog shift registers typically operate at frequencies between 1MHz and 10MHz for linear sensors, and between 5MHz and 25MHz for area sensors.

[0543] [Table 34]

[0544] [Table 35]

[0545] [Table 36]

[0546] A typical CCD output stage is shown in Figure 5.37 with the associated voltage waveforms. The output stage of a CCD is connected to a sensing capacitor C S At the beginning of each pixel period, the charge of each pixel is converted to a voltage through C S The voltage at the reference level V REFwhich generates a reset glitch. The amount of light sensed by each pixel is measured by the difference ΔV between the reference level and the video level. The charge on a CCD can be as low as 10 electrons, and a typical CCD output has a sensitivity of 0.6 μV / electron. Most CCDs have a saturation output voltage of about 500 mV to 1 V for area sensors and 2 V to 4 V for linear sensors. The DC level of the waveform is 3 V to 7 V.

[0547] CCDs are typically fabricated in a CMOS process and therefore have limited ability to perform on-chip signal conditioning. Therefore, the CCD output is typically processed by external conditioning circuitry. The nature of the CCD output requires it to be clamped before being digitized by the ADC. In addition to this, offset and gain functions are typically part of the analog signal processing.

[0548] The output voltage of a CCD is small and very often buried in noise. The largest noise source is the thermal noise in the resistor of the FET reset switch. This noise can have a typical value of 100 electrons rms to 300 electrons rms (approximately 60 mVrms to 180 mVrms). This...

Claims

1. A first analyte, e.g., O ２ a sensing system comprising an array of sensor units configured to generate a first signal in response to at least one analyte proportional to its content and to generate a second signal in response to at least one analyte proportional to a second analyte, e.g., a pH value, wherein each sensor unit of the array of sensors is positioned to correspond to a corresponding well on a sample carrier comprising an array of wells; a stage configured to receive the sample carrier; a motion actuator assembly configured to position the stage and / or the sensing system relative to one another in one or more of the x-axis, z-axis, and y-axis; a sample temperature control element, e.g. a heating element, configured to control the temperature of a sample in at least one well (e.g. each well) of the sample carrier to be within a predetermined amount of a sample in another well of the sample carrier; a signal processing module operably connected to the sensing system, the signal processing module being configured, for example, to receive and / or amplify the first signal and the second signal; An analytical instrument comprising:

2. The instrument of claim 1 , wherein the sensing system, the stage, the motion actuator assembly, the sample control element, and the signal processing module are contained within a housing.

3. The instrument of claim 2 , wherein the housing includes an opening in a sidewall dimensioned to allow passage of the stage and the sample carrier.

4. The instrument of claim 1 further comprising the sample carrier.

5. The instrument of claim 4 , wherein the sample carrier is positioned on the stage.

6. The apparatus of claim 1 , wherein the movement actuator assembly includes at least one axial actuator assembly, for example at least one x-axis actuator assembly.

7. 7. The instrument of claim 6, wherein the at least one axis actuator assembly, e.g., an x-axis actuator assembly, is configured to position the stage relative to the sensing system on at least one axis, e.g., the x-axis, e.g., to align at least one well (e.g., each well) of the sample carrier arranged on the stage with a corresponding sensor unit on the x-axis.

8. 7. The apparatus of claim 6, wherein the at least one axis actuator assembly, e.g., an x-axis actuator assembly, is configured to position the stage relative to the housing on at least one axis, e.g., the x-axis, e.g., within the housing or external to the housing through the opening.

9. The apparatus of claim 1 , wherein the movement actuator assembly includes at least one y-axis actuator assembly.

10. 10. The instrument of claim 9, wherein the at least one axis actuator assembly, e.g., a y-axis actuator assembly, is configured to position the stage relative to the sensing system on at least one axis, e.g., the y-axis, for example, to align at least one well (e.g., each well) of the sample carrier arranged on the stage with a corresponding sensor unit on the y-axis.

11. The apparatus of claim 1 , wherein the movement actuator assembly includes at least one z-axis actuator assembly.

12. The instrument of claim 11, wherein the at least one axis actuator assembly, e.g., a z-axis actuator assembly, is configured to position the sensing system relative to the stage on at least one axis, the z-axis, e.g., to position each sensor unit in fluid communication with the corresponding well.

13. The device of claim 1 , wherein the sensing system is incorporated into or on a cartridge.

14. The apparatus of claim 1 , further comprising a dispensing system including at least one syringe configured to dispense at least one target agent into one or more wells of the sample carrier.

15. 15. The instrument of claim 14, further comprising an injector movement actuator assembly positioned to actuate the at least one injector to dispense the at least one target agent across a plurality of wells of the sample carrier.

16. 10. The instrument of claim 1, wherein the dispensing system includes an array of injectors configured to dispense at least one target agent, each injector positioned to correspond to a corresponding well on the sample carrier.

17. 17. The instrument of claim 16, wherein the array of injectors comprises two or more injectors, for example at least two, three, four, five, six, seven, eight, nine, or ten injectors, positioned to correspond to at least two wells (e.g., each well) on the sample carrier.

18. 15. The apparatus of claim 14, further comprising a manifold temperature control element, e.g., a heating element, configured to control the temperature of the dispensing system, e.g., the target agent, the sensing system, and / or the cartridge.

19. 20. The instrument of claim 18, wherein the manifold temperature control element and the sample temperature control element are configured to independently control temperature.

20. 19. The instrument of claim 18, wherein the manifold temperature control element is configured to control the temperature of the target agent and / or the sensing system and / or the cartridge, and the sample temperature control element is configured to control the temperature of the sample in the array of wells of the sample carrier to be within 3°C, for example within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the temperature of the corresponding target agent and / or corresponding sensor unit.

21. 2. The instrument of claim 1, wherein the sample temperature control element and / or the manifold temperature control element are configured to control evaporation of the sample in the array of wells to less than 25%, e.g., less than 20%, less than 15%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

22. 15. The instrument of claim 14, wherein the at least one axial actuator assembly, e.g., at least one z-axis actuator assembly, is configured to position the dispensing system relative to the stage on at least one axis, e.g., the z-axis, e.g., to position each injector in communication, e.g., fluid communication, with the corresponding well, enabling delivery of the target agent to the sample.

23. 15. The device of claim 14, wherein the at least one injector or each injector of the array of injectors is configured to dispense the same target agent.

24. 15. The device of claim 14, wherein each injector is configured to independently dispense a selected targeted agent, e.g., a first injector configured to dispense a first targeted agent, a second injector configured to dispense a second targeted agent, optionally a third injector configured to dispense a third targeted agent, a fourth injector configured to dispense a fourth targeted agent, and an nth injector configured to dispense an nth targeted agent.

25. 25. The apparatus of claim 24, wherein a plurality of injectors or an array of injectors are configured to independently dispense two or more target agents into at least one well of the sample carrier.

26. The device of claim 14 , wherein the sensing system and the dispensing system are integrated into or on the cartridge.

27. The device of claim 14 , further comprising at least one target agent loaded into the dispensing system.

28. The instrument of claim 1 , wherein the sample temperature control element and / or the manifold temperature control element are formed from a temperature-conductive material.

29. 30. The instrument of claim 28, wherein the sample temperature control element is fixed to the stage.

30. 30. The instrument of claim 28, wherein the sample temperature control element is configured for close proximity or direct contact with the sample carrier.

31. 30. The apparatus of claim 28, wherein the manifold temperature control element is configured for close proximity or direct contact with the dispensing system.

32. 2. The instrument of claim 1, wherein the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g. each well) of the sample carrier to be between 0°C and 70°C above ambient temperature, for example between 8°C and 20°C above ambient temperature, for example 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 30°C, 40°C, 50°C, 60°C, or 70°C above ambient temperature.

33. 10. The instrument of claim 1, wherein the sample temperature control element is configured to maintain the temperature of the sample in at least one well (e.g., each well) of the sample carrier within a predetermined range.

34. 2. The instrument of claim 1, wherein the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier so that a sensor signal corresponding to the level, production, or consumption of a target analyte, e.g., the first analyte or the second analyte, does not differ by more than a predetermined amount between two identical or substantially identical samples, e.g., does not differ significantly between two identical or substantially identical samples.

35. The target analyte is O ２ and wherein the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) such that the sensor signal, as a function of the level, production, or consumption of the target analyte, does not differ by more than 10% between two identical or substantially identical samples, e.g., by more than 5%, 3%, 1%, or 0.1%.

36. 35. The instrument of claim 34, wherein the target analyte is a analyte proportional to a pH value, and the sample temperature control element is configured to control the temperature of the sample in at least one well (e.g., each well) so that the sensor signal corresponding to the level, production, or consumption of the target analyte does not differ by more than 10% between two identical or substantially identical samples, e.g., by more than 5%, 3%, 1%, or 0.1%.

37. 2. The instrument of claim 1, wherein the sample temperature control element is configured to control the temperature of a sample in at least one well (e.g., each well) of the sample carrier to be within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the sample in another well of the sample carrier.

38. 10. The instrument of claim 1, wherein the manifold temperature control element is configured to control the temperature of a sensor to be within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​another sensor.

39. 2. The instrument of claim 1, wherein the sample temperature control element and the manifold temperature control element are configured to control the temperature of the sample in at least one well (e.g., each well) of the sample carrier to within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C of a corresponding sensor.

40. 2. The instrument of claim 1, wherein the sample temperature control element is configured to control the temperature of a sample in a first well to be within a predetermined amount of a sample in a second well, the first well being a boundary well, the second well being an interior well of the sample carrier, and the boundary well being a well with no other wells located between the boundary well and an edge or boundary of the sample carrier.

41. 2. The instrument of claim 1, wherein the manifold temperature control element is configured to control the temperature of a sensor corresponding to a first well to be within a predetermined amount of a sensor corresponding to a second well, the first well being a boundary well, the second well being an interior well of the sample carrier, and the boundary well being a well with no other wells located between the boundary well and an edge or boundary of the sample carrier.

42. 41. The instrument of claim 40, wherein the sample temperature control element and / or the manifold temperature control element are configured to maintain the temperature of the sample in the first well and the second well and / or the sensors corresponding to the first well and the second well within a predetermined range.

43. 41. The instrument of claim 40, wherein the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well, such that, when the samples are analyzed under the same or substantially the same conditions, a sensor signal corresponding to the level, production, or consumption of a target analyte, e.g., the first analyte or the second analyte, does not differ by more than a predetermined amount between each sample, e.g., does not differ significantly between each sample.

44. The target analyte is O ２ and / or the target analyte is proportional to a pH value, and the sample temperature control element and / or the manifold temperature control element are configured to control the temperature of the samples in the first well and the second well (e.g., two identical or substantially identical samples) and / or the sensors corresponding to the first well and the second well so that, e.g., when the samples are analyzed under the same or substantially the same conditions, the sensor signal corresponding to the level, production, or consumption of the target analyte does not differ by more than 10% between each sample, e.g., by more than 5%, 3%, 1%, or 0.1%.

45. 2. The instrument of claim 1, wherein the sample temperature control element is configured to control the temperature of the sample in the first well to within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the sample in the second well, and / or the manifold temperature control element is configured to control the temperature of the sensor corresponding to the first well to within 3°C, e.g., within 2°C, within 1°C, within 0.6°C, within 0.5°C, within 0.4°C, within 0.3°C, within 0.2°C, or within 0.1°C, of ​​the sensor corresponding to the second well.

46. 43. The instrument of claim 42, wherein the first well is a boundary well and the second well is an internal well of a sample carrier having one or more wells, for example 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384 or more wells.

47. The instrument of claim 1 , wherein the sample temperature control element comprises a heating element.

48. The instrument of claim 1 , wherein the sample temperature control element forms a controlled temperature zone that includes the array of wells of the sample carrier.

49. 49. The device of claim 48, wherein the controlled temperature zone does not include a headspace of the housing.

50. 49. The instrument of claim 48, wherein the volume of the controlled temperature zone does not exceed the volume of the sample carrier by more than 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x.

51. 49. The device of claim 48, wherein the volume of the controlled temperature zone does not exceed 10% of the volume of the housing, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

52. 49. The instrument of claim 48, wherein the temperature of components outside the controlled temperature zone is not substantially changed, e.g., not increased or decreased, by actuation of the sample temperature control element.

53. 2. The instrument of claim 1, wherein the sample temperature control element is configured to bring the temperature of the sample in at least one well (e.g., each well) of the sample carrier to within a predetermined range of a target temperature within about 5 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, or 1 minute of activation of the sample temperature control element and / or introduction of the sample carrier into the controlled temperature zone.

54. 2. The instrument of claim 1, wherein the sample temperature control element and / or the manifold temperature control element are configured to control temperature so as to control, e.g., reduce, limit, or inhibit, diffusion of gas in the controlled temperature zone.

55. 55. The instrument of claim 54, wherein the sample temperature control element and / or the manifold temperature control element are configured to control temperature in a manner that reduces, limits, or inhibits diffusion of gas in the controlled temperature zone so that the composition of the gas in the controlled temperature zone does not change significantly, for example, by no more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20%.

56. The device of claim 1 , wherein the signal processing module is capable of operating at elevated relative humidity (RH), for example at least 75% RH, 85% RH, 95% RH, or 99% RH.

57. The device of claim 1 , wherein the signal processing module is configured to simultaneously receive and amplify the first signal and the second signal.

58. The device of claim 1 , wherein the signal processing module is configured to receive and amplify the first signal and the second signal separately, e.g., sequentially.

59. 10. The apparatus of claim 1, wherein the signal processing module is configured to detect one or more of the first signal and the second signal using time-based detection, such as decay rate, phase shift, or anisotropy detection.

60. 10. The apparatus of claim 1, wherein the signal processing module is configured to detect one or more of the first signal and the second signal using intensity-based detection, optionally including ratiometric measurements.

61. 10. The apparatus of claim 1, wherein the signal processing module includes a printed circuit assembly formed of an insulating material having a high dielectric constant.

62. 10. The apparatus of claim 1, wherein the signal processing module includes a printed circuit assembly having a transimpedance amplifier including a grounded guard trace.

63. 10. The apparatus of claim 1, wherein the signal processing module comprises a printed circuit assembly formed of surface mount components, e.g., substantially free of secondary hand-soldered high gain components.

64. The apparatus of claim 1 , wherein the signal processing module includes a printed circuit assembly including a thermally conductive excitation source, and optionally the thermally conductive excitation source is in thermal communication with, e.g., thermal contact with, a heat sink.

65. 65. The apparatus of claim 64, wherein the sensing system does not include a reference signal detector.

66. 2. The apparatus of claim 1, wherein the signal processing module is configured to operate with reduced parasitic currents, such as reduced interference, dark current, or noise, associated with detecting and / or amplifying at least one of the first signal and the second signal.

67. the sensing system is constructed and arranged to form a measurement chamber between a sample-facing surface of each sensor unit and a sensor-facing surface of at least one well (e.g., each well) when the movement actuator assembly is deployed to position each sensor unit in fluid communication with the sample in a corresponding well; evaporation of the sample, flow of the sample, or diffusion of components of the sample, e.g., analyte components, out of the measurement chamber is reduced; 10. The device of claim 1.

68. 68. The instrument of claim 67, wherein the sample temperature control element is configured to control the temperature of the sample in each measurement chamber.

69. The device of claim 1 , wherein the sensing system includes one or more optical sensors, such as photoluminescence sensors.

70. The device of claim 1 , wherein the sensing system includes one or more electrochemical sensors.

71. The sensing system detects O in the sample. ２ 10. The instrument of claim 1, configured to generate a signal in response to a rate of change of an analyte proportional to its content, for example, a signal proportional to the oxygen consumption rate (OCR) of the sample.

72. 2. The instrument of claim 1, wherein the sensing system is configured to generate a signal in response to a rate of change of the analyte that is proportional to the pH value of the sample, e.g., a signal proportional to the extracellular acidification rate (ECAR) and / or the proton efflux rate (PER) of the sample.

73. The instrument of claim 1 , wherein the sensing system is configured to generate a signal in response to one or more electrochemical properties of the sample, such as impedance.

74. 10. The apparatus of claim 1, wherein the signal processing module is operably connected to a computing network or computer device programmed to calculate one or more of mitochondrial respiration, glycolysis, adenosine triphosphate (ATP) production rate, and a mitochondrial toxicity (mitotox) index value of the sample in response to one or more of the first signal and the second signal.

75. 75. The apparatus of claim 74, wherein the signal processing module is operably connected to a cloud-based computing network.

76. 75. The apparatus of claim 74, wherein the signal processing module is operably connected to a data storage module that stores historical values ​​of the first signal and the second signal.

77. 77. The apparatus of claim 76, wherein the data storage module is a local memory storage device.

78. 77. The apparatus of claim 76, wherein the data storage module is a cloud-based memory storage device.

79. The array of sensor units is X ｓ１ The sensor unit includes X ｓ１ 2. The apparatus of claim 1, wherein is equal to or greater than 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, or 384.

80. The array of injectors is X ｓ２ Including X injectors ｓ２ 2. The apparatus of claim 1, wherein is equal to or greater than 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384, 768, or 1536.

81. Sensor unit X ｓ１ and X on the syringe ｓ２ 2. The device of claim 1, wherein the ratio of:

82. Injector X ｓ２ and the X of the sensor unit ｓ１ 2. The device of claim 1, wherein the ratio of:

83. The apparatus of claim 1 , wherein each sensor unit of the array of sensor units is configured to independently generate one or more of the first signal and the second signal.

84. The apparatus of claim 1 , wherein each sensor unit of the array of sensor units is configured to simultaneously generate the first signal and the second signal.

85. 2. The apparatus of claim 1, further comprising a light source, such as a fluorescent lamp, a light emitting diode (LED), or a laser, configured to excite a sensor of the sensor unit to generate one or more of the first signal and the second signal.

86. 86. The apparatus of claim 85, wherein the light source is configured to generate a reference signal, and wherein fluctuations in intensity from the light source are corrected proportional to drift by monitoring the reference signal generated by the light source.

87. 86. The apparatus of claim 85, wherein the light source is positioned on a thermally conductive printed circuit assembly configured to minimize drift from the light source, and optionally the thermally conductive printed circuit assembly is formed from a material configured to minimize drift caused by thermally induced variations from the light source by at least 20%, e.g., at least 15%, 10%, 5%, or 1%.

88. The apparatus of claim 1 , further comprising an electric motor configured to actuate one or more of the movement actuator assemblies, e.g., the x-axis actuator assembly, the z-axis actuator assembly, and the y-axis actuator assembly.

89. 10. The apparatus of claim 1, further comprising a stall sensing module programmed to generate a notification signal, optionally pausing the protocol, e.g., stopping motor movement, if a predetermined protocol step is not completed within a predetermined time interval.

90. 2. The instrument of claim 1, further comprising a proximity sensor configured to generate a notification signal and optionally pause a protocol when a component is positioned within a predetermined distance of another component, e.g., when a sensor unit is positioned within a predetermined distance of a corresponding well of the sample carrier.

91. 10. The device of claim 1, further comprising a proximity sensor configured to generate a notification signal, and optionally pause a protocol, when the opening in the side wall of the housing is ajar and / or when external light is detected within the housing.

92. 10. The instrument of claim 1, having an OCR detection range of 2000 pmol / min to 0.01 pmol / min, such as 700 pmol / min to 0.01 pmol / min, for example 50 pmol / min to 0.01 pmol / min.

93. 10. The instrument of claim 1, having an OCR lower detection limit of less than 50 pmol / min, e.g., less than 40 pmol / min, less than 30 pmol / min, less than 20 pmol / min, less than 10 pmol / min, less than 5 pmol / min, less than 3 pmol / min, less than 1 pmol / min, less than 0.1 pmol / min, or less than 0.01 pmol / min.

94. 10. The instrument of claim 1, further comprising an optical module positioned to image or scan one or more samples in the array of wells of the sample carrier.

95. 95. The instrument of claim 94, wherein the optical module is operably connected to the computer, optionally configured to display and / or record images or scans of the sample in real time.

96. 10. The apparatus of claim 1, further comprising a transfer module formed of multiplexed fiber optic material configured to transfer optical signals from the array of sensor units to the signal processing module.

97. 97. The apparatus of claim 96, wherein the transfer module is configured to transfer one or more of an excitation optical signal, a reference optical signal, and an emission optical signal.

98. 97. The apparatus of claim 96, wherein the transfer module is configured to interface directly with one or more sensor units.

99. 97. The apparatus of claim 96, wherein the sensing system includes a homogenized fiber optic waveguide optically connected to the transfer module, optionally the homogenized fiber optic waveguide configured to uniformly distribute light to one or more sensor units.

100. 10. The instrument of claim 1, further comprising an environmental control module configured to control the environment of the sample in at least one well (e.g., each well) of the sample carrier, e.g., configured to control the environmental gas and / or relative humidity (RH).

101. The environmental control module controls the N ２ Concentration, O ２ concentration, and CO ２ 101. The device of claim 100, configured to control one or more of the concentrations.

102. The environmental control module may include a gas, such as N, in fluid connection with the sample carrier. ２ , O ２ , and CO ２ 102. The device of claim 101, comprising one or more sources of:

103. 101. The instrument of claim 100, wherein the environmental control module forms a controlled environmental zone that includes the array of wells of the sample carrier.

104. 104. An apparatus as claimed in claim 103, wherein the controlled environment zone is formed in a sealed container, e.g. a hermetically sealed container.

105. 10. The device of claim 1 configured for use in a gas-controlled environment.

106. A method of using the analytical instrument of claim 1, comprising: loading a sample carrier containing one or more cell samples, each sample disposed in a corresponding well of the sample carrier, into the analytical instrument; A method comprising:

107. 107. The method of claim 106, wherein the cell sample comprises live cells.

108. 107. The method of claim 106, wherein loading the sample carrier comprises fixing the sample carrier on the stage.

109. Providing an analytical instrument according to claim 1; loading a sample carrier containing one or more cellular samples into the analytical instrument, each sample being disposed in a corresponding well of the sample carrier; The first analyte, e.g., O ２ obtaining a first plurality of values ​​from signals responsive to at least one analyte proportional to its content, each signal being generated by a corresponding sensor unit of the sensing system; Optionally, obtaining a second plurality of values ​​from signals responsive to the second analyte, e.g., at least one analyte proportional to a pH value, each signal being generated by a corresponding sensor unit of the sensing system; processing the first plurality of values; Optionally, processing the second plurality of values; and a method for analyzing a cell sample.

110. 110. The method of claim 109, further comprising controlling the temperature of a sample in at least one well (e.g., each well) of the sample carrier to be within a predetermined amount of the sample in another well of the sample carrier.

111. 110. The method of claim 109, wherein the sample carrier is loaded into the controlled temperature zone and / or controlling the temperature of the sample comprises forming the controlled temperature zone.

112. 110. The method of claim 109, further comprising controlling the temperature of the sensing system.

113. 110. The method of claim 109, further comprising dispensing a target agent into each sample in the array of wells of the sample carrier.

114. 114. The method of claim 113, further comprising loading the target agent into the dispensing system of the analytical instrument.

115. 114. The method of claim 113, further comprising controlling the temperature of the target agent.

116. 110. The method of claim 109, wherein the same sample is present in at least one well (e.g. each well) of the array of wells of the sample carrier.

117. 110. The method of claim 109, wherein a first sample is present in a first well of the array of wells of the sample carrier and a second sample is present in a second well of the array of wells of the sample carrier.

118. 118. The method of claim 117, wherein the first sample is a test sample and the second sample is a control.

119. 110. The method of claim 109, wherein the sample comprises living cells.

120. 110. The method of claim 109, wherein the sample comprises one or more of free cells, cell constructs, free tissue, tissue constructs, organelles, enzymes, cell products or by-products, and conditioned medium.

121. 110. The method of claim 109, wherein the sample comprises mammalian cells or tissue.

122. 110. The method of claim 109, wherein the sample comprises a non-mammalian cell or tissue.

123. 110. The method of claim 109, wherein the sample comprises a single-cell organism, such as a microorganism.

124. 110. The method of claim 109, wherein the sample comprises whole animal model tissue, e.g., zebrafish, C. elegans, or Drosophila.

125. 110. The method of claim 109, wherein the sample comprises whole plant model tissue or whole plant model cells.

126. The first test component is O ２ The method of claim 109, wherein the amount is proportional to the content.

127. 110. The method of claim 109, wherein the second test component is proportional to pH value.

128. 110. The method of claim 109, wherein the first value and the second value are obtained independently.

129. 110. The method of claim 109, wherein the first value and the second value are obtained simultaneously.

130. 110. The method of claim 109, further comprising obtaining an image or scan of the sample during or after the analysis.

131. 110. The method of claim 109, further comprising measuring one or more electrochemical properties of the sample, such as impedance, during or after the analysis.

132. 110. The method of claim 109, further comprising obtaining or calculating a mitochondrial toxicity (mitotox) index value for the sample during or after the analysis.

133. 110. The method of claim 109, further comprising controlling the environment of the sample in at least one well (e.g. each well) of the sample carrier, e.g. controlling the ambient gas and / or relative humidity (RH).

134. Controlling the environment may involve controlling the N gas surrounding the sample. ２ Concentration, O ２ concentration, and CO ２ 134. The method of claim 133, comprising controlling one or more of the concentrations.

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