Multi-mode system and method for analyzing cells

Abstract

Long-term cell analysis is provided by a device comprising: a chamber configured to receive a sample carrier having a plurality of wells with electrodes in electrical communication with an impedance meter; a cartridge having a compound and at least one delivery port for the compound; a camera configured to capture images of the cell culture in each well through an associated window in the well when the sample carrier is positioned at a first position; a fluid handler configured to communicate with the sample carrier and the cartridge and deliver a compound from the cartridge to a given well based on impedance measurements of the sample in the given well when the sample carrier is positioned at a second position; and a motion stage configured to move the sample carrier between the first position and the second position.

Description

[Technical Field]

[0001] Aspects and embodiments disclosed herein generally relate to measuring samples of living cells.

[0002] [CROSS-REFERENCE TO RELATED APPLICATIONS] This disclosure claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 483,218, entitled "MULTIMODE SYSTEMS AND METHODS FOR ANALYZING CELLS," filed February 3, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Rates such as the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are important indicators of mitochondrial respiration and glycolysis, and these measurements provide a system-level overview of cellular metabolic function in cultured cells and ex vivo samples. In addition, live-cell impedance measurements have been widely accepted as a label-free, noninvasive, quantitative analytical method for assessing cellular health.

[0004] Therefore, there is a need to develop new systems and methods for performing long-term metabolic and / or impedance-based measurements of living cells and imaging these cells in real time. Summary of the Invention [Means for solving the problem]

[0005] According to one aspect, a sensing system includes an array of sensor units configured to generate a first signal in response to a first analyte over time and generate a second signal in response to a second analyte over time, wherein each sensor unit of the array of sensor units 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 movement actuator assembly configured to position at least one of the stage and the sensing system relative to one another on one or more of an x-axis, a z-axis, and a y-axis; and a movement actuator assembly configured to move at least one of the stage and the sensing system relative to one another on at least one well of the sample carrier. A device capable of long term measurements is provided, comprising: a liquid handling system for dispensing a substance into a well; a sample control element configured to control a characteristic of a sample in at least one well of a sample carrier over said long term to be within a predetermined amount of another sample in another well of the sample carrier; and a controller operably connected to the sensing system and the sample control element, the controller being configured to control one or more of the temperature, humidity and gas content of an environment surrounding the sample carrier over said long term, and to acquire data corresponding to a first signal and a second signal for at least two points spanning said long term.

[0006] In some embodiments, the long-term measurement is performed in a microchamber having a reduced volume of 3 microliters or less, which is created by moving a sensor unit of an array of sensor units down a predetermined position into a corresponding well in a sample carrier.

[0007] In some embodiments, the long-term measurements are performed non-continuously between a single modality selected from the group consisting of flux measurements, impedance measurements, and imaging.

[0008] In some embodiments, the long-term measurements are performed non-continuously between at least two modalities selected from the group consisting of flux measurements, impedance measurements, and imaging.

[0009] In some embodiments, the control element controls the sample environment to maintain environmental parameters at target levels for associated wells within the sample carrier.

[0010] In some embodiments, the target level for the environmental parameter is programmatically changed over the course of the extended measurement.

[0011] In some embodiments, the control element controls the sample environment to achieve a target cellular microenvironment for the biological model in the sample via at least one of direct cellular, intracellular, pericellular, and proximal measurements of sample parameters.

[0012] In some embodiments, the cellular microenvironment is controlled for each sample.

[0013] In some embodiments, the target level for the sample parameter is programmatically changed over the course of the extended measurement.

[0014] In some embodiments, the device further comprises a ventilation system configured to alter the headspace gas composition in the cellular microenvironment.

[0015] In some embodiments, the sample control element comprises one or both of a sample temperature control element configured to control the temperature of the sample, or a sample environment control element comprising one or both of a gas control element configured to control the gas content of one or more of the O2, CO2, and N2 content of the sample, or a humidity control element configured to control the humidity of the environment.

[0016] In some embodiments, the sample control element comprises a heater.

[0017] In some embodiments, the first signal is a measurement of a first analyte proportional to the O2 content in a given well, and the second signal is a measurement of a second analyte proportional to the pH value in a given well.

[0018] In some embodiments, the first signal is measured in parallel with the second signal.

[0019] In some embodiments, the long period of time is 6 to 72 hours, 6 to 170 hours, 6 to 168 hours, 12 to 60 hours, 24 to 48 hours, 12 to 36 hours, 24 to 48 hours, 36 to 60 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 60 to 72 hours, 48 ​​to 72 hours, 36 to 72 hours, 24 to 72 hours, 12 to 72 hours, 12 to 24 hours, 24 to 36 hours, 36 to 48 hours, or 48 to 60 hours.

[0020] In some embodiments, the device further comprises an image capture element configured to image a sample or a feature of a sample in each well of a plurality of wells defined in the sample carrier through an opening or window, the image capture element configured to capture and process at least one image from each well of the sample carrier.

[0021] In some embodiments, the sample carrier comprises a plurality of wells configured to hold a predetermined amount of sample, each well of the plurality of wells comprising an opening or window that allows an image capture element to capture at least one image from each well of the sample carrier.

[0022] In some embodiments, the device further comprises an electrode surface comprising a non-conductive carrier on the base of the sample carrier, a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a single plane and having substantially the same surface area, and a plurality of connection pads located on the sample carrier, each connection pad in electrical communication with at least one of the electrode structures in each well of the plurality of wells.

[0023] In some embodiments, a plurality of wells configured to hold a predetermined amount of sample are positioned above a plurality of electrode arrays, and each well of the plurality of wells comprises an opening or window that allows an image capture element to capture at least one image from each well of the sample carrier.

[0024] In some embodiments, the device further comprises an impedance measuring device configured to measure impedance changes due to the attachment of a sample in each well of a sample carrier or to stimulate a sample in each well of the sample carrier with an electrical signal, wherein the electrode surface is at the base of the sample carrier, the electrode surface comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a shared plane and having substantially the same surface area; and a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures, wherein the impedance element detects changes in electrical impedance between the electrode structures or stimulates the sample with the electrical signal, and the impedance element detects changes in electrical impedance between the electrode structures or stimulation output of the sample with the electrical signal.

[0025] In some embodiments, a plurality of wells configured to hold a predetermined amount of sample are positioned above a plurality of electrode arrays, and each well of the plurality of wells comprises an opening or window that allows an image capture element to capture at least one image from each well of the sample carrier.

[0026] According to one aspect, a sensing system includes an array of sensor units configured to generate a first signal in response to a first analyte over an extended period of at least six hours and to generate a second signal in response to a second analyte over said extended period, wherein each sensor unit of the array of sensor units 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 movement actuator assembly configured to position one or both of the stage and the sensing system relative to one another on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system that dispenses agents into samples in each well of the sample carrier; and a sample control element, wherein the sample control element is a sample temperature control element configured to control the temperature in each well of the sample carrier to be within a predetermined temperature of each other, or a sample environment control element, wherein the gas control element is configured to control the O2, CO2, and N2 content of each well of the sample carrier to be within a predetermined ratio of each other, or a humidity control element configured to control the humidity in each well of the sample carrier to be within a predetermined ratio of each other. a sample control element comprising one or both of a sample environment control element, a humidity control element configured to control the humidity to be within a certain amount; an image capture element configured to image the sample or a feature of the sample in each well of the sample carrier through an opening, the image capture element configured to capture at least one image from each well of the sample carrier; an impedance element comprising an electrode surface configured to measure impedance changes due to sample deposition or stimulate the sample in each well of the sample carrier with an electrical signal, the electrode surface being at a base of the sample carrier, the electrode surface comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a common plane and having substantially the same surface area; and a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures, the impedance element detecting changes in electrical impedance between the electrode structures oror stimulating the sample with an electrical signal; and a signal processing module operably connected to the sensing system, the signal processing module configured to receive and condition a first signal and a second signal from the sensor unit, process at least one image from the image capture element, and measure a change in electrical impedance between the electrode structures with an impedance element or measure a stimulation output of the sample with the electrical signal.

[0027] According to one aspect, a sensing system includes an array of sensor units configured to generate a first signal in response to a first analyte over an extended period of at least six hours and to generate a second signal in response to a second analyte over said extended period, wherein each sensor unit of the array of sensor units 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 movement actuator assembly configured to position at least one of the stage and the sensing system relative to one another on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system configured to dispense an agent to a sample in each well of the sample carrier; a sample control element, wherein the sample temperature control element is configured to control a temperature in each well of the sample carrier to be within a predetermined temperature range of each other, or a sample environment control element, wherein the gas control element is configured to control the O2, CO2, and N2 content of each well of the sample carrier to be within a predetermined ratio of each other; a sample control element comprising one or both of a sample environment control element, a humidity control element configured to control humidity within each well of the sample carrier to be within a predetermined amount of each other; an impedance element comprising an electrode surface configured to measure impedance changes due to deposition of a sample or to stimulate a sample in each well of the sample carrier with an electrical signal, the electrode surface being at a base of the sample carrier, the electrode surface comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on the same plane and having substantially the same surface area; a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures, the impedance element detecting changes in electrical impedance between the electrode structures or stimulating a sample held in the sample carrier with an electrical signal; and a signal processing module operatively connected to the sensing system,A device having long-term measurement capabilities is provided, comprising: a signal processing module configured to receive and condition a first signal and a second signal from the sensor unit; and measure a change in electrical impedance between the electrode structure by an impedance element or measure a stimulation output of the sample by the electrical signal.

[0028] In some embodiments, the device further comprises an image capture element configured to image samples or sample features in each well of the sample carrier through the opening, the image capture element configured to capture at least one image from each well of the sample carrier.

[0029] According to one aspect, a sensing system includes an array of sensor units configured to generate a first signal in response to a first analyte over an extended period of at least six hours and to generate a second signal in response to a second analyte over said extended period, wherein each sensor unit of the array of sensor units 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 movement actuator assembly configured to position at least one of the stage and the sensing system relative to one another on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system configured to dispense an agent to the sample in each well of the sample carrier; and a sample temperature control element or sample environment control element configured to control temperatures in each well of the sample carrier to be within a predetermined temperature of each other. A device capable of long term measurements is provided, comprising: a sample control element comprising one or both of a sample environment control element, the sample control element comprising one or both of a gas control element configured to control the O2, CO2, and N2 content of each well of a sample carrier to be within a predetermined ratio of each other, or a humidity control element configured to control the humidity in each well of the sample carrier to be within a predetermined amount of each other; an image capture element configured to image a sample or a feature of a sample in each well of the sample carrier through an opening, the image capture element configured to capture at least one image from each well of the sample carrier; and a signal processing module operably connected to a sensing system, the signal processing module configured to receive and condition first and second signals from the sensor unit and to process at least one image from the image capture element.

[0030] In some embodiments, the device further comprises an impedance element comprising an electrode surface configured to measure impedance changes due to attachment of a sample or stimulate the sample in each well of the sample carrier with an electrical signal, the electrode surface being at a base of the sample carrier, the electrode surface comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on the same plane and having substantially the same surface area; and a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures, wherein the impedance element detects changes in electrical impedance between the electrode structures or stimulates the sample with an electrical signal.

[0031] According to one aspect, there is provided an impedance element comprising an electrode surface configured to one or both measure impedance changes due to attachment of a sample or stimulate with an electrical signal a sample in each well of a plurality of wells defined in a sample carrier, the electrode surface being at a base of the sample carrier, the electrode surface comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a single plane and having substantially the same surface area; and a plurality of connection pads located on the sample carrier, each connection pad being electrically connected to at least one of the electrode structures. a signal processing module operably connected to the impedance element, the signal processing module being configured to measure the changes in electrical impedance by the impedance element and to process the at least one image from the image capture element; a plurality of connection pads in communication with the electrode structure, the impedance element detecting changes in electrical impedance between the electrode structures or stimulating the sample with an electrical signal; an image capture element configured to image the sample or a feature of the sample in each well of the sample carrier through the opening, the image capture element being configured to capture at least one image from each well of the sample carrier; and

[0032] According to one aspect, a sample carrier is provided comprising: a plurality of wells configured to hold a predetermined amount of sample, each well of the plurality of wells comprising an opening that allows an image capture element to capture at least one image from each well of the sample carrier; an electrode surface comprising a non-conductive carrier on a base of the sample carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a single plane and having substantially the same surface area; a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures in each well of the plurality of wells; and a plurality of structures that, when mated with sensor units of the array of sensor units, create microchambers having reduced volumes.

[0033] In some embodiments, the reduced volume is 3 microliters or less.

[0034] In some embodiments, a feature of the plurality of features is a ledge, lip, ridge, or stop configured to control the distance that the sensor unit can protrude downward into the plurality of wells up to a predetermined distance.

[0035] In some embodiments, the sample carrier is a microtiter plate, a flow chip, or a 3D tissue or spheroid formation / measurement plate.

[0036] In some embodiments, one or more wells of the sample carrier are made from a material that restricts gas diffusion.

[0037] In some embodiments, one or more wells of the sample carrier are provided with a window through the electrode that allows viewing or imaging of the cell sample from the bottom of the well.

[0038] In some embodiments, one or more wells of the sample carrier do not include a window through the electrode, such that viewing or imaging of the cell sample is performed from the top of the well opposite the location where the electrode is defined.

[0039] In some embodiments, the sample carrier comprises a lid.

[0040] In some embodiments, the lid comprises one or more sensors that measure at least one of O2, pH, and CO2.

[0041] In some embodiments, the sample carrier comprises a cartridge.

[0042] In some embodiments, the cartridge comprises one or more sensors or compound / substance ports.

[0043] According to one aspect, there is provided an analytical device comprising: a sample carrier having a plurality of wells, each well of the plurality of wells being fluidly isolated from each other well of the plurality of wells, the sample carrier comprising an electrode configured to measure the impedance of a sample disposed in a given well and to define a window through which the sample is visible from outside the sample carrier; a flux detector individually corresponding to each well of the plurality of wells of the sample carrier and configured to detect a first analyte; an image capture element configured to image a sample disposed in one of the plurality of wells of the sample carrier through the window and capture images from each well of the plurality of wells of the sample carrier; and an environmental control module configured to maintain environmental parameters around the sample carrier within predetermined ranges for a duration of at least six hours at a time.

[0044] In some embodiments, the environmental control module maintains CO 2 concentration, O 2 concentration, and N 2 concentration in the atmosphere of the environment surrounding the plurality of wells.

[0045] In some embodiments, the device further comprises a flux cartridge movable relative to the sample carrier on an axis substantially perpendicular to a plane intersecting each well of the multiple wells of the sample carrier, the flux cartridge comprising a plurality of heads, the flux cartridge configured such that each well of the multiple wells of the sample carrier can be addressed by a head of the multiple heads, and each head of the multiple heads that addresses a given well comprises a surface proximal to the sample carrier that defines a reaction chamber within the well and is configured to limit the amount of liquid or evaporation from the reaction chamber.

[0046] In some embodiments, each well of the plurality of wells comprises a volume-defining member configured to limit the range of motion between the sample carrier and a second element of the device or to define a minimum non-zero distance between the sample carrier and a second element of the device.

[0047] In some embodiments, the volume-defining member comprises at least one of a shelf, a ridge, a lip, and a position control for a motor that moves the sample carrier relative to the sensor array and stops at a certain distance above the base of the corresponding well.

[0048] In some embodiments, the sample carrier is movable relative to the flux detector and image capture element so that the flux detector and imaging element can sequentially access the sample carrier.

[0049] In some embodiments, the device further comprises a liquid handling module configured to deliver liquid to the sample carrier.

[0050] In some embodiments, the sample carrier is movable relative to the liquid handling module.

[0051] In some embodiments, the liquid handling module is configured to deliver liquid to individual wells of the plurality of wells.

[0052] In some embodiments, the flux detector is configured to detect a second analyte.

[0053] According to one aspect, a device is provided that includes: a chamber configured to receive a sample carrier having a plurality of wells and a cartridge containing a compound; a camera positioned within the chamber below where the sample carrier is received in the chamber, the camera configured to capture images of the contents of each well of the plurality of wells; a sensor positioned within the chamber and configured to monitor cell growth in each well of the plurality of wells; a temperature controller configured to adjust the temperature of the sample and sensor held in each well of the plurality of wells; and a fluid handler in communication with the sample carrier and the compound, the fluid handler configured to deliver the compound from the cartridge to a given well based on one or more of the pH of the contents of the given well of the plurality of wells and an image of the given well captured by the camera.

[0054] In some embodiments, the device further comprises an environmental controller configured to regulate the temperature of the atmosphere, the CO 2 concentration of the atmosphere, and the O 2 concentration of the atmosphere within the chamber.

[0055] According to one aspect, a device is provided that includes a sample carrier having a plurality of wells, each well of the plurality of wells having a first electrode in contact with a first side of the well and a second electrode in contact with a second side opposite the first side of the well, the first electrode and the second electrode each being in electrical communication with an electrical measurement module on the sample carrier; a chamber configured to receive a sample carrier; a cartridge having a compound and at least one delivery port for the compound; a temperature controller configured to adjust the temperature of the sample held in each well of the plurality of wells and the electrical property measurement module; and a fluid handler in communication with the sample carrier and the cartridge, the fluid handler configured to deliver at least one compound from the cartridge to a given well of the plurality of wells based on a measurement value of the sample in the given well of the plurality of wells obtained by the electrical measurement module between the first electrical contact and the second electrical contact.

[0056] In some embodiments, the sample measurement is a measurement of cell growth in a given well as an impedance value, or a measurement of cell stimulation.

[0057] In some embodiments, the device further comprises an environmental controller configured to regulate the temperature of the atmosphere, the CO 2 concentration of the atmosphere, and the O 2 concentration of the atmosphere within the chamber.

[0058] According to one aspect, there is provided a sample carrier comprising a plurality of wells, each well of the plurality of wells having a first electrode in contact with a first side of the well and a second electrode in contact with a second side of the well opposite the first side, the first electrode and the second electrode each being in electrical communication with an impedance meter on the sample carrier; a chamber configured to receive the sample carrier; a cartridge comprising a compound and at least one delivery port for the compound; and a camera disposed within the chamber below where the sample carrier is received, the camera viewing an associated window in each individual well when the sample carrier is positioned in a first position within the chamber. a camera configured to capture images of the cell culture in each individual well of the plurality of wells; a fluid handler in communication with the sample carrier and the cartridge when the sample carrier is positioned at a second position in the chamber, the fluid handler configured to deliver a compound from the cartridge to a given well of the plurality of wells based on an impedance measurement of the sample in the given well of the plurality of wells obtained by an impedance meter between the first and second electrical contacts; and a motion stage in contact with the sample carrier, configured to move the sample carrier between the first and second positions.

[0059] In some embodiments, the device further comprises a temperature controller configured to regulate the temperature of a sample held in an individual well of the plurality of wells.

[0060] In some embodiments, the device further comprises an environmental controller configured to regulate the temperature of the atmosphere, the CO 2 concentration of the atmosphere, and the O 2 concentration of the atmosphere within the chamber.

[0061] According to one embodiment, a method of using the device is provided, comprising loading a sample carrier containing one or more cell samples into the device, with each sample disposed in a corresponding well of the sample carrier.

[0062] In some embodiments, samples are analyzed over an extended period of time, from 6 hours to 72 hours.

[0063] In some embodiments, the cell sample comprises live cells.

[0064] According to one embodiment, a method of analyzing a cell sample is provided, comprising preparing a device, loading a sample carrier containing one or more cell samples into the device, with each sample disposed in a corresponding well of the sample carrier, and analyzing the cell sample.

[0065] In some embodiments, samples are analyzed over an extended period of time, from 6 hours to 72 hours.

[0066] In some embodiments, the method further includes positioning one or both of the stage and the sensing system relative to one another on one or more of an x-axis, a z-axis, and a y-axis.

[0067] In some embodiments, the method further comprises dispensing a compound into the sample in each well of the sample carrier.

[0068] In some embodiments, the method further comprises controlling the temperature in each well of the sample carrier to be within a predetermined temperature of each other.

[0069] In some embodiments, the method further comprises controlling the gas content of each well of the sample carrier to be within a predetermined ratio of each other.

[0070] In some embodiments, the method comprises generating a first signal in response to a first analyte over an extended period of time, and generating a second signal in response to a second analyte over said extended period of time.

[0071] In some embodiments, the method further includes receiving and conditioning the first signal and the second signal from the sensor unit.

[0072] In some embodiments, the method further comprises calculating one or more metabolic flux parameters, including at least one of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and / or proton efflux rate (PER).

[0073] In some embodiments, the method further comprises imaging the sample or sample features in each well of the sample carrier through the opening.

[0074] In some embodiments, the method further comprises processing at least one image from the image capture element.

[0075] In some embodiments, the method further comprises measuring a change in impedance of the sample.

[0076] In some embodiments, the sample comprises living cells.

[0077] In some embodiments, 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.

[0078] In some embodiments, the sample comprises mammalian cells or tissues.

[0079] In some embodiments, the sample comprises stem cells.

[0080] In some embodiments, the sample comprises cells of the cardiovascular system.

[0081] In some embodiments, the sample comprises non-mammalian cells or tissues.

[0082] In some embodiments, the sample comprises a single-cell organism.

[0083] In some embodiments, the sample comprises whole animal model tissue.

[0084] In some embodiments, the sample comprises whole plant model tissue or plant model cells.

[0085] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0086] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like reference numeral. For clarity, not every component may be labeled in every drawing. [Brief explanation of the drawings]

[0087] [Figure 1] FIG. 1 is a front perspective view of a device for analyzing live cells, according to one embodiment. [Figure 2] FIG. 1 is a rear perspective view of a device for analyzing live cells, according to one embodiment. [Figure 3] FIG. 1 is a side view of a device for analyzing live cells, according to one embodiment. [Figure 4] FIG. 1 is a top view of a device for analyzing live cells, according to one embodiment. [Figure 5] FIG. 1 is a side view of selected components of a device 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] FIG. 1 is a schematic diagram of selected components of a system for analyzing live cells, according to one embodiment. [Figure 9] FIG. 1 is a schematic diagram of selected components of a device for analyzing live cells, according to one embodiment. [Figure 10A] 1 is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein, according to one embodiment, and in particular the OCR readings from a system disclosed herein and a comparative system, showing improved and stable performance from 0 to 15 minutes for the present system compared to the comparative device. [Figure 10B] 1 is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the methods disclosed herein, according to one embodiment, and in particular the OCR readings from a system disclosed herein and a comparative system, showing improved and stable performance from 0 to 15 minutes for the present system compared to the comparative device. [Figure 10C] 1 is a graph showing the extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein, according to one embodiment. [Figure 10D] 1 is a graph showing the extracellular acidification rate (ECAR) of live cells analyzed by the methods disclosed herein, according to one embodiment. [Figure 11A] 1 is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment, particularly OCR readings from the system disclosed herein and a comparative system, demonstrating significantly lower variability in results, particularly for metformin-treated cells (lower line). [Figure 11B] 1 is a graph showing the oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment, in particular the OCR readings from the system disclosed herein and a comparative system, demonstrating significantly lower variability in results, particularly for metformin-treated cells (lower line). [Figure 12] 1 is a table showing the average evaporation of materials (eg, culture media) contained within sample carriers analyzed by the methods disclosed herein, according to one embodiment. [Figure 13A] A comparison of spheroids. [Figure 13B]A comparison of spheroids. [Figure 14] FIG. 1 is a block diagram illustrating a multi-detection system according to one embodiment. [Figure 15] FIG. 1 is a block diagram illustrating a multi-detection system according to one embodiment. [Figure 16] FIG. 1 is a block diagram illustrating a multi-detection system according to one embodiment. [Figure 17] FIG. 1 is a block diagram illustrating a multi-detection system according to one embodiment. [Figure 18] FIG. 1 is a block diagram illustrating a multi-detection system according to one embodiment. [Figure 19] FIG. 2 is a diagram of a non-imaging analysis subsystem according to one embodiment. [Figure 20] FIG. 1 illustrates an injection subsystem according to one embodiment. [Figure 21] FIG. 1 illustrates a multi-detection system according to one embodiment. [Figure 22A] FIG. 2 is a perspective view of an environmental control subsystem according to one embodiment. [Figure 22B] FIG. 2 is a rear view illustrating an environmental control subsystem according to one embodiment. [Figure 22C] FIG. 2 is a front view illustrating an environmental control subsystem according to one embodiment. [Figure 23] FIG. 1 is a functional block diagram illustrating control of modalities of a device according to one embodiment. [Figure 24] FIG. 1 is a flow diagram of a method for controlling a multi-detection system according to an exemplary embodiment. [Figure 25A] FIG. 1 is a first diagram illustrating an immersion objective according to an embodiment. [Figure 25B] FIG. 2 is a second diagram illustrating an immersion objective according to an embodiment. [Figure 26] FIG. 1 illustrates a fluid pump system according to one embodiment. [Figure 27] FIG. 1 illustrates an objective lens coupling according to one embodiment. [Figure 28A] FIG. 1 is a perspective view showing an immersion objective lens according to a first embodiment. [Figure 28B]FIG. 1 is a top view showing an immersion objective lens according to a first embodiment. [Figure 28C] 28C is a first cross-sectional view taken along the line AA in FIG. 28B, showing the immersion objective according to the first embodiment provided with a liquid sphere. [Figure 28D] 28C is a second cross-sectional view taken along the line AA in FIG. 28B, showing the immersion objective according to the first embodiment with a sample carrier (eg, a microplate) provided thereon. [Figure 29A] FIG. 4 is a top view showing an immersion objective lens according to a second embodiment. [Figure 29B] 29B is a first cross-sectional view taken along the line BB in FIG. 29A, showing the immersion objective according to the second embodiment provided with a liquid sphere. [Figure 29C] 29B is a second cross-sectional view taken along line BB in FIG. 29A, showing an immersion objective according to a second embodiment with a sample carrier (eg, a microplate) provided thereon; [Figure 30A] FIG. 10 is a top view showing an immersion objective lens according to a third embodiment. [Figure 30B] 30B is a first cross-sectional view taken along the line CC in FIG. 30A, showing the immersion objective according to the third embodiment provided with a liquid sphere; FIG. [Figure 30C] 30B is a second cross-sectional view taken along line CC in FIG. 30A, showing an immersion objective according to a third embodiment with a sample carrier (eg, a microplate) provided thereon; [Figure 31A] FIG. 10 is a top view showing an immersion objective lens according to a fourth embodiment. [Figure 31B] 31B is a first cross-sectional view taken along the line DD in FIG. 31A, showing the immersion objective according to the fourth embodiment provided with a liquid sphere. [Figure 31C]31B is a second cross-sectional view taken along the line DD in FIG. 31A, showing an immersion objective according to a fourth embodiment with a sample carrier (eg, a microplate) provided thereon; [Figure 32A] FIG. 1 is a first diagram of a multi-detection system of a laser point-scanning confocal modality according to one embodiment. [Figure 32B] FIG. 2 is a second diagram of a multi-detection system for wide-field or spinning disk confocal modality according to one embodiment. [Figure 33] FIG. 2 is a diagram of an exemplary user interface according to one embodiment. [Figure 34] FIG. 1 is a schematic diagram showing a cross-sectional view of a transfer module according to one embodiment. [Figure 35] 1 is a drawing of a side view of a transfer module, according to one embodiment. [Figure 36] 1 is a graph illustrating the accuracy of measurements made on a device having a thermally conductive excitation source, according to one embodiment. [Figure 37A] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 37B] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 37C] FIG. 1 shows Mitotoxicity results showing negative MTI values. [Figure 38A] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Figure 38B] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Figure 38C] FIG. 1 shows Mitotoxicity results showing positive MTI values. [Figure 39] 1 is an exemplary MTI detection graph. [Figure 40] 1 is a graph showing dynamic dose-response OCR data for three test compounds. [Figure 41] 1 is a graph showing Z' values ​​achieved using a MitoTox assay and an MTI-based analysis, according to one embodiment. [Figure 42] FIG. [Figure 43] FIG. 2 is a diagram showing a relay optical system. [Figure 44] FIG. 1 is a diagram showing the structure of an excitation-emission emission separation device. [Figure 45] FIG. 1 shows a holder and associated optical attachment elements. [Figure 46] FIG. 1 is a diagram of a sample carrier and its imaging. [Figure 47A] FIG. 1 is a schematic diagram of a device having two electrode structures. [Figure 47B] FIG. 1 is a schematic diagram of a device having two electrode structures. [Figure 47C] FIG. 1 is a schematic diagram of a device having two electrode structures. [Figure 48] FIG. 1 illustrates an exemplary multimodal analytical device. [Figure 49] FIG. 1 is a schematic system diagram of one embodiment of a system for analyzing live cells. [Figure 50] FIG. 50 is an exploded view of the sample carrier and cartridge of FIG. 49. [Figure 51] FIG. 1 is a plan view of the stage without the sample carrier. [Figure 52] FIG. 1 is a side view of the stage without the sample carrier. [Figure 53] FIG. 2 is a perspective view of the bottom of a well of a sample carrier; [Figure 54] FIG. 10 is a top view of the bottom of the well of the sample carrier. [Figure 55] FIG. 10 shows a feedback mechanism for controlling gas concentrations in the wells of a sample carrier. [Figure 56A] Graph showing oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein according to one embodiment, specifically three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), particularly demonstrating significantly less variability in results at 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 56B] Graph showing oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein according to one embodiment, specifically three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), particularly demonstrating significantly less variability in results at 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 56C] Graph showing oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein according to one embodiment, specifically three experimental replicates of OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), particularly demonstrating significantly less variability in results at 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 57A] Graphs of the same data showing basal oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment, specifically basal OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), particularly demonstrating significantly less variability in the lower OCR rates when cells are plated at lower densities compared to the comparative instrument. [Figure 57B] Graphs of the same data showing basal oxygen consumption rate (OCR) of live cells analyzed by the method disclosed herein, according to one embodiment, specifically basal OCR readings from an analytical instrument disclosed herein (left) and a comparative analytical instrument (right), particularly demonstrating significantly less variability in the lower OCR rates when cells are plated at lower densities compared to the comparative instrument. [Figure 58]FIG. 58 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, across three experimental replicates. In particular, FIG. 58 shows the standard deviation of basal OCR readings from an analytical instrument disclosed herein (gray) and a comparative analytical instrument (black), demonstrating significantly less variability in results compared to the comparative instrument. DETAILED DESCRIPTION OF THE INVENTION

[0088] The present disclosure provides, at least in part, systems, methods, and consumables that enable long-term measurements of cellular samples. Also disclosed herein are multi-mode systems, methods, and consumables that perform bioenergetic-based measurements (e.g., metabolic flux measurements), electronics-based excitation or stimulation, electronics-based signal measurements (e.g., field potential recording, impedance measurements), and imaging. The present disclosure also provides, at least in part, temperature / environmental control of parameters such as temperature, gases (O, CO, N, etc.), humidity, and atmospheric pressure. Any combination of these components is also disclosed herein.

[0089] The systems (e.g., instruments, apparatus, and devices) and methods (e.g., assays) described herein can include or use one or more components, such as a flux measurement system with long-term measurement capabilities, an impedance measurement system, an imaging measurement system, or any combination thereof. In some embodiments, the flux measurement system can include elements for temperature / environment control, fluid handling, or both. Conventional systems and methods may not be suitable for long-term measurements, at least in part, due to significant evaporation from wells / sample media and compound / substance ports. The systems and methods described herein can control the sample environment or microenvironment to enable long-term measurements. Such control can include, for example, liquid handling, temperature control, gas control, humidity control, or any combination thereof. In some embodiments, evaporation of compound / substance ports can be addressed by injecting compounds via liquid handling instead of loading them into the compound / substance ports in a cartridge prior to the assay. In some embodiments, calibration can be shortened or even eliminated when a time-based detection approach is employed. In some embodiments, the systems or methods described herein can control CO2 levels to allow for cell sample growth. In some embodiments, the systems and methods described herein include or use an impedance measurement system suitable for use in combination with the flux measurement systems described herein.

[0090] Conventional systems and methods that separately measure extracellular flux, impedance, and imaging may not be suitable for long-term measurements of cell samples, including simultaneous measurements of extracellular flux, impedance, and imaging. For example, conventional systems and methods may cause hypoxic shock to the cell sample. For long-term extracellular flux assays, evaporation of the compound / substance port volume within the cartridge is a limiting factor, preventing assays from exceeding six hours without additional fluid handling. Imaging may be interfered with by the electrodes.

[0091] In one aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample in a sample carrier in real time at specified intervals; injecting one or more chemical compounds, exchanging cell growth or running medium, and controlling temperature and / or environmental conditions (e.g., gas, humidity) via pre-loaded cartridges (i.e., temperature or environmental conditions (e.g., gas, humidity) or both); simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by measuring impedance in real time; and facilitating long-term measurements.

[0092] In another aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample within a sample carrier in real time at specified intervals, injecting one or more chemical compounds via an embedded fluid handling device, exchanging cell growth or running medium, controlling temperature and / or environmental conditions (e.g., gas, humidity), simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by measuring impedance in real time, and facilitating long-term measurements.

[0093] In yet another aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample within a sample carrier in real time at specified intervals; injecting one or more chemical compounds via pre-loaded cartridges, exchanging cell growth or running medium, controlling temperature and / or environmental conditions (e.g., gas, humidity); simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by measuring impedance in real time; and simultaneously imaging the cell sample by visualization through a specified area at the bottom of the sample carrier that is free of electrodes and gaps in the impedance conductors.

[0094] In yet another aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample within a sample carrier in real time at specified intervals, injecting one or more chemical compounds via pre-loaded cartridges, exchanging cell growth or running medium, controlling temperature and / or environmental conditions (e.g., gas, humidity), simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by measuring impedance in real time, and simultaneously imaging the cell sample by visualization through a transparent impedance conductor at the bottom of the sample carrier.

[0095] In one aspect, the present disclosure provides a device capable of measuring, in real time at specified intervals, one or more metabolic parameters of a suspension cell sample flowing from a biological growth chamber or processing unit into a measurement chamber; exposing the sample to one or more chemical compounds; refreshing the sample with growth or running medium; controlling temperature and / or environmental conditions (e.g., gas, humidity); and simultaneously imaging the cell sample and performing image-based fluorescence and / or impedance measurements (i.e., fluorescence or impedance measurements, or both).

[0096] In another aspect, the present disclosure provides a device capable of measuring in real time at specified intervals one or more metabolic parameters of a suspension cell sample flowing from a biological growth chamber or bioprocessing unit into a measurement chamber; exposing or not exposing the sample to one or more chemical compounds; refreshing the sample with growth or running medium; controlling temperature and / or environmental conditions (e.g., gas, humidity); simultaneously imaging the cell sample and performing image-based fluorescence measurements; and re-flowing the cell sample into the biological growth chamber or bioprocessing unit at specified intervals for further cell growth, which can be repeated up to a time specified by a user.

[0097] In yet another aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample in a sample carrier in real time at specified intervals by monitoring one or more analytes in a medium at a specified height above the sample, injecting one or more chemical compounds, exchanging cell growth or running medium, controlling temperature and / or environmental conditions (e.g., gas, humidity) by specific controllers and medium via an embedded fluid handling device, and simultaneously measuring cellular function via attachment / detachment of the cell sample by measuring impedance in real time.

[0098] In yet another aspect, the present disclosure provides a device capable of measuring one or more metabolic parameters of a cell sample in a sample carrier in real time at specified intervals by monitoring the analyte using a lid with a spine having an analyte sensor extending into the medium at a specified height above the sample; removing the lid with the spine using a robotics / handler system and placing a cover or cartridge on the sample carrier; measuring one or more metabolic parameters of a cell sample in real time at specified intervals by creating a microchamber with a cartridge having an analyte sensor at its distal end; injecting one or more chemical compounds, exchanging cell growth or running medium, controlling temperature and / or environmental conditions (e.g., gas, humidity), and optionally adding medium and / or washing medium in a well plate via an embedded fluid handling device or via pre-filled / or user-filled cartridge compound / substance ports via a specific controller and medium; removing the cartridge and placing an analyte sensing monitoring lid with a spine on the sample carrier; and simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by measuring impedance in real time.

[0099] [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.

[0100] 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.

[0101] 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.

[0102] As used herein, the terms "acquire" or "obtain" refer to obtaining possession of a physical entity or value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" that physical entity or value. "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly obtaining" refers to receiving the physical entity or value from another person or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting material. Exemplary changes include creating 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. Directly obtaining a value includes performing a process involving a physical change of a sample or another substance, such as performing an analytical process (also referred to herein as "physical analysis") involving a physical change of a substance, such as a sample, analyte, or reagent, and also includes performing an analytical method, such as separating or purifying a substance, such as an analyte, or a fragment or other derivative thereof, from other substances; combining an analyte, or a fragment or other derivative thereof, with another substance, such as a buffer, solvent, or reactant; or altering the structure of an analyte, or a fragment or other derivative thereof, for example, by severing or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or altering the structure of a reagent, or a fragment or other derivative thereof, for example, by severing or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent. In one embodiment, directly obtaining includes direct measurement. In one embodiment, indirectly obtaining includes inference.

[0103] As used herein, the term "obtaining a sample" refers to taking possession of a sample, e.g., a sample described herein, by "directly obtaining" or "indirectly obtaining" the sample. "Directly obtaining a sample" means performing a process to obtain the sample (e.g., performing a physical method such as surgery or extraction). "Indirectly obtaining a sample" refers to receiving a sample from another person or source (e.g., a third-party laboratory that directly obtained the sample). Directly obtaining a sample includes performing a process that involves a physical change in a starting material, such as a physical substance, e.g., tissue, e.g., tissue from a human patient or tissue previously isolated from a patient. Exemplary changes include creating 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 involves breaking or forming a covalent or non-covalent bond.

[0104] The term "ambient temperature," as 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.

[0105] 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 one 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) prior to 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 between 2.45 and 2.86 (referred to as the P / O ratio) × 2 (to convert oxygen atoms to oxygen molecules). In one embodiment, the constant is 2.75.

[0106] As used herein, the term "bioenergetic capacity" refers to the level of increased glycolysis and / or mitochondrial activity (i.e., glycolysis or mitochondrial activity, or both) that a cell can affect, utilize, and / or induce (i.e., can affect, utilize, or induce, or two or more of these). In one embodiment, bioenergetic capacity is determined in response to increased energy demand and / or in response to inhibition / perturbation of energy production. In one embodiment, bioenergetic capacity includes a value for oxygen consumption (e.g., oxygen consumption rate (OCR)) and a value for proton efflux (e.g., proton efflux rate (PER)). In one embodiment, the oxygen consumption value (e.g., OCR) is a function of mitochondrial uncoupling. In one embodiment, the proton efflux value (e.g., PER) is a function of ATPase inhibition. In one embodiment, PER is glycolytic PER (glycoPER), which mathematically removes the contribution of CO2.

[0107] As used herein, the term "bioenergetic balance" refers to the balance between aerobic and glycolytic energy production. In one embodiment, bioenergetic balance describes the proportion of ATP produced by glycolysis of oxidative phosphorylation. In one 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.

[0108] "Bioenergetic work" as the term is used herein refers to the amount of ATP produced by cells.

[0109] As used herein, the term "cell sample" refers to a sample containing cells or cell products or by-products. In one embodiment, the cell sample comprises a plurality of cells. In one embodiment, the cells are disposed in a medium. The cell sample can be or comprise one or more of cells, tissues, cell or tissue constructs, organelles, enzymes, and / or conditioned medium.

[0110] As used herein, the term "cellular metabolic function" refers to the ability of an organism to carry out chemical reactions necessary to sustain life. In embodiments, the cellular metabolic function of a cell sample can be monitored by measuring the OCR and ECAR.

[0111] As used herein, the term "extracellular acidification rate (ECAR)" refers to the measurement of proton extrusion in the extracellular medium over time. The ECAR can be reported as the rate of change in pH units over the assay run time, e.g., millipH / minute (mpH / min).

[0112] As used herein, the term "glycolysis" or "glycolytic activity" refers to the cellular metabolic function that converts glucose into lactate.

[0113] "Mitochondrial respiration," as the term is used herein, refers to the metabolic reactions and processes that require oxygen and occur in mitochondria to convert energy stored in macronutrients into ATP.

[0114] 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 0-1) identify mitochondrial toxicity due to uncoupling, while negative MTI values ​​(typically 0--1) identify mitochondrial toxicity due to inhibition.

[0115] 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 several 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.

[0116] As used herein, the term "oxygen consumption rate (OCR)" refers to a quantitative measurement of oxygen consumption by a sample over time. Thus, OCR can provide a measure of cellular and mitochondrial respiration over time. OCR values ​​can be reported as the rate of change of O content over the assay run time, e.g., picomoles / minute (pmol / min).

[0117] In one embodiment, OCR includes scenarios in which 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 the oxygen consumption is the oxygen depletion in the sample corrected for oxygen back-diffusion into the sample, or the oxygen consumption is the oxygen depletion uncorrected for oxygen back-diffusion into the sample, or scenarios in which 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 the oxygen consumption is equal to or substantially equal to the oxygen depletion in the sample.

[0118] In one embodiment, oxygen consumption is determined directly or indirectly, e.g., in a test well or across a capillary, e.g., by inferring from a measured oxygen gradient, or by measuring oxygen at preselected time points.

[0119] In one embodiment, oxygen consumption is reported in units other than rate of change of O2 content, such as sensor response per unit time (microseconds / minute, relative fluorescence units / minute, etc.).

[0120] As used herein, the term "primary cells" 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 proliferation. 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).

[0121] The term "proton efflux rate (PER)" as used herein refers to a quantitative measure of extracellular acidification that takes into account medium 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.

[0122] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest. In one embodiment, 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 tissue such as from a fresh, frozen, and / or preserved (i.e., frozen or preserved, or both) organ, tissue, biopsy, resection, smear, or aspirate, or cells from any stage of a subject's pregnancy or development. In one embodiment, the source of the sample is blood or a blood component. In one embodiment, the sample is a primary sample, e.g., obtained directly from the source of interest by any suitable means. In one embodiment, 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).

[0123] As used herein, the term "sample carrier" refers to a substrate on which a sample can be carried. In one 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 various 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.

[0124] [Systems with long-duration capabilities and multi-mode systems] Without wishing to be bound by theory, it is believed that in some embodiments the systems, consumables, and methods described herein are particularly suited for long-term measurements of live cell samples.

[0125] In one aspect, the present disclosure provides components for measuring cellular bioenergetic parameters, e.g., metabolic flux parameters such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and / or proton efflux rate (PER), in real time. In another aspect, the present disclosure provides components for measuring cellular electrical properties, such as cellular impedance, and enabling electronic excitation of signals interfacing with a cellular sample. In yet another aspect, the present disclosure provides optical and / or imaging components, such as a microscope or inverted microscope equipped with a high-definition camera for bright-field and / or fluorescent imaging of cells with or without labels. In yet another aspect, the present disclosure provides real-time temperature / environment control, optionally with monitoring / feedback typically desired to create a more physiologically relevant measurement / sample environment. This temperature / environment control can include, for example, temperature, humidity, atmosphere control, and gas control (oxygen, carbon dioxide, nitrogen, etc.). In another aspect, the present disclosure provides compound infusion or fluid control to measure and quantify interactions between target compounds and cellular activity.

[0126] In another aspect, the present disclosure provides multi-mode systems that include combinations of the above-described components in different pairs. Exemplary components of multi-mode systems are further described below.

[0127] [Bioenergetics measurements, e.g., components of extracellular flux measurements] The bioenergetics measurement, e.g., extracellular flux measurement, components of the system can include, for example, microchamber formation, electro-optical systems, and / or fluorescent analyte sensors that respond in response to analyte concentration. In some embodiments, an LED can be used to excite the analyte sensor, and a detection device can be used to measure signal change over time. The signal can be measured in any of the available detection modes, including, for example, intensity, DLR, TRF, ratiometric, Time of Fluid (ToF), etc. In some embodiments, the system can move a z-axis component to form a microchamber between the cartridge consumable and the sample carrier (e.g., a microwell plate). In some embodiments, a consumable with an impedance component microelectrode is used, and both parameters are measured simultaneously.

[0128] Other exemplary extracellular flux components of the system are described, for example, in the section entitled "Bioenergetics Measurement Module" herein.

[0129] [Components for electrical measurements, e.g., electronic excitation and cell impedance measurements] The electrical measurement, e.g., electronic excitation and impedance measurement, components of the system can include consumables, such as microelectrodes embedded in the cell-seeded surface. These microelectrodes can be made from materials that are easily formed and compatible with cell growth. In some embodiments, the microelectrodes are optimized for low impedance characteristics to maximize the accuracy of the measurement and excitation pulses. In some embodiments, the microelectrodes are positioned in a manner that minimizes the space between opposite polarity connections while increasing the length of the electrodes or connected electrodes to increase the potential between the microelectrodes. To read the impedance measurement, a low-power signal can be excited at either end of the microelectrode-seeded surface. The real and imaginary components of the impedance can then be measured. In some embodiments, the microelectrodes are excited with a very low alternating current and measured with the microelectrode of the opposite polarity. Measuring the opposite current and voltage compared to the current and voltage measured at the source electrode can present an opportunity to measure the real and imaginary components of the impedance of the cell sample. This electronic pulse can also be used to stimulate cells in response to the electrical pulse.

[0130] Other exemplary electronic excitation and cell impedance measurement components of the system are described, for example, in the section entitled "Electrical Measurement Module" herein.

[0131] [Imaging and Optical Components] The imaging and optical components of the system can enable detection modes such as fluorescence intensity, luminescence, fluorescence polarization, time-resolved fluorescence, alpha, UV-visible absorbance, fluorescence, phase contrast, bright field, high contrast bright field, color bright field, and phase contrast. System components can include, for example, filter cubes, image processing elements, cameras such as CMOS or CCD devices, objective lenses, LEDs and lasers, and routines that enable optimal image or measurement collection.

[0132] Other exemplary imaging components of the system are described, for example, in the sections entitled "Imaging Module" and "Optics Module" herein.

[0133] [Consumable parts] In some embodiments, the system includes an interface for interacting with a consumable. The consumable can be any cell sample-holding consumable. Exemplary consumables include, but are not limited to, flow chips, microtiter plates with any number of wells, 2D cell cultures, and 3D tissue or spheroid formation / measurement plates. In some embodiments, the consumable includes microelectrodes if impedance measurements or electrical excitation are desired. In some embodiments, the consumable can form microchambers to enable flux measurements. In some embodiments, the consumable is made of a material that limits gas diffusion to increase flux sensitivity. To image a cell sample, components comprising the imaging system can be configured to read from below or above the consumable. When imaging from below, the consumable can have an opening or window through the microelectrode for viewing the cell sample. The opening or window does not include a microelectrode and is made of clear, translucent plastic or glass that can be imaged through the bottom or top. When imaging from above, the consumable can have an optional feature above the sample, such as a flux measurement cartridge that can be removed to view the sample.

[0134] Other exemplary consumable parts are described, for example, in the section entitled "Consumables" herein.

[0135] [System Functions] The system can be set up by the user to measure in a specified mode (imaging, flux, impedance, etc.). The user can also configure the length of the measurement, number of injections, incubation time, imaging settings, etc. The system can be configured to automate the movement of consumables within the system or the measurement of consumables.

[0136] [Bioenergy measurement module] The devices and methods described herein provide a comprehensive overview of cellular metabolic function within cultured cell samples and ex vivo samples over longer periods of time than previously available devices could provide. Unlike previous devices, which suffer from significant evaporative loss and the accumulation of metabolites from the sample that are not present in vivo over the corresponding time period, the described long-term devices control various sample characteristics, including temperature, humidity, atmospheric composition, etc., to enable the analysis of metabolic function within cultured cell samples and ex vivo samples for continuous periods of more than six hours (e.g., up to 72 hours, 150 hours, etc.), without the need for human operator intervention. The devices can include various analytical systems, including flux, impedance, and imaging systems, used to identify sample characteristics over time to researchers and a control device (e.g., a computer) used to monitor and manage intracellular conditions to maintain one or more characteristics within predetermined ranges over the extended time period. Thus, the described devices can facilitate long-term measurements by measuring metabolic parameters in real time to control the injection of various chemical compounds, change cell growth or running media, and control environmental conditions.

[0137] In various embodiments, measurements in "incubator-like" conditions are achieved by controlling CO2 in the sample chamber, eliminating the need for additional buffers (e.g., HEPES) typically used for long-term measurements. These buffers can be problematic in some biological models. CO2 control facilitates long-term metabolic analysis. Gas control also facilitates metabolic analysis at lower O2 concentrations (e.g., lower O2 achieved via N2 purging) used to replicate in vivo conditions or model certain disease states (e.g., hypoxic stress and / or cycling ischemia / reperfusion modeling, long-term tumor modeling, etc.). Gas levels are controlled by a feedback mechanism that controls the gas purging, which can include a fan to more rapidly change CO2 and / or O2 concentrations (i.e., CO2 concentration, O2 concentration, or both) at desired times.

[0138] Bioenergetic capacity drives 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. As such, the devices and methods disclosed herein can be used to determine and / or quantify (i.e., determine and / or quantify) key indicators of healthy cellular function, predict cellular performance in in vitro disease models, and discover compounds / substances through modulation of metabolic targets, signaling, and substrates, with the goal of better understanding disease states and enabling insights into appropriate therapies to alter disease states, optimize and improve healthy phenotypes, and / or cellular performance.

[0139] The devices and methods disclosed herein can be used to measure two major metabolic pathways for living cells, mitochondrial respiration and glycolysis, in real time to provide a functional kinetic measure of cellular bioenergetic capacity.

[0140] The devices and methods disclosed herein can facilitate the testing of disease models and key cellular processes, including activation, proliferation, differentiation, cell death, cell homeostasis, and / or disease progression, therapeutic discovery by identifying and validating targets for potential therapeutic compounds / substances, and providing for optimizing the engineering and manufacturing of cell therapies.

[0141] In one embodiment, mitochondrial respiration, glycolic activity, and / or metabolic balance (i.e., mitochondrial respiration, glycolic activity, and / or metabolic balance) are temporal measurements of cellular activity independent of the media / buffer surrounding the cells. Creating microchambers allows for sensitive measurement of the cellular activity being detected. Changes in cellular mitochondrial respiration and / or glycolytic activity result in real-time minute changes in O2, CO2, and lactate in the environment surrounding the cells, and these changes in the environment are detected by the device via OCR, ECAR, and / or PER measurements.

[0142] In one embodiment, changes in cellular mitochondrial respiration, glycolytic activity, and / or metabolic balance have a feedback loop that facilitates the maintenance of or transition to a desired metabolic phenotype. This can be achieved, for example, by adding nutrients via an on-board liquid handling device or by altering sample environmental conditions (e.g., by changing O2 concentration). For example, FIG. 55 shows a feedback mechanism 5700 in which oxygen concentration 5710 in cell growth medium 5720 (in which various cells 5730 are growing) is measured by oxygen sensor 5754 attached to the distal tip of sensor spine 5752 in region 5722 proximal to oxygen sensor 5754. While oxygen sensor 5754 is discussed herein as a non-limiting example, various other sensors for measuring different gas compositions can be used in addition to or in place of oxygen sensor 5754. Growth medium 5720 is held in a well 5760 or other compartment that is sealed or semi-sealed by a lid 5750 (which defines a sensor spine 5752) to a measurement chamber 5770, which is also sealed or semi-sealed from the outside environment. Measurement device 5750 receives a signal from sensor spine 5752, which is communicated to a signal processor 5790, which calculates the measured O concentration 5710 in region 5722 in real time, determines whether (and how much) to supply N from N blower 5780 to chamber 5770, and adjusts the amount of O or other gases in chamber 5770 and / or well 5760 (i.e., chamber 5770, well 5760, or both) (e.g., by allowing N to flow in and pushing out undesired gases).

[0143] In another embodiment, mitochondrial respiration and / or glycolytic 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 ATP synthase inhibitors, mitochondrial uncouplers, or ETC inhibitors, to the medium that affects the cells.

[0144] 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 multi-well sample carriers. OCR and ECAR or PER can be used to determine, i.e., mitochondrial respiration and glycolysis, as well as ATP production rates. 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.

[0145] It should be noted that the cell samples described herein can include free cells, cell constructs, free tissues, and tissue construct samples. 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, greater precision and consistency, improved temperature control, and improved automation compared to conventional devices and methods.

[0146] Conventional systems are susceptible to moisture and contamination caused by factors such as the laboratory environment, storage, and manufacturing process, tend to experience operational errors over time including inconsistencies in debris movement / accumulation, and are susceptible to evaporation, edge-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, providing lower O2 detection limits and improved measurement accuracy.

[0147] The combination of hardware and analytical software provided in the devices disclosed herein enables real-time monitoring of live cells using rare cells, ex vivo cells, and genetically engineered cells in fields such as immunology and disease, and allows for the construction of more suitable disease models. The enhancements disclosed herein improve measurement performance. These enhancements generally facilitate identifying targets for novel compounds / agents, validating target effects on cellular function, optimizing disease models, and determining the safety and anti-tumor potential of T cell therapy compounds / agents moving from the laboratory to pharmaceutical therapeutic development and toxicology programs.

[0148] The devices 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.

[0149] The devices and methods disclosed herein provide the ability to analyze live cells over an extended temperature range, for example, controlled temperature zones smaller than the headspace of the temperature control element and housing, which represent an improvement over conventional devices.

[0150] The devices and methods disclosed herein provide more uniformity in the heating of temperature control elements, which can improve cell biological characteristics at consistent temperatures and sensing using device sensors, and reduce edge effects in the system.

[0151] The devices and methods disclosed herein may provide temperature control with faster start-up times than conventional devices.

[0152] The devices and methods disclosed herein include electro-optic substrates capable of functioning at humidity levels as high as 95%. Previous devices often perform suboptimally at 70%-80% humidity. Therefore, the devices can be shipped, stored, or used in geographic regions with high humidity or when it is desirable to control higher humidity levels within the device.

[0153] The devices and methods disclosed herein provide improved performance and detection at lower levels of OCR, previously manifesting as noise, thereby enabling the analysis of damaged or impaired immune cells, thereby expanding the range of different cell types that can be analyzed by the device.

[0154] 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 analytes and measuring the rates of cellular respiration, glycolysis, and ATP production. The devices described herein can be employed to determine extracellular, intracellular, and pericellular analytes.

[0155] According to certain embodiments, disclosed herein is a system, also referred to herein as a device. The device can include a stage adapted to support a multi-well sample carrier, also referred to herein as a sample carrier or sample carrier cartridge. The device can include a sensor adapted to sense a cellular component associated with a cell sample in a well of the multi-well sample carrier. The device can include a dispensing system adapted to introduce fluid into the well. The device 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 adapted for insertion into the well by relative movement of the stage and the plunger.

[0156] In particular, the device can include a plurality of sensors, each sensor adapted to sense cellular components in a corresponding well of the multi-well sample carrier. Thus, the device can include an array of sensors. The sensors can independently and selectively sense cellular components in each well. The dispensing system can include one or more injectors. The dispensing system can be configured to independently and selectively introduce a fluid or agent into each well. The plunger can be adapted to be independently and selectively inserted into each well.

[0157] The device 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.

[0158] 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.

[0159] 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 multi-well sample carrier 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 the wells of the multi-well sample carrier.

[0160] In one exemplary embodiment, one or more sensors can be adapted to sense changes in oxygen level and pH (proton concentration) of the cell culture medium associated with metabolic activity of the cell sample in the wells of the multi-well sample carrier. The stage, sensor, and dispensing system can cooperate to simultaneously measure the basal oxygen consumption rate and basal extracellular acidification rate of the cell sample using the sensors. The dispensing system can then be used to sequentially administer one or more drugs to the cell sample. In one exemplary embodiment, the one or more drugs can include a mitochondrial ATP synthase inhibitor (oligomycin A), a mitochondrial uncoupler BAM15, and / or a mixture of mitochondrial complex I and complex III inhibitors (rotenone and antimycin A, respectively). The sensor can optionally substantially simultaneously measure the oxygen consumption rate and extracellular acidification rate after each dispensing of the one or more drugs. Additional drugs, such as a modulator reagent, can optionally be dispensed prior to dispensing the described reagent, or the extracellular ionophore monensin can be injected after injection of rotenone / antimycin A into the cells. The same measurements of oxygen consumption rate and extracellular acidification rate can be performed before and after each aliquot.

[0161] Components of this device 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 device and method," each of which is incorporated herein by reference in its entirety for all purposes.

[0162] One or more of the following features may be included: the sensor may be configured to analyze the component without disturbing the cells, the well may include a step, and the plunger or barrier may be adapted to agitate the medium prior to analysis of the component.

[0163] 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 the 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.

[0164] The device can include a light source, such as a fluorescent lamp, light-emitting diode (LED), or 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 can be configured to generate a reference signal. Fluctuations in intensity from the light source can be corrected proportionally to the drift by monitoring the reference signal generated by the light source. The light source can 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 can be formed of a material configured to minimize drift caused by thermally induced fluctuations of the light source by at least 20%, e.g., at least 15%, 10%, 5%, or 1%.

[0165] In certain embodiments, the one or more sensors measure CO, O, Ca ++ , H + The wells can be adapted to analyze (determine the presence or concentration of) extracellular components, such as ATP, ATP, or cellular metabolites consumed or secreted. Analytes proportional to O2 content include, for example, CO2, O2. Analytes proportional to the pH of the sample include, for example, Ca. ++ , H + More than one analyte, for example at least one analyte, can be measured to analyze extracellular components.

[0166] The one or more sensors can be adapted to analyze a first extracellular component. In some embodiments, the one or more sensors can be adapted to analyze multiple extracellular components, e.g., more than one, more than two, more than three, more than four, or more components. Each sensor can analyze multiple components simultaneously. Each sensor can analyze multiple components individually, e.g., sequentially. The present disclosure generally describes sensor units configured to analyze a first target analyte, e.g., at least one analyte proportional to O2 content, and a second target analyte, e.g., at least one analyte proportional to pH value. However, it should be understood that the sensor unit can be configured to analyze additional or alternative target analytes.

[0167] In certain embodiments, the sensor is an optical sensor. The optical sensor can be a fluorescence- or phosphorescence-based sensor. Alternatively, the sensor can 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, while 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 may include optical, O2, pH, temperature, CO2, or a combination thereof.

[0168] Additionally, in one embodiment, the sensor may be an electrochemical sensor or a 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 embodiment, the well or chamber may also include one or more reference probes, in the form of any of the sensors discussed above, that generate signals of known values ​​for device calibration.

[0169] One exemplary sensor unit is an oxygen-sensitive photoluminescent dye. The photoluminescent dye can be selected from any oxygen-sensitive photoluminescent dye. Suitable dyes 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, and azaporphyrins, as well as long-decay luminescent complexes of iridium(III) or osmium(II).

[0170] Typically, in such embodiments, the hydrophobic oxygen-sensitive photoluminescent dye can be formulated with a suitable oxygen-permeable, 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 the 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.

[0171] Regardless of the type of solid-state sensor selected, in one embodiment, by way of example only, the sensor can 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 the distal end of at least one of the protrusions into a mixture of a fluorescent indicator in the medium.

[0172] However, it should be understood that in certain embodiments, the sensor may be spotted or immersed in whole or in part onto one or more of the protrusions. Furthermore, 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. Furthermore, 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 achieved by one or more techniques, such as vapor deposition, chemical coating, spin coating, immersion, and robotic spotting.

[0173] The dispensing system can include one or more injectors configured to selectively introduce fluids or agents into each well independently. In some embodiments, the dispensing system can include an array of injectors, e.g., at least one injector positioned to correspond to each well of the sample carrier. In some embodiments, the dispensing system can include one or more movable injectors, each configured to introduce fluids or agents into multiple wells of the sample carrier.

[0174] In certain embodiments, to actuate the movement of one or more injectors, for example 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.

[0175] Thus, the device can have one, two, three, four, five, six, seven, eight, nine, or ten movable injectors positioned to be driven across multiple wells, rows of wells, or columns of wells. Alternatively, the dispensing system can have an array of one or more fixedly positioned injectors corresponding to each well, e.g., one, two, three, four, five, six, seven, eight, nine, or ten injectors. 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.

[0176] The assemblies and processes according to exemplary embodiments of the present disclosure can be suitable for measuring components in any of a variety of sample types, such as biological samples. In one embodiment, for example, the systems and processes according to exemplary embodiments of the present disclosure can 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 can include cellular material derived from cells, such as organelles, mitochondria, cell extracts, cell products or by-products, or conditioned medium. Measurements can be completed in a label-free manner.

[0177] An exemplary system is shown in Figures 1-4. As shown in Figures 1-4, the device or device 100 includes a housing 10 having an opening in a sidewall of the housing 10. The opening may optionally be closable by a door 12. Within the housing 10 is a stage 20 adapted to receive a multi-well sample carrier 30. The stage 20 may be movable by an x-axis actuator assembly so as 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 (i.e., a computer, a power source, or both).

[0178] The electronic port 14 may be compatible with one or more of USB, mini-USB, HDMI, DVI, dual-DVI, mini-DVI, micro-DVI, DisplayPort, mini-DisplayPort, VGA, mini-VGA, RS-232, Ethernet / LAN, or any other electronic port capable of transmitting data. While the devices shown in FIGS. 1-4 include electronic port 14, it should be noted that the device may be connectable to an external computer by any means known in the art, such as wireless fidelity networking (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.

[0179] An exemplary assembly 110 is shown in FIG. 5. The assembly 110 can be housed within the housing 10 shown in FIGS. 1-4. The assembly 110 includes components of a sensing system 40 (e.g., fiber optic) 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 to force pressurized air through injector ports on the sensor cartridge that have corresponding substance / material ports in the manifold, and the manifold holes are "sealed" by a gasket and a force applied to the manifold. One or more components of the manifold can be independently movable in the z-axis as directed by a z-axis actuator assembly 54 of the motion actuator assembly. The temperature of the manifold and / or cartridge (i.e., the manifold, the cartridge, or both) can be controlled by a manifold temperature controller 52. The assembly 110 includes a stage 20 adapted to receive a multi-well sample carrier 30 (on which the cartridge is shown). The temperature of the samples in the multi-well sample carrier 30 can be controlled by a sample temperature control element 22. The stage 20 is movable along the x-axis as directed 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.

[0180] The device may include an automated measurement system. The device may also include a computer or may be connectable to a computer, with the automated 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 microplate. The controller may operate the sensor to perform sensing of one or more components in one or more wells of the 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 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.

[0181] In certain embodiments, the graphical user interface can include a plurality of display areas, each area associated with one of the wells. The graphical user interface can be configured to receive instructions written in each area associated with one of the wells for designing a multi-well experiment and to receive data acquired by the sensor in response to execution of the multi-well experiment for display in each area associated with one of the wells. Thus, a method executable by the controller can be independently and selectively applied to one or more wells through instructions from the graphical user interface.

[0182] FIG. 6 illustrates an exemplary system including a system (laboratory device) connectable to a cloud-based computing network and a computer through the cloud-based network. The system 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 system components can be provided through a user interface accessible on the computing device or the cloud-based computing network. The user interface can be provided on a web browser software platform and / or 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 system. Other mechanisms for connecting to the cloud, such as desktop software or driver software, can also be used. The system may also include 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, which may store historical data, protocols, data processing algorithms, and / or controller-executable instructions.

[0183] 7 and 8 are schematic diagrams of the systems disclosed herein, showing the electronic components in more detail. FIG. 7 is a diagram of the system operably connected to a central control computer. The substrate includes a microcontroller or system controller operably connected to the temperature control elements for the manifold and tray (i.e., sample temperature control elements) and the pipetting system or injection unit. An additional 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 devices described herein can include stepper motors with higher torque, which improves the accuracy of measurements over the life of the device and reduces the need to provide maintenance and / or replace motor components.

[0184] Also included as part of the movement actuator assembly are proximity and / or encoder sensors (i.e., proximity sensors and / or encoder sensors) configured to sense the relative positioning of the stage or multi-well sample carrier and other device components, such as the sensor unit and injectors of a pipetting system. The proximity sensors can be configured to generate a notification signal, and optionally pause a protocol, when a component is positioned within a predetermined distance of another component, for example, when a sensor unit is positioned within a predetermined distance of a corresponding well of a sample carrier. Additionally or alternatively, the proximity sensors can be configured to generate a notification signal, and optionally pause a protocol, when an opening in a sidewall of the housing is ajar and / or when external light is detected within the housing.

[0185] The system may also include a stall detection module programmed to generate a notification signal, optionally pausing the protocol and, for example, stopping motor movement, if a predetermined protocol step is not completed within a predetermined time interval. The stall detection 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 encoder movement and flag a stall.

[0186] The diagram of Figure 7 also includes sensing units for the O2 and pH analytes operably connected to a signal processing module including a microcontroller and amplifiers configured to receive and amplify signals from the sensor units. The signal processing module is further operably connected to the system controller and central control computer. The system further includes a barcode scanner configured to scan barcodes encoding information operably transmittable to the central control computer.

[0187] 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 consumables or samples in the multi-well sample carrier. The system controller is also operably connected to an emission amplifier or signal processing module. The signal processing module is operably connected to an optical fiber or 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 dispense system. Optionally, a separate system controller operably connected to the manifold heater or manifold temperature control element can be provided.

[0188] 9 shows an exemplary sensor unit 41 deployed in a well 31. The exemplary sensor unit 31 is a fluorescent sensor. A fluorophore having fluorescent properties that depend on at least one of the presence and concentration of a component in the well 31 may be disposed on the surface of 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 fluorescent emission from the fluorophore.

[0189] The present disclosure provides methods, devices, and measurement systems for adding test compounds to wells and measuring the contents of the wells using sensors. The methods can be implemented as high-throughput assays by adding one or more test compounds to one or more wells, or by adding the same or different test compounds to multiple wells in a microplate. In certain embodiments, the test compounds are introduced while the sensor probe remains in equilibrium with the liquid contained within each well, e.g., submerged therein. In such embodiments, equilibration time 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.

[0190] In certain embodiments, the present devices and methods store and deliver one or more test compounds or target agents per well. Test compounds can be delivered using a supply of compressed gas from a remote source to actuate compound delivery. 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 test compound delivery from multiple ports using a supply of compressed gas from a remote source.

[0191] In one embodiment, a cartridge is provided that is adapted to mate with a multi-well sample carrier having a plurality of wells. The cartridge can include a substantially planar element having a plurality of regions corresponding to respective openings of a common number of wells in the multi-well sample carrier. At least one port can be formed in the cartridge in at least one region, the port adapted to deliver a test fluid, such as an aqueous solution of a candidate compound / substance or other agent, to each well. The cartridge can also include at least one of: a) a sensor or portion thereof adapted to analyze a component 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.

[0192] The components and features of the cartridge are further described, for example, in U.S. Pat. No. 9,170,255, entitled "Cell analysis device and method," which is incorporated herein by reference in its entirety for all purposes.

[0193] The device may include an elevator mechanism adapted to move the cartridge relative to the stage or plate to place sensors in the wells, typically for simultaneously placing multiple sensors in multiple wells. The device may include a pressure source adapted to be fluidly engaged with the cartridge to deliver test fluid from ports in the cartridge to the wells. The device may also include a multiplexer disposed between the pressure source and the cartridge, the multiplexer adapted to be in fluid communication with multiple ports formed in the cartridge. The multiplexer may be in selective fluid communication with an exclusive set of ports formed in the cartridge. The device may include a controller for controlling 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 placed in the well. The controller may be in communication with a computer or a graphical interface, as described above.

[0194] In certain exemplary embodiments, the cartridge opening adapted to receive the sensor can include a sensor sleeve structure having a surface adjacent to the well of the multi-well sample carrier. 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.

[0195] The array of sensors corresponding to the array of wells can be integral with 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 adapted to be mounted relative to the sample carrier.

[0196] Methods for analyzing cells using the devices disclosed herein are provided. The methods can be employed to measure cells disposed in a medium in a multi-well sample carrier. The methods can include one or more of: disposing at least a portion of a sensor in the medium in a well of the multi-well sample carrier; analyzing a component associated with 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 component to determine any changes therein. In certain embodiments, one or more components can be analyzed substantially simultaneously. In particular, changes in the rate of one or more components can be measured over an assay time period, e.g., to determine metabolic or other activity of a cell sample.

[0197] The analyzing step can include analyzing a respective component for each cell in the medium in each well. The respective components can be the same component. 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 fluids or agents can include the same test fluid or agent.

[0198] The analyzing step can include analyzing each component for each cell 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.

[0199] The method can include controlling the temperature and / or environment of the cell sample before, during, and / or after the analysis step (i.e., before, during, and / or after the analysis step). In certain embodiments, the method can include controlling the temperature and / or environment of the cell sample throughout the performance of the analysis method. Controlling the environment can include, for example, controlling the relative humidity (RH) and / or the composition of the environmental gas, such as the concentration of N, O, and / or CO. For example, in certain embodiments, controlling the environment can include creating a hypoxic environment by purging air with N gas.

[0200] The method may further include imaging or scanning the sample during the analyzing step, during the delivering step, and / or following the analyzing and / or delivering steps.

[0201] 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 structures, free tissues, tissue structures, 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 unicellular organisms, such as microorganisms. In certain exemplary embodiments, the sample can include whole plant or animal model tissues, such as zebrafish, C. elegans, or Drosophila.

[0202] The biological material to be analyzed can include cellular material. The biological material can include living cells, including bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, or insect cells. The cells can be animal cells, human cells, immune cells, or immortal cells.

[0203] 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 liver cancer cells (e.g., HepG2), human epidermoid carcinoma cells (e.g., A431), and whole organisms such as zebrafish, C. elegans, and Drosophila. Certain embodiments of the devices and methods disclosed herein enable the analysis of live cells requiring temperatures between 28°C and 40°C without the need to place the device in a temperature-controlled room.

[0204] The devices and methods disclosed herein can be employed to facilitate research in the areas of cancer, immunology, toxicology, compound / agent discovery, and immunotherapy, among others.

[0205] In one embodiment, the cell sample is obtained or derived from a subject, such as a human or non-human animal. In one embodiment, the subject is a mouse, which in one embodiment has or is at risk of having a disorder. Nevertheless, in one embodiment, 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 embodiment, the cell sample includes modified cells genetically engineered for heterologous expression of a gene of interest and / or engineered for inhibition of expression of a gene, such as, for example, cells from a knockout mouse or a CRISPR KO library. Nevertheless, in one embodiment, the cell sample includes stem cells or stem cell-derived cells. Nevertheless, regardless of the cells used, in one embodiment, the cell sample includes medium, such as a culture medium or growth medium, and the cells may be disposed in the medium. It will further be appreciated that in one embodiment, the cell sample comprises a plurality of cells, such as a plurality of cells described herein.

[0206] 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, and three-dimensional cell samples, such as tissue samples, cell spheroids, organoids, biopsy samples, cell scaffolds, and organs-on-a-chips. Examples of parameters related to cellular function that can be measured include carbon dioxide concentration, oxygen concentration or oxygen tension, calcium ions, and hydrogen ions. However, in one embodiment, the parameter measured is oxygen concentration, such as oxygen consumption. Through these tests, an understanding of what drives cellular phenotype and function and / or an accurate depiction of the cellular environment or microenvironment can be obtained.

[0207] The assemblies and processes according to exemplary embodiments of the present disclosure can be used to measure metabolic data of living cells or the (micro)environmental conditions of any living cells. Cellular materials to be tested can include, for example, bacterial cells, fungal cells, yeast cells, prokaryotic cells, and eukaryotic cells. Cells that can be tested include mammalian cells, including animal cells and human cells. Specific cells that can be tested include cancer cells, immune cells, immortal cells, primary cells, induced pluripotent stem cells, and cells infected with viral or bacterial pathogens.

[0208] For example, in one embodiment, the assembly and process according to the exemplary embodiment of the present disclosure can be used to support immunotherapy. Immunotherapy is a type of treatment that strengthens a patient's immune system to fight cancer, infectious diseases, and other diseases. The immunotherapy process can 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 a patient's blood. The T cells are then transferred to a bioreactor and expanded or cultured. In addition, T cells may be modified to possess specific proteins called receptors. Receptors on T cells are designed to recognize and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in a bioreactor to achieve a certain cell density and then delivered to the patient 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 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 significant success in combating hematological disorders. 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 materials and modified products.

[0209] NK cells are a type of cytotoxic lymphocyte that can seek out and destroy infected cells in the body. NK cells can exhibit extremely rapid 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 grown to a relatively high cell density in a bioreactor.

[0210] Culturing cells, such as T cells, NK cells, or other mammalian cells, typically requires a somewhat complicated process from inoculation to patient use. The disclosed assemblies and processes can be used to monitor the metabolism of cells during any point during the culturing process to ensure that the cells are healthy and / or have a desired metabolic phenotype, and that the medium in which the cells are growing contains optimized levels of nutrients. The present systems and processes can be used, for example, to make adjustments to ensure the metabolic fitness of the cells as they are growing.

[0211] In addition to immune cells, the metabolism of cancer cells can also be monitored to provide an understanding of which nutrients are nourishing 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 growth. 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 liabilities of potential therapeutics. The risk of mitochondrial toxicity, for example, can be assessed with high specificity and sensitivity. In this manner, the mechanism of action of several mitochondrial toxicants can be determined.

[0212] [Electrical measurement module] According to certain embodiments, the system further comprises an electrical measurement module configured to measure various electrical properties of samples held in the wells of the sample carrier. In various embodiments, the electrical measurement module monitors one or more of the impedance, inductance, resistance, or capacitance of the samples held in each well and provides electrical signals of the measured properties to the control module to track changes in the electrical properties over time (e.g., 6 to 72 hours). In other embodiments, the electrical measurement module stimulates the samples held in the wells of the sample carrier and measures the electrical signals of the stimulated cells.

[0213] FIG. 47A is a schematic diagram of a consumable 4900 in which two electrode structures of the same or similar area are deposited on a substrate (e.g., a sample carrier) in which one or more wells are formed. The first electrode structure includes electrode elements 4910a-4910c, and the second electrode structure includes electrode elements 4910d-4910f (collectively, electrode elements 4910). The electrode elements within the electrode structures are connected to each other by arc-shaped connecting electrode buses 4925. Like electrode elements 4910, such connecting buses 4925 are also made of a conductive material (e.g., gold film, platinum film, gold film on chromium or titanium film). These conductive connecting paths or connecting buses 4925 may have an insulating coating. The electrode elements 4910 include electrode wires with connected circular portions. The total area of ​​the electrode elements 4910 and the gaps between them can correspond to, or be slightly larger or smaller than, the bottom of a well (e.g., a cylindrical well, a conical well, or a cubic well), such as the bottom of a commonly used 24-well, 96-well, or 384-well sample carrier. To ensure that molecular interactions occurring at the bottom of the wells can contribute to impedance changes, the entire surface of the wells can be covered with electrodes. This arrangement has the advantage that non-uniform molecular interactions occurring at the bottom of these wells result in only small variations in the impedance measured between the electrode elements 4910. Although three electrode elements 4910 are shown extending from each connection bus 4925, various embodiments may use more or fewer electrode elements 4910 of different lengths, widths, and surface characteristics.

[0214] Connection pads 4950 that can be connected to external impedance measurement circuitry. 4930 are electrical connection traces that connect the connection pads to the electrode elements 4910. Such connection traces can extend in any direction within the plane of the electrode.

[0215] One or more gaps or windows 4920 are defined between the electrode elements 4910 to allow imaging of various contents of the well in which the consumable 4900 is placed. In various embodiments, the window 4920 can be centrally located within the consumable 4900 to correspond to the center of the well, but various sub-windows 4920 can also be defined such that the electrode structure 4910, connection bus 4925, or contraction pads 4950 do not occupy space. These sub-windows 4920 can be aligned with microwells or other subcompartments defined within the well, or with various features of the sample to be imaged.

[0216] FIG. 47B is a schematic diagram of a consumable 4900 with two electrode structures of similar area deposited on a substrate. As shown in FIG. 47B, electrode elements 4910a-4910f are rectangular lines that together form an interdigitated electrode structure unit, although other shapes and sizes can be used in various embodiments. As in FIG. 47A, the electrode elements 4910 within each electrode structure are connected via arc-shaped conductive paths or electrode buses 4925. Connection pads 4950 are connected to the electrode structures via electrical connection traces 4930. One or more gaps or windows 4920 are defined between the electrode elements 4910 to allow imaging of various contents of the well in which the consumable 4900 is placed. In various embodiments, the window 4920 can be centrally located within the consumable 4900 to correspond to the center of the well, although various sub-windows 4920 can also be defined so that the electrode structures 4910, connection buses 4925, or contract pads 4950 do not occupy space. These sub-windows 4920 may be aligned with microwells or other subcompartments defined within the well, or with various features of the sample to be imaged.

[0217] FIG. 47C is a schematic diagram of a consumable 4900 having electrode structures 4930a-4930d of similar dimensions deposited on a substrate. Electrode structures 4930a-4930d include a plurality of interconnected electrode elements 4910a-4910h. Electrode elements 4910 are rectangular lines that together form an interdigitated electrode structure unit, although other shapes and sizes can be used in various embodiments. Unlike FIGS. 47A and 47B, electrode structures including electrode elements 4910a-4910c and 4920a-4920d are connected to connection pads 4950. One or more gaps or windows 4920 are defined between electrode elements 4910 to enable imaging of various contents of the well in which consumable 4900 is placed. In various embodiments, the window 4920 can be centrally located within the consumable 4900 to correspond to the center of the well, but various sub-windows 4920 can also be defined such that space is not taken up by the electrode structure 4910, connection bus 4925, or contraction pads 4950. These sub-windows 4920 can be aligned with microwells or other subcompartments defined within the well, or with various features of the sample to be imaged.

[0218] Examples of electrical measurement modules are further described, for example, in U.S. Pat. No. 7,470,533, entitled "Impedance-based devices and methods for use in assays," which is incorporated herein by reference in its entirety for all purposes.

[0219] [Temperature control] The devices described herein include one or more temperature control elements designed to reduce temperature gradients between outer (e.g., boundary) wells and inner wells of a multiwell sample carrier. 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 a multiwell sample carrier, a sensor unit, and / or an injector. For example, a sample temperature control element can be dimensioned to fit into a multiwell sample carrier. A manifold temperature control element can be dimensioned to fit into a sensor, an injector, and / or a cartridge, and can optionally be dimensioned to cover the multiwell sample carrier when the cartridge is positioned to mate with the multiwell sample carrier, e.g., when the sensor unit and / or injector are in fluid communication with the wells of the multiwell sample carrier. In some embodiments, a microenvironment is formed that includes the manifold and heater, the heated components surrounding the sensor cartridge, and the tray heater in direct contact with the sample carrier, allowing for temperature maintenance over an extended period of time. The manifold temperature control element can be configured to mate with the sample temperature control element and cover the multi-well sample carrier.

[0220] The design of the temperature control elements creates controlled temperature zones or microenvironments within the device. The controlled temperature zones typically include the 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., temperature control does not extend to the entire internal chamber of the device, such that the temperature of components outside the controlled temperature zones is not substantially changed, e.g., increased or decreased, by actuation of the temperature control elements. In some embodiments, 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. In some embodiments, the volume of the controlled temperature zone does not exceed 10% of the volume of the housing, e.g., not exceeding 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0221] 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, 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 supply, circuit board, and light source. The low operating temperature allows the device to be used to test sample types that previously could not 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 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.

[0222] The creation of a controlled temperature zone or microenvironment generally enables the device to bring the temperature of the sample in 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.

[0223] Furthermore, the design of the temperature control element allows the device to achieve temperature uniformity and a wider operating temperature range than previous designs. The wider operating temperature range allows the device to be used with a wider variety of cell types, such as non-mammalian cells, which may require lower or higher temperatures than previously achievable, improving viability during assays. The greater operating temperature can improve the sensitivity of the sensing unit, for example, allowing the device to have a lower OCR detection limit than previous devices. In some embodiments, measurement uniformity and / or accuracy are improved.

[0224] The manifold temperature control elements can be configured to control the temperature of a 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 temperatures of samples in an array of wells of a 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 sample carrier, e.g., between samples within the interior and boundary of a sample carrier, and / or between cartridge components and corresponding samples in the sample carrier.

[0225] In some embodiments, the sample temperature control element is configured to control the temperature of the sample in 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 in each well of a sample carrier, e.g., two identical or substantially identical samples, such that, when the samples are analyzed under the same or substantially the same conditions, a sensor signal responsive to target analyte level, production, or consumption does not differ by more than a predetermined amount between the two identical or substantially identical samples, e.g., does not differ by more than 10% between the two identical or substantially identical samples, e.g., does not differ 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, e.g., photoluminescence sensor readings, cellular metabolism and other functions, and / or analyte concentrations, that may occur as a result of temperature differences.

[0226] The design of the temperature control elements reduces sample evaporation during protocol execution. Evaporation, if severe enough to alter the concentration of analytes in the medium, can affect cellular function. The temperature uniformity achieved by the sample temperature control elements and / or manifold temperature control elements results in reduced sample evaporation compared to conventional devices. In some embodiments, the temperature control elements can be configured to control sample evaporation within an 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 longer duration, such as 6-hour assays, 8-hour assays, 10-hour assays, or longer. Furthermore, the multi-well sample carrier can be designed to reduce evaporation during cell culture and incubation processes.

[0227] Surprisingly, it has been found that the design of the temperature control element provides a lower O2 detection limit and improved measurement accuracy. For example, the systems 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 systems can have an improved 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.

[0228] Additionally, the design of the temperature control element can reduce, limit, or inhibit differential (gradient) diffusion of gas within 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, the diffusion of gas within the controlled temperature zone, cartridge, sample carrier, so 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.

[0229] [Environmental Control] The devices described herein can include or be associated with one or more environmental control modules designed to control the environment surrounding the multi-well sample carrier. The environmental control module can be designed to control the environmental gas and / or relative humidity (RH) of the environment surrounding the sample. For example, the environmental control module can be configured to control one or more of the N2 concentration, O2 concentration, and CO2 concentration of the gas surrounding the sample. The RH can be increased or decreased by the environmental control module. For example, the RH can be decreased to less than 75%, less than 65%, less than 55%, less than 45%, or less than 35%, or the RH can be increased to more than 65%, more than 75%, more than 85%, or more than 95%. The environmental control module can enable the use of the device for ischemia / reperfusion modeling and other controlled gas experiments.

[0230] The environmental control module can include one or more sources of gas, e.g., N2, O2, and CO2, fluidly connected to the sample carrier. The environmental control module can form a controlled environment zone that includes an array of wells in the sample carrier. The controlled environment zone can be open or closed to the ambient environment. The environmental control module can include a pump or fan configured to direct gas to or remove gas from the sample carrier.

[0231] In certain embodiments, the environmental control module is integrated into the device. A controlled environmental zone can be formed within a sealed container, e.g., a sealed vessel. The environment can be formed by moving a heated component to surround, cover, or encase a heated sample carrier. The heated component can be made of a thermally conductive material, e.g., 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 so that there is minimal airflow. The container can house a sample carrier, e.g., a stage that holds the sample carrier. In some embodiments, the container can house a cartridge with a sample carrier. To form a controlled environmental zone, the sealed container can be fluidly connected to a gas source and appropriately purged with one or more selected gases.

[0232] 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 with low gas solubility.

[0233] The environmental control modules can be integrated with system software, e.g., operably connected to a controller and / or system processor, and the software can be programmed to cycle the environmental control modules according to a selected protocol.

[0234] The environmental control module can be integrated with system software, e.g., operably connected to a controller and / or system processor, and receives input from measurements of the cellular microenvironment (e.g., intracellular O2, pericellular O2, or O2 measurements proximate to the cell sample), thereby enabling the environmental control to provide a target cellular microenvironment. The software can be programmed to cycle the environmental control module to provide the target microenvironment according to a selected protocol.

[0235] [Image capture module] According to certain embodiments, the system further comprises an imaging module configured to capture and process images of samples held within the sample carrier. One exemplary imaging module comprises an image capture element, such as a camera or array of cameras, and associated accessory optical components that assist the image capture element in imaging samples or sample features within each well of the sample carrier through a window in the sample carrier opposite the side on which the multiple wells are defined. The image capture element or sample carrier can be moved relative to one another by a motion stage to align the camera or camera array with the window and position it within the well so that the contents can be imaged.

[0236] In various embodiments, a light source is associated with the imaging module to illuminate the sample, fluorescent labels, etc. The light source can be located on the same side of the sample carrier as the image capture element (e.g., below the sample carrier, as a flashlight or direct lighting), on the opposite side of the sample carrier (e.g., as a backlight), or on another portion of the cavity (e.g., ambient lighting). Further, the light source can be configured to generate light within the visible spectrum, infrared spectrum, ultraviolet spectrum, and combinations thereof, which the image capture element is configured to detect. In various embodiments, the image capture element or controller can color shift portions of the captured image outside the visible spectrum into the visible spectrum, apply grayscaling, color correction, etc. Exemplary light sources 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.

[0237] 42 shows a light source 4400 according to one exemplary embodiment of the present disclosure. In some embodiments, the light source 4400 comprises two light-generating devices: a xenon flash lamp 4410 and a tungsten lamp 4420. In other embodiments, the light source 4400 can comprise a xenon continuous wave lamp, a light emitting diode (LED), a laser, or any other light-generating device.

[0238] Tungsten light sources are very stable, and their emission spans the visible spectrum from the blue to the far infrared, peaking at approximately 1 μm. Tungsten light sources are best suited for measurements in the visible and infrared regions of the spectrum. In contrast, xenon flash light sources deliver most of their radiation in the deep UV, UV, and short visible spectral ranges. Furthermore, xenon flash light sources provide very fast bursts of light that last for several microseconds and decay rapidly, making them suitable for time-resolved measurements with current multi-detector systems.

[0239] The xenon flash lamp 4410 has a parabolic reflector 4411 positioned such that the arc 4412 of the lamp 4410 is located near the focal point of the reflector 4411, providing an essentially collimated beam from the reflector 4411. The tungsten lamp 4420 has a parabolic reflector 4421 positioned such that the filament 4422 of the lamp 4420 is located near the focal point of the reflector 4421, providing an essentially collimated beam from the reflector 4421. FIG. 42 shows that a lens 4423 can be used to focus the beam from the reflector 4421 onto the exit portal 4430 of the light source 4400. As shown in FIG. 43, according to an embodiment of the present disclosure, relay optics can be used to focus the beam onto the entrance to an optical fiber. Alternatively, lens 4423 can focus the beam from reflector 4421 directly onto the entrance of an optical fiber within excitation spectrum device 4500 .

[0240] In various embodiments, the excitation spectrum device 4500 has two spectrally selective devices that differ in their physical techniques for separating different wavelengths of light: the first device is a filter selection device 4520 with a variety of user-interchangeable filters 4521. The second device is a double monochromator 4530.

[0241] The first path directs the light through one of filters 4521 in filter selection device 4520 that transmits a narrow band of light. The light then propagates through optical fiber 4522 to exit port 4540. The second path bypasses filter 4521 by directing the light through hole 4523 in filter selection device 4520. The light then continues through optical fiber 4531, which receives the circular image of the arc or filament spot from light source 4400 formed at entrance port 4510 and shapes the light spot into a slit shape to fit the input slit of double monochromator 4530. After monochromator 4530 selects the narrow band of light, optical fiber 4521 changes the shape of the light from the exit slit shape of monochromator 4530 to a circular shape that resembles the shape of the well in sample carrier 4700.

[0242] The light path selector 4550 can move relative to the filter selection device 4520 and can direct light spectrally selected by the filter 4521 or the monochromator 4530 to the exit port 4540 .

[0243] The movable off-axis parabolic reflector 4440 has two operating positions. In the first position, shown in solid lines in FIG. 42, the reflector 4440 reflects and focuses the light from reflector 4411. In the second position, shown in dashed lines in FIG. 42, the reflector 4440 does not obstruct the light from reflector 4421. This arrangement allows light from both lamps to be focused at the same location. Additionally, a fan 4417 directs air across the fins 4415 of the cooling extrusion for the xenon light source 4410 and onto the tungsten light source 4420. This arrangement allows both light sources to share a single cooling system.

[0244] Placing two light sources close to each other and offsetting their optical axes, preferably at approximately a 90-degree angle from each other, allows for a very compact lighting system with a shared cooling system. The use of a parabolic reflector around the light source, combined with an off-axis parabolic reflector, results in very efficient coupling of light from the arc and filament into the system, where the final focal point of both light sources is the same. By utilizing separate light source compartments with separate exit light points for each compartment, the system allows for a compact arrangement compared to systems that require the mechanical movement of an optical relay system to switch between light sources.

[0245] 44 shows the structure of an excitation-emission emission separation device 4600 according to one exemplary embodiment of the present invention. The general purpose of the excitation-emission emission separation device 4600 is to irradiate a sample with excitation light and / or collect emission light from the sample. The excitation-emission emission separation device 4600 can be positioned above or below the sample carrier 4550 relative to the surface on which the wells are defined, or one can be positioned above the sample carrier 4450 and the other below.

[0246] The sample carrier 4450 comprises a substrate having a plurality of wells defined therein to receive samples for analysis. Each well comprises a volume-defining member configured to limit the range of motion between the substrate and a second element of the system and / or to define a minimum non-zero distance between the substrate and a second element of the system (e.g., to prevent the second element from touching a sample held in the well). In various embodiments, these volume-defining members comprise shelves, raised ridges, and stops at certain points above the base of the corresponding well that define the volume and shape of the well and the separation distances between the well, other wells in the sample carrier 4450, and other elements of the system that operate with the sample carrier 4450.

[0247] In some embodiments, several light paths can be used, depending on the measurement technique. In absorbance measurements, the excitation and emission light are preferably collinear. As shown in Figure 44, absorbance measurements are performed in block 4640, where the well is illuminated from below at point G with excitation light. This excitation light can come from a monochromator 4530 or a filter selection device 4520. On the other side of the well, a detector 4650 is positioned to capture the emission light that has passed through the sample.

[0248] In luminescence measurements, no excitation light is required and only emission light from the sample is collected by the excitation-emission separation device 4600. In block 4630, a single optical fiber bundle 4735 is used to maximize the light gathering capabilities of the system and thus improve the signal.

[0249] In fluorescence measurements, two optical paths are available to deliver excitation light to the sample and collect emission light from the sample, which can be optimized to further improve the overall system performance.

[0250] Block 4620 represents the first optical path for fluorescence measurement, which can use a partially reflecting mirror or a dichroic mirror to ensure that the excitation light and emission light are collinear as they enter and exit the sample. Light is delivered to block 4620 by optical fiber 4532. A variable aperture 4601, preferably having several apertures with diameters ranging from approximately 1.5 mm to 4 mm, is positioned in front of guide fiber 4522. Within well 4555, an image of the aperture positioned in front of optical fiber 4522 is formed by lenses 4621 and 4622. The size of the aperture of variable aperture 4601 is selected to fill the well as completely as possible with light while preventing light from entering adjacent wells and causing crosstalk.

[0251] The light is reflected by a partially transmitting mirror 4623 on a movable holder 4627. More than one mirror can be placed on holder 4627. Some mirrors can be dichroic mirrors to improve the signal, as all excitation light is reflected toward the well and all emission light is transmitted toward the exit fiber. The dichroic mirror can also improve the signal-to-noise ratio of the measurement system, as residual excitation light that reaches the well and is reflected by the meniscus lens is prevented from reaching the exit fiber. Emission light from the well is collected by lenses 4621, 4622, and 4670 into fiber optic bundle 4731. Collection lens 4670 in front of fiber optic bundle 4731 ensures that emission light from the entire depth of the well is collected, thus increasing the system signal.

[0252] The high energy collection characteristics of the present system ensure low detection limits and allow for a variety of fluid levels to produce acceptable results without the need to refocus the optics based on fluid volume, in contrast to confocal style measurements described, for example, in U.S. Patent No. 6,097,025 (incorporated herein by reference in its entirety), which use confocal optics that collect light from only a small portion of the well.

[0253] In some embodiments, linear polarizers 4624 and 4625 are contained in holder 4627, and the same movement that positions the appropriate mirror in the optical path can also be used to select the polarizer for fluorescence polarization measurements, eliminating the need for a separate mechanism for switching polarizers and improving the reliability of the system.

[0254] Block 4610 shows a second optical path for fluorescence measurements, which uses a tilted-V arrangement of optics for direct well illumination and collection. This allows the system to transmit the entire amount of light from optical fiber 4532 into well 4555. For this purpose, the numerical apertures of optics 4611 and 4612 are matched to optical fiber 4532. A cone of excitation light enters the well and excites the well contents through the first leg of the V. Emission light is collected by the second leg of the V. The numerical apertures of lenses 4614 and 4613 are matched to exit optical fiber 4732. The V is tilted relative to the vertical plane, directing excitation light specularly reflected from the well surface directly away from the light-collecting leg of the V. This arrangement therefore introduces spatial separation between emission and excitation light in addition to spectral separation, significantly improving the signal-to-noise ratio. This tilted-V arrangement can also be used to perform fluorescence polarization measurements.

[0255] Input ports A and B of excitation-emission separation device 4600 accept fiber bundles from excitation spectrum device 4500. The fibers can be positioned to direct light spectrally separated by filters in excitation spectrum device 4500 to input B of block 4620. Fibers can also be positioned to direct light spectrally separated by monochromators in excitation spectrum device 4500 to input A of block 4610. Alternatively, the inputs can be reconfigured by switching fibers 4522 and 4532. This switching can be accomplished manually. Emission light is collected from ports C and D by fibers 4731 and 4732. The placement of fibers 4731 and 4732 at exit ports C and D determines the origin of the emission light in the fibers.

[0256] Figure 45 shows holder 4627 with associated dichroic mirrors 4623, 4628, and 4629 and linear polarizers 4624, 4625, and 4626, according to an exemplary embodiment of the invention. Holder 4627 is fixed to slider 4650, which slides along rails 4651 by force applied from motor 4652 through belt 4653. Holder 4627 moves in a direction perpendicular to the plane defined by the optical axes of the excitation and emission lights. Two different fibers 4522 and 4532 could occupy the fiber positions shown in Figure 45, but for clarity, only fiber 522 is shown.

[0257] In the illustrated design, there are five possible positions for holder 4627 relative to fiber 4522, which delivers excitation light. The first position is shown in FIG. 45 and corresponds to the situation where the center of mirror 4628 is aligned with the optical axis of fiber 4522. In this position, fluorescence polarization-based assays cannot be performed. If holder 4627 is moved to the left a distance equal to the distance between the centers of mirror 4628 and mirror 4629, holder 4627 reaches the second position. In the second position, mirror 4629 plays an active role, and fluorescence polarization-based assays cannot be performed.

[0258] The other three positions of holder 4627 correspond to three different situations. First, when the right third of mirror 4623 is positioned in front of fiber 4522, fluorescence polarization-based assays cannot be performed. Second, when the middle third of mirror 4623 is positioned in front of fiber 4522, linear polarizer 4624 is in the optical path of the excitation light, and linear polarizer 4626 is in the optical path of the emission light. In this case, the polarization vectors of the excitation light and the emission light intersect. Third, when the left third of mirror 4623 is positioned in front of fiber 4522, linear polarizer 4624 remains in the optical path of the excitation light, and the other linear polarizer 4625 is in the optical path of the emission light. In this case, the polarization vectors of the excitation light and the emission light are parallel. Therefore, the linear movement of holder 4627 not only selects which mirror is positioned in the optical path, but also enables fluorescence polarization measurements.

[0259] As shown in Figure 45, linear polarizers 4625 and 4626 have parallel surface orientations and perpendicular polarization axis orientations. Linear polarizers 4625 and 4626 have active regions of equal size, each corresponding to the size of the cross section of the emitted light. The polarization axis of linear polarizer 4624 is parallel to the polarization axis of linear polarizer 4625 and perpendicular to the polarization axis of linear polarizer 4626. The area of ​​linear polarizer 4624 is at least twice the area of ​​linear polarizer 4625. The area of ​​mirror 4623 is at least three times the area of ​​linear polarizer 4625. Mirror 4623 is partially reflective and partially transmissive.

[0260] 46 shows a top view of sample carrier 4550 along the vertical axis toward sample carrier 4450 in block 4610 of excitation-emission emission separation device 4600. Points A and B' are input portals of excitation-emission emission separation device 4600. Lenses 4611, 4612, 4663, and 4664 focus excitation light onto well 4455 in sample carrier 4550. Lenses 4613, 4614, 4673, and 4674 collect and focus emission light onto points C and D', which are exit portals of excitation-emission emission separation device 4600. The optical axes of lenses 4611, 4612, 4663, 4664, 4613, 4614, 4673, and 4674 are directed along the diagonal of well 4555 defined in sample carrier 4550. Using this arrangement, simultaneous readings can be taken from the same well 4555 via a filter-based or monochromator-based spectral system. Because excitation light from point A is reflected towards point B' and vice versa, very little excitation light is reflected towards exit portals C and D'. Thus, the emission light is spatially separated from the excitation light.

[0261] [Optical module] The device can further include an optical module positioned to image or scan samples in the multi-well sample carrier. The optical module can be positioned within the housing. The optical module can be operably connected to the controller. The optical module can be controlled or operated via a graphical user interface. Furthermore, images or scans acquired by the optical module can be viewed and / or recorded (i.e., viewed or recorded, or both) via the graphical user interface, optionally in real time. Thus, in some embodiments, the optical module is operably connected to a computer, and the computer is configured to display and / or record images or scans of the samples in real time.

[0262] Cell-based assays, especially live cell assays, are becoming more common in life science research. Microplates are increasingly being used as vessels for the investigation of cell growth processes by qualitative and quantitative means. In many cases, researchers work with cells using multiple specialized devices.

[0263] Photoluminescence, e.g., fluorescence and / or phosphorescence, readings can be achieved using dedicated conventional fluorescence readers or multi-detector readers, using instruments with a light beam diameter large enough to obtain a representative measurement of fluorescence throughout the well, or to perform area scanning and mapping of the signal across the well. Most devices provide for plate incubation, fluid injection, and also allow the option of gas control (CO2 and / or O2) similar to tissue culture incubators.

[0264] With wide-field imaging modalities, more information can be obtained from cells than just the fluorescent signal level in a well. Laboratory microscopes are commonly used, using bright-field and phase contrast for unstained cells and fluorescent imaging for stained cells. Some devices 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 device option.

[0265] Typically, these devices are purchased from various vendors, forcing users to physically transfer vessels, e.g., microplates, from one device to another as needed, and may also be forced to track the entire sample analysis process and collate and combine data from several devices to obtain a complete, comprehensive analysis of a cell sample. Without robotics, it can be nearly impossible to properly perform lengthy, complex experiments or assays. The use of robotics further increases both the cost and complexity of the analysis. The combination of non-imaging analytical modalities (fluorescence, absorbance, and chemiluminescence), wide-field fluorescence imaging at the cell level, confocal fluorescence imaging, environmental control, and reagent injection in a single device may provide a complete, comprehensive analytical approach, freeing users from tedious microplate handling, microplate tracking, and data transfer. Approaches for a combined system capable of storing, collating, and analyzing data acquired from individual devices are described herein.

[0266] [consumables] The present disclosure provides consumables that can be used to analyze cell samples according to the systems and methods described herein.

[0267] In some embodiments, the system includes an interface for interacting with a consumable. The consumable can be any cell sample-holding consumable. Exemplary consumables include, but are not limited to, flow chips, microtiter plates with any number of wells, 2D samples, and 3D tissue or spheroid formation / measurement plates. For example, microtiter plates can have 6, 12, 24, 48, 96, 384, or more wells. In some embodiments, the consumable includes microelectrodes if impedance measurements or electrical excitation are desired. In some embodiments, the consumable can form microchambers to enable flux measurements. In some embodiments, the consumable is made of a material that limits gas diffusion to increase flux sensitivity. To image the cell sample, components comprising the imaging system can be configured to read from below or above the consumable. When imaging from below, the consumable can have a window through the microelectrode for viewing the cell sample. When imaging from above, the consumable can remove any features above the sample, such as a flux measurement cartridge, to view the sample.

[0268] Consumables include, but are not limited to, impedance electrodes, lids, and sample carriers (e.g., cell culture plates) with or without cartridges. In some embodiments, the lid can have one or more sensors, e.g., O2 / pH / CO2 sensors. In some embodiments, the cartridge can have one or more sensors and / or compound / substance ports. Consumables can be sequenced through various steps of an automated workflow.

[0269] In some embodiments, the sample carrier is a cell culture plate. In some embodiments, the sample carrier comprises a plurality of wells. In certain embodiments, a well of the plurality of wells comprises an impedance electrode. In other embodiments, a well of the plurality of wells does not comprise an impedance electrode. For example, the impedance electrode can be wired to detect real and imaginary impedance components of a cell sample during growth and / or measurement. In some embodiments, the wells are fabricated with a uniform electrode at the bottom. In some embodiments, the wells are fabricated with a window at the bottom for imaging the cells. When a well is fabricated with a window at the bottom for imaging the cells, normalization can be performed and applied to the measurements. In some embodiments, the wells comprise a ridge to facilitate the formation of a microchamber that does not interfere with the impedance electrode. A cartridge can be loaded into the well and rest on the ridge during measurement. The microchamber can then be refreshed by removing the cartridge from the ridge.

[0270] [Sample Control Module] According to certain embodiments, the system further comprises a sampling control module. In various embodiments, the sampling control module can operate in conjunction with an environmental control module, as described herein. The sampling control module includes one or more of a sample environment temperature control element (such as a temperature control module described herein) configured to control the temperature of the sample and / or sample carrier, a gas control element configured to control the gas content of one or more of the O, CO, and N content of the sample, a humidity control element configured to control the humidity of the environment surrounding the sample carrier (e.g., to prevent / reduce / enhance evaporation), and a measurement device control element configured to control the temperature of sensors that interface with the sample and / or well to determine various properties thereof.

[0271] Additionally, the sampling control module can operate in conjunction with the fluid handler or cartridge to control the temperatures of various compounds / substances (e.g., reagents, drugs under test, other media) to be within certain predetermined ranges. By controlling the temperature of compounds / substances added to the wells, the controller can reduce the effect of temperature shock of the introduced material on the samples in those wells and store the compounds / substances at a temperature different from the temperature at which they are delivered (e.g., refrigerating the compound to extend its shelf life, heating the compound to reduce its viscosity). In various embodiments, the controller can maintain the compounds / substances at a standby temperature different from the rest of the environment within the device while waiting to be introduced into the wells. The controller can additionally or alternatively adjust the temperature of the compound / substance from the temperature of the environment (or standby temperature) prior to introduction into the wells. For example, a compound can be stored at X degrees as a standby temperature (in an environment where temperature T=X or T≠X) and then heated (or cooled) to Y degrees for introduction into a well maintained at Z degrees (where X≠Y≠Z, X≠Y≧Z, or X≠Y≦Z).

[0272] In various embodiments, the sample temperature environment control element and / or the measurement device control element are heaters that generate heat via electrical resistance to electrical current passing through the various heating elements.

[0273] In various embodiments, the gas control element communicates with one or more gas canisters containing gas to control the atmosphere of an individual sample well or a cavity in the system into which a sample carrier is inserted. The gas control element can include various sensors that detect the balance of gas content and / or the pressure of the gas therein within the well and / or cavity. Based on the sensor readings, the gas control element can evacuate, apply negative pressure, or otherwise remove a portion of the gas atmosphere from the well and / or cavity and replace the removed portion with a desired composition of at least one of O, CO, and N at a desired pressure to maintain the desired atmosphere composition. Additionally or alternatively, the gas control element can inject at least one of O, CO, and N at a desired pressure to adjust the existing atmosphere without evacuating, aspirating, or otherwise removing a portion of the existing atmosphere.

[0274] In various embodiments, the humidity control element includes a dehumidifying element for removing moisture from the atmosphere of the well and / or cavity into which the sample carrier is inserted, and / or is in communication with a water source for injecting additional water into the sample well or its atmosphere (e.g., via a sprayer or humidifier element).

[0275] In various embodiments, the gas control and humidity control elements operate by opening and closing covers to one or more wells in the sample carrier to release undesired atmospheres, and are connected to liquid handling elements or flux / consumable cartridges, which contain various consumable growth gas supplies used to adjust or re-establish the desired atmospheric composition within a given well, in addition to various growth media, stimulants, modifiers, etc., supplied to samples held within the wells.

[0276] [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 moisture absorption in the dielectric materials used to insulate the conductive paths.

[0277] The devices disclosed herein are designed to reduce parasitic current paths by including a signal processing module capable of operating at high relative humidity, such as 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 device for longer time frames. Thus, real-time cellular 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, or 12 hours.

[0278] The signal processing module is a processor operably 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 signals 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 the detection and / or amplification of signals from the sensor units.

[0279] The signal processing module can be configured to detect signals 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 detecting and / or processing ratiometric measurements. Briefly, the measurements can include an analyte-sensitive signal measurement and an analyte-insensitive or largely analyte-insensitive reference measurement. A ratio between the references can be incorporated to facilitate ratiometric evaluation of analyte flux or concentration.

[0280] 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 includes a printed circuit assembly having a transimpedance amplifier including a grounded guard trace. In some embodiments, the signal processing module can include one or more photosensitive components, such as a semiconductor diode, a photomultiplier tube, an avalanche photodiode, a CMOS sensor, a CCD, or the like. In some embodiments, these photosensitive components can be connected to the transimpedance amplifier. In some embodiments, the signal processing module includes a printed circuit assembly formed from surface-mounted components, e.g., substantially free of secondary, hand-soldered high-gain components. 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., thermal contact with, a heat sink. The thermally conductive excitation source can be any excitation source that changes intensity with temperature, e.g., a laser diode or a light-emitting diode (LED). In some embodiments, the signal processing module includes a printed circuit assembly with an integrator design. In some embodiments, the signal processing module includes a printed circuit assembly having an operational amplifier design.

[0281] Surprisingly, it has been discovered that the design of the thermal conductivity excitation source significantly reduces thermal drift, such that less reference correction is generally required, which can reduce correction errors and thereby improve measurement accuracy ( FIG. 36 ). The data shown in the graph of FIG. 36 demonstrates the reduction in thermal drift after including the thermal conductivity excitation source. In some embodiments, the improved design of the thermal conductivity excitation source can reduce (or eliminate) the need to include a reference signal detector, reducing the complexity of 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 reference signal detector-less.

[0282] The components and features of the signal processing module are further described in "Section 5: High Impedance Sensors" by Kester et al., which is incorporated herein by reference in its entirety for all purposes.

[0283] [Transportation Module] According to certain embodiments, the system 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.

[0284] The transfer module can be formed of multiplexed fiber optic material. Figures 34 and 35 show several views of an exemplary transfer module 60, including a side view (Figure 35) and a cross-sectional view (Figure 34) of the transfer module 60. In an embodiment, the transfer module 60 can include an array of fiber optic bundles, each in communication with a corresponding sensor unit in the array of sensor units. The fiber optic bundles can be positioned and configured to interface directly with one or more sensor units. Each fiber optic bundle can be formed of an array of fiber optic cables housed within a fiber probe housing, e.g., a metal fiber probe and / or a plastic probe housing, as shown in the cross-sectional view of Figure 34.

[0285] 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 uniformly distribute light to one or more sensor units. The homogenizer can improve mechanical and optical shuffling.

[0286] [Device combination] In certain embodiments, cells can be analyzed serially by performing successive measurements of the same cell sample. Samples can be analyzed in any order to measure cellular bioenergetic work, such as O2, CO2, pH, etc. Data can be stored in a cloud-based storage unit and, optionally, analyzed on a cloud-based data processing and visualization system. Samples from the same cell sample, different samples, or the same cell line can be analyzed using electrochemical measurements, e.g., impedance measurements. Data can be stored in a cloud-based system. Samples from the same sample, different samples, or the same cell line can be visually observed for cell growth and morphology. Data can be stored in a cloud-based system. Data obtained from independent measurements can be correlated with corresponding samples / measurements, for example, by labeling the samples with barcodes or other digital identification systems. Data can be collected and collated in a cloud-based storage unit and, optionally, processed in a cloud-based data processing and visualization system. Collated data from analyzing the same cell sample can be queried for patterns and information.

[0287] Each of the measurements can be performed within a device described herein or a combination of devices each operably connected to a data storage and processing system, e.g., a cloud-based system or computer.

[0288] 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. 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.

[0289] In certain embodiments, a sample or an aliquot of a sample can be analyzed to measure cellular bioenergetic work by measuring parameters such as O, CO, pH, or other metabolically related parameters, and can be visually observed for cell growth and morphology simultaneously, e.g., in parallel, substantially in parallel, or after an extended period of time. In some embodiments, a sample or an aliquot of a sample can be analyzed to measure cellular bioenergetic work by measuring parameters such as O, CO, pH, or other metabolically related parameters, and can be analyzed simultaneously, e.g., in parallel, substantially in parallel, or after an extended period of time, for electrochemical measurements (e.g., impedance). In some embodiments, a sample or an aliquot of a sample can be visually observed for cell growth and morphology, and can be analyzed simultaneously, i.e., in parallel, substantially in parallel, or after an extended period of time, for electrochemical, e.g., impedance, measurements, and can be visually observed for cell growth and morphology simultaneously, e.g., in parallel, substantially in parallel, or after an extended period of time.

[0290] It is understood that the cell sample is transferred between modalities over an extended period of time, and the cell sample is normalized between modalities. The sample is not measured continuously with one modality over the entire extended period of time; rather, the sample is measured with a modality, normalized, and then measured again with the same modality. In one embodiment, the sample is measured with a first modality, normalized, and then measured again with the first modality at multiple different time points over the extended period of time for investigation, such as measuring the extracellular flux of the sample with a measurement time of 3 minutes and collection and normalization times of 5, 10, 15, 30, 60, or more minutes, followed by a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and Nth measurements of extracellular flux over a 72-hour period. Additionally or alternatively, the sample can be measured in one modality and then moved to a second or third modality for a second or third measurement, or any variation thereof. In one embodiment, the sample can be measured over time, starting with a measurement in a first modality (e.g., measuring extracellular flux in response to an analyte (e.g., measuring O2 loss or pH changes)), moving the sample to a second modality (e.g., impedance measurement) and / or a third modality (e.g., imaging), and then returning the sample to the first modality (e.g., flux measurement).

[0291] In one embodiment, each of the multiple cell samples is monitored separately by each modality, e.g., one sample of the same cell line is monitored for bioenergetic metabolism, another sample of the same cell line is monitored for impedance, and yet another sample of the same cell line is monitored visually for cell growth. In another embodiment, the same cell samples can be analyzed in parallel or substantially in parallel, e.g., the bioenergetic metabolism of the cell samples can be monitored in parallel or substantially in parallel by imaging the cell samples. In another embodiment, the same cell samples can be analyzed in parallel or substantially in parallel, e.g., the bioenergetic metabolism of the cell samples can be monitored in parallel or substantially in parallel by impedance measurements. In another embodiment, the same cell samples can be analyzed in parallel or substantially in parallel, e.g., the bioenergetic metabolism of the cell samples can be monitored in parallel or substantially in parallel by impedance measurements and imaging.

[0292] In another embodiment, the same cell sample can be analyzed at an extended time period, for example, the bioenergetic metabolism of the cell sample can be monitored for a first time period with respect to imaging, and the same cell sample can be monitored for a second time period, a third time period, a fourth time period, a fifth time period, a sixth time period, a seventh time period, an eighth time period, a ninth time period, a tenth time period, an eleventh time period, a twelfth time period, a thirteenth time period, a fourteenth time period, a fifteenth time period, a sixteenth time period, a seventeenth time period, an eighteenth time period, a nineteenth time period, a twentieth time period, and subsequent time periods with respect to bioenergetic metabolism and imaging. For example, analysis can be performed after 6 to 72 hours, e.g., 12 to 60 hours, 24 to 48 hours, 12 to 36 hours, 24 to 48 hours, 36 to 60 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 12 hours, 60 to 72 hours, 48 ​​to 72 hours, 36 to 72 hours, 24 to 72 hours, 12 to 72 hours, 12 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 ​​to 60 hours, or up to one week (e.g., 168 to 170 hours).

[0293] In another embodiment, the same cell sample can be analyzed at an extended time period, for example, the bioenergetic metabolism of the cell sample can be monitored for impedance in a first time period, and the same cell sample can be analyzed for bioenergetic metabolism and impedance in a second time period, a third time period, a fourth time period, a fifth time period, a sixth time period, a seventh time period, an eighth time period, a ninth time period, a tenth time period, an eleventh time period, a twelfth time period, a thirteenth time period, a fourteenth time period, a fifteenth time period, a sixteenth time period, a seventeenth time period, an eighteenth time period, a nineteenth time period, a twentieth time period, and so on. The analysis can be performed after a period of time, for example, 6 hours to 72 hours, e.g., 12 hours to 60 hours, 24 hours to 48 hours, 12 hours to 36 hours, 24 hours to 48 hours, 36 hours to 60 hours, 6 hours to 60 hours, 6 hours to 48 hours, 6 hours to 36 hours, 6 hours to 24 hours, 6 hours to 12 hours, 60 hours to 72 hours, 48 ​​hours to 72 hours, 36 hours to 72 hours, 24 hours to 72 hours, 12 hours to 72 hours, 12 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 ​​hours to 60 hours, or up to 1 week (e.g., 168 hours to 170 hours).

[0294] In another embodiment, the same cell sample can be analyzed after an extended period of time, for example, the bioenergetic metabolism of the cell sample can be monitored in parallel for impedance measurements and imaging during a first period of time. The same cell sample can be monitored for bioenergetic metabolism, impedance, and imaging during a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and subsequent periods, e.g., 6 hours to 72 hours, e.g., 12 hours to 60 hours, 24 hours to 48 hours. , 12 hours to 36 hours, 24 hours to 48 hours, 36 hours to 60 hours, 6 hours to 60 hours, 6 hours to 48 hours, 6 hours to 36 hours, 6 hours to 24 hours, 6 hours to 12 hours, 60 hours to 72 hours, 48 ​​hours to 72 hours, 36 hours to 72 hours, 24 hours to 72 hours, 12 hours to 72 hours, 12 hours to 24 hours, 24 hours to 36 hours, 36 hours to 48 hours, 48 ​​hours to 60 hours, or up to one week (e.g., 168 hours to 170 hours) after analysis.

[0295] While this disclosure generally refers to measuring metabolism, it should be noted that similar methods can also be used to measure or detect cellular microenvironment features, such as the environmental conditions experienced by a sample. These conditions can be manipulated toward desired microenvironmental conditions, possibly through environmental control. These conditions can be manipulated to correlate with cellular responses. As an exemplary embodiment, impedance, specific imaged cellular parameters, cellular oxygenation, fluorescence-measured parameters (e.g., cellular metabolism) that vary in response to oxygen or pH can be controlled to implement models to characterize the impact of tumor microenvironmental conditions on cellular function. As a further example, such characteristics can be controlled to analyze cardiac muscle cell beating rate and / or metabolism in response to reduced oxygen and / or nutrient availability, where beating rate is controlled pharmacologically or using electrical pacing via a device.

[0296] The embodiments described herein overcome the above-described disadvantages, as well as other disadvantages not described above. Also, an embodiment is not required to overcome the above-described disadvantages, and an example embodiment may not overcome all of the above-described problems.

[0297] According to an aspect of one 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.

[0298] According to an aspect of an exemplary embodiment, there is provided a method of sample analysis, the method including selecting at least one subsystem from 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 analysis subsystem to analyze the one or more samples, and controlling the selected at least one 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 analysis operation of the analysis subsystem to analyze the one or more samples.

[0299] According to an aspect of an exemplary embodiment, there is provided a non-transitory computer-readable medium having embodied thereon a program that, when executed by a computer, causes the computer to perform a sample inspection method, 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 that images the one or more samples and an analysis subsystem that analyzes the one or more samples; and controlling the selected at least one subsystem to perform an inspection on the one or more samples, the inspection including an imaging operation of the imaging subsystem that images the one or more samples and an analysis operation of the analysis subsystem that analyzes the one or more samples.

[0300] According to one aspect of an exemplary embodiment, a device for analyzing a sample is provided. The device can include a receptacle support configured to support a microplate containing microplate wells configured to hold samples, also referred to herein as a multi-well sample carrier, plate, or sample carrier. In one embodiment, imaging of the sample is performed using an automated cell imaging reader, such as those disclosed in U.S. Pat. No. 10,072,982 (incorporated by reference in its entirety for all purposes), e.g., Cytation® 5, Cytation® 7, or the like. In one embodiment, imaging of the sample is performed using a confocal imaging device comprising: a receptacle support configured to support a microplate including microplate wells configured to hold the 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.

[0301] It will be appreciated that the cell sample may be viewed using any type of imaging modality that allows for visual examination of the cells.

[0302] In certain embodiments, the cell sample can be observed using phosphorescence lifetime imaging microscopy (PLIM) and / or fluorescence lifetime imaging microscopy (FLIM), including two-photon excitation imaging.

[0303] In certain embodiments, an imaging modality known as confocal imaging may be suitable for imaging cellular samples, e.g., 3D cellular structures such as spheroids. In confocal imaging, a point or portion of a sample can be illuminated at a time. Light can 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 can be performed using a spinning disk, also known as a scan disk or Nipkow disk.

[0304] Confocal imaging is an imaging modality particularly suited for use with spheroids, as it allows spheroids to be sectioned layer by layer and 3D models to be generated in silico for both accurate cell counting and 3D image manipulation to view the spheroids from various angles.

[0305] Figures 13A and 13B are comparative images of spheroids. Figure 13A shows a spheroid photographed at 20x magnification using wide-field imaging. Figure 13B shows a spheroid photographed at 20x magnification using confocal imaging. While the size of the spheroid can be assessed using the image in Figure 13A, individual cells and spheroid structure only become visible with the confocal imaging in Figure 13B.

[0306] The resolution advantage resulting from confocal imaging in Figure 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 Figure 13A.

[0307] The addition of confocal fluorescence imaging to a device 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 most versatile single device for analyzing microplate-based assay formats, including those aimed at studying 3D cell spheroids.

[0308] In one example, there may be a workflow where widefield imaging is performed for faster screening, while confocal imaging is performed for public images of O2, CO2, and pH measurements taken from the sample.

[0309] Widefield imaging can be performed for HCS-type assays, where widefield imaging offers faster throughput and the resulting image analysis remains statistically robust. Confocal imaging can then be employed to acquire representative wells of "hits" compared to "controls" for publication or presentation purposes.

[0310] In one example, there may be a workflow in which widefield imaging is performed for a more rapid initial screening of spheroids based on size, and then confocal imaging is used for a deeper assessment of the size of each "hit" well based on nuclei counts, which is more accurate when using confocal imaging.

[0311] Typically, wide-field imaging cannot "see" deep enough into the 3D spheroid to reliably count individual nuclei, but wide-field imaging 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 obtain improved image analysis for counting all nuclei in the spheroid, which could not be done with wide-field imaging alone.

[0312] In one example, there can be a proliferation assay (3D endothelial cell spheroid assay) to identify candidate wound healing compounds / substances. Primary compound / substance screening can be performed in microplates, where small endothelial spheroids are treated with a library of unknown compounds to determine which compounds induce increased cell growth / proliferation. Compounds that induce increased growth can be candidates for further wound healing studies.

[0313] In the analysis workflow, a plate reader can be used to rapidly screen microplates using GFP fluorescence intensity to identify wells with increased spheroid size. Wells that meet a GFP intensity threshold (the threshold is statistically determined during assay development) are considered "hits" and selected for further imaging. Control wells are always imaged as reference wells for comparison with hit wells. Confocal imaging of 3D spheroids can be performed to acquire two-channel z-stack image sets (Hoescht33342 nuclear marker and GFP marker) of the entire spheroid sample. Upon image processing and analysis of the maximum projection of the z-stack, the cell count of the spheroid is determined to quantify its size. Visual inspection of the distribution of nuclear masks in the images is performed to determine the presence or absence of cell death within the spheroid. Results from image analysis of hit wells are then compared to controls to determine the percentage of growth relative to the control.

[0314] In one exemplary workflow, a 3D tumoroid cytotoxicity immune response assay (3D tumoroid assay from surgical specimens to determine immune and cytotoxic treatment responses) is performed. This assay involves culturing tumoroids obtained from animal models or surgical specimens from patients. Because these tumoroids are animal / patient-derived, tumor-derived immune cell responses can be evaluated in vitro, allowing for analysis of tumor response to various treatments. This assay can use heterogeneous multicellular tumor models to evaluate the efficacy of novel treatments in a microplate-based format.

[0315] For example, tumoroids can be stained for nuclei counting (e.g., blue) and for immune cell markers (e.g., red). A microplate reader can be used to evaluate wells with high cytotoxicity, indicated by a low blue signal, and wells with high immune response, indicated by 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 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 marker 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 hit wells are compared to controls to determine the percentage of cytotoxicity or immune response relative to the control.

[0316] Some of the above examples take advantage of the ability of a single device to perform assays as "hit picking." An initial, rapid read identifies samples of particular interest using a fast 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 the results of particular interest. This process is particularly important when the final result is high-resolution confocal imaging, which requires large data storage, and collecting vast amounts of information about only a few samples of interest provides significant savings in data storage 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 disparate processing steps can streamline analysis.

[0317] Other applications of the capabilities of a single device 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 negatively 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 optimal image quality, the exact location of the spheroid in the well is no longer known. In a preferred embodiment, wide-field imaging with a lower magnification but a wider field of view may be performed to locate the spheroid (region of interest) and position the well so that the location of the found spheroid (region of interest) is aligned with the optical axis. Then, a higher-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 is moved along its focal axis perpendicular to the well bottom. The spheroid (region of interest) can be identified using the device's non-imaging analysis modality by performing a fluorescent area scan and selecting the region of maximum fluorescent signal for imaging.

[0318] FIG. 14 is a block diagram illustrating a multi-detection system according to one embodiment.

[0319] 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 microplate 300, a carriage 310, an incubation chamber 320 for incubating samples in wells 200, an environmental control subsystem 2000, and a confocal imaging subsystem. The multi-detection system may also include an external subsystem 2100.

[0320] Samples are placed into wells 200 (e.g., microwells) of a microplate 300. The microplate 300 is transported in and out of an assay incubation chamber 320 by a carriage 310. When positioned so as to be exposed to the external environment of the multi-detection system, the microplate 300 can be accessible outside of the incubation chamber 320 and / or housing 1900 for access by a technician or robotic arm. Once the microplate 300 is positioned within the chamber, a variety of supported imaging and non-imaging analytical modalities can be performed.

[0321] Carriage 310 is part of a microplate transport subsystem for positioning microplate 300 and may include any suitable combination of belts, platforms, microplate holders, motors, and positioning software running under hardware control for positioning. Once microplate 300 is placed in incubation chamber 320, the entire microplate 300 remains incubated. The incubation system and incubation chamber 320 are described in detail below.

[0322] The non-imaging analytical subsystem 1300 can 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 sample in the well 200. The non-imaging analytical subsystem 1300 can be implemented as a filter-based subsystem or as a hybrid of any or all of the above.

[0323] 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.

[0324] External subsystem 2100 can be an external confocal illumination subsystem for confocal imaging that can be modularly connected to and disconnected from an imaging subsystem within housing 1900 via optical fibers to add flexibility to the physical arrangement of external subsystem 2100 relative to the device. Alternatively, the confocal imaging illumination subsystem can be arranged to be integrated within housing 1900.

[0325] Fluid injection subsystem 1100 injects reagents into wells 200 as required by the assay. 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.

[0326] Environmental control subsystem 2000, shown disposed externally to housing 1900, can include a gas control module that provides control of atmospheric conditions within housing 1900. Other control modules can include modules for control of temperature, humidity, and other conditions that can be controlled within housing 1900 under the control of environmental control subsystem 2000. The environmental control subsystem can include any combination of pumps, reservoirs, lines or tubing, fans, and heating and cooling elements to control all conditions within housing 1900. 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 environmental control subsystem 2000.

[0327] 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.

[0328] FIG. 15 is a block diagram illustrating a multi-detection system according to one embodiment.

[0329] The multi-detector system allows for several imaging modalities. In addition to confocal imaging modalities, 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. 15.

[0330] The microplate 300 can be disposed on a carriage 310 (e.g., a carrier for a sample carrier) that positions the wells 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 an objective turret 1232. The relative position of the imaging illumination subsystem 1600 is shown in FIG. 15, and the imaging illumination subsystem 1600 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, in which some elements of FIG. 15 are omitted for clarity.

[0331] FIG. 16 is a block diagram illustrating a multi-detection system according to one embodiment.

[0332] 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.

[0333] A spinning disk confocal system is developed as an exemplary embodiment of a confocal imaging system. This system is based on the use of a spinning disk (Figure 18) optical path. The disk is positioned at an intermediate image plane conjugate to the sample and detection planes. Thus, the disk is in both the excitation and emission optical paths. In one exemplary embodiment, the disk is typically about 2 mm thick and made of glass or quartz. The disk can be opaque or coated to a given transparency or opacity, except for transparent areas left as a pattern of pinholes or slits. Ideally, the disk surface is made non-reflective to incident light. The sample to be imaged is illuminated by excitation light transmitted through pinholes. Only radiation emitted by the sample, originating from these illuminated spots on the sample, reaches the detector through pinholes in the disk. The pinholes or slits are numerous but spaced apart from each other so that they act optically independently. Ideally, energy from neighboring pinholes 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.

[0334] 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 one exemplary embodiment, the output tip of an optical fiber can be the light (radiation) source. Radiation is used as an embodiment because the excitation spectrum can be outside 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. The input tip of the fiber can be illuminated from a light source module external to the device, providing flexibility in selecting the best light source match for sample imaging needs. The fiber also provides the flexibility to split 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 can be sent through excitation filter 1531, then reflected from dichroic mirror 1533 and focused onto spinning disk 1504 by tube lens 1520. Here and throughout this specification, the term "lens" can 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. Field lens 1519 minimizes light loss and directs light exiting the disk to be collected by tube lens 1250. Tube lens 1250 directs excitation radiation via mirror 1220 to objective lens 1230, which illuminates a small spot on the sample near the bottom of the well. Sample components are dyed with a dye corresponding 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:

[0335] Light emitted by the sample is collimated by objective lens 1230, reflected by mirror 1220, and collected onto spinning disk 1504 by tube lens 1250 and field lens 1519. 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 on detector 1560, which is typically a pixelated digital camera such as a charged couple device (CCD) camera or a complimentary metal-oxide semiconductor (CMOS) camera. The sample image captured by the camera can be stored in the multi-detector system's memory or an external computing system, where it may be enhanced and analyzed for various properties, and / or presented to a user on a visual display.

[0336] 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 creates a bandpass for excitation, the emission filter 1532 creates a bandpass for emission, and the dichroic mirror 1533 separates the excitation and emission to fully utilize the available energy and suppresses the amount of excitation light reflected from multiple optical surfaces that reaches the detector as the excitation light travels toward the sample, including the disk surface. While the lens 1521 (e.g., the emission filter) provides the majority of the excitation light suppression, the dichroic mirror 1533 also plays a suppression role. An alternative configuration to 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 for 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 labeled electronically or optically with a code that is automatically read via a barcode or some other automatically available method.

[0337] The surface of the spinning disk, along with the sample, is imaged onto the detector. Therefore, dust particles adhering to the disk surface can appear as artifacts in the image, such as bright streaks of light caused by the disk's rotation. Small particles can easily adhere to the disk surface with sufficient force to resist centrifugal force. The spinning disk 1504 and 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 ambient environment to prevent dust particles from accumulating on the disk. Windows 1551 and 1550 in the module allow light to pass through but not dust. Ideally, these dust-proof windows should be located as far 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 completely contained within the disk modules 1502 and 1553. Therefore, users should not open the modules to avoid introducing dust particles into the disks.

[0338] 16 shows two disk modules 1502 and 1553 mounted on a multi-detector device. 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.

[0339] A significant advantage of allowing users both confocal and wide-field imaging options in the same device is the ability to overlay images from various imaging modalities, such as a wide-field image and the same image from a confocal imaging modality. Alternatively, a bright-field image can be used to locate the region of interest and then imaged confocally. For this configuration to properly acquire an image, the magnification of both modalities must match exactly; otherwise, the images will not be properly overlaid. The light in the section between tube lens 1520 and tube lens 1250 is not parallel. In the confocal modality, there are several flat windows in this section of the optical path, such as a confocal disk and a dust window. 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 to the space 1501 between the confocal disks where wide-field imaging occurs. This ensures that the sample remains in focus relative to a fixed objective lens position even when the imaging 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.

[0340] The size of the pinhole on the confocal disk is ideally selected based on the parameters of the imaging objective 1230. In one 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 on the disk = 1.2 × magnification of the objective lens × emission wavelength / numerical aperture of the objective lens

[0341] Both the numerical aperture (NA) and magnification of the objective are part of this equation. If the pinhole is too small, excessive light will be lost, lengthening the time it takes 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 50-70 μm range. This works reasonably well as a compromise for the range of high-magnification objectives typically deployed in confocal microscopy. However, it is preferable that a disk with the appropriate pinhole can be matched to the objective being used.

[0342] Some spinning disk embodiments do not have a spiral pattern of round holes, but instead employ a slit aperture. A slit aperture can provide relatively brighter illumination of the sample and a stronger luminescence signal, while a pinhole aperture can provide relatively better axial resolution. Therefore, for some imaging applications, including biological fluorescence applications, a slit can be preferable as it can shorten image acquisition time, which is another reason for modifying the disk even with a fixed objective lens.

[0343] Multiple discs may be deployed in the imaging device and selection among the discs may be performed by the user or automatically by a multi-detection system.

[0344] 16 shows an example of two disk modules 1502, 1553 used in a multi-detection device. 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 labeled electronically or optically with a code that is automatically read via a barcode or some other available method.

[0345] 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.

[0346] Identifying the module allows for automated software setup and automatic resetting and calibration of the module's axial position 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 one another. 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 and sample planes 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. Ideally, the disk should then be positioned at 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 disk's axial position 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. Multiple 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.

[0347] 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.

[0348] The two concepts of user-interchangeable disk modules and automated axial disk positioning work best in tandem, but can also be implemented separately. If automated axial disk positioning is not available, disk modules can be configured to be interchangeable with some criterion on the module ensuring disk position and proper placement in the device. The concept of an easily interchangeable disk module that does not require user opening and therefore is 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 objective and sample.

[0349] Even if the device is limited to one or two disk modules, automatic axial adjustment may be used to alleviate the need for strict control of the location of the detector image sensor sensitive surface in the detector 1560 (such as a camera). This allows maximum user flexibility in camera selection and allows for 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.

[0350] FIG. 17 is a block diagram illustrating a multi-detection system according to one embodiment.

[0351] FIG. 17 shows wide-field imaging deployed in one exemplary embodiment. As explained above, the optical section (with elements labeled 15xx) allows for both confocal imaging (with spinning disk 1504 or 1503 in the optical path) and wide-field imaging (through the space 1501 between the disks). However, for wide-field modalities that researchers may want to deploy in a single, versatile device, using this optical system and confocal light source 1540 and confocal cube 1530 can have drawbacks. For confocal imaging, excitation radiation should be directed onto the disk through multiple optical elements positioned prior to the disk surface (e.g., dichroic mirror 1533, tube lens 1520, window 1551). 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 reflected light remains as far away from the detector as possible, and an emission filter 1532 to suppress unwanted reflected light. 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 high levels of excitation radiation that are partially reflected. Any dust particles may also be excited and fluoresce. Despite the designer's best intentions, some of the light reaches the detector, reducing the signal-to-noise ratio. Thus, a non-fluorescent sample that would otherwise appear very dark on an image may appear less 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. Wide-field microscopy using the confocal partial excitation element described above in FIG. 16 can involve significant compromises in image quality and system capabilities.

[0352] In one exemplary embodiment, an alternative subsystem that can be used for wide-field fluorescence imaging is included in the same device. The confocal cube 1530 of the confocal subsystem is positioned out of the path, and a spinning disk module is positioned in space 1501 for wide-field imaging. This transforms the configuration of FIG. 15 into the configuration of FIG. 17. The dedicated wide-field subcomponents 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 to the LED cube 1201 for best signal-to-noise performance. Several pairs of these cubes, corresponding to the particular chemistry being investigated, may be included on the slider.

[0353] This design has several advantages.

[0354] 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.

[0355] 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 can provide sufficient excitation when placed near the sample as shown in Figure 17.

[0356] Third, especially important when samples need to be excited in the UV range, some objectives are rated as UV objectives, transmit UV light, and exhibit very low fluorescence when excited by UV. However, there is no guarantee that commonly available optical elements in the remaining optical path, such as tube lenses, will not fluoresce when illuminated by UV light. When wide-field images of samples stained with the common DAPI nuclear stain are required, a common approach in the confocal optical path is to use a wavelength of approximately 400 nm, thus avoiding strong excitation of optical elements in addition to the sample. However, shifting excitation from 360 nm, the ideal wavelength for DAPI stain excitation, toward 400 nm significantly reduces the emitted light. Researchers must either add a higher concentration of dye to the sample or increase the detector gain, thereby reducing the signal-to-noise ratio of the image. Ideally, excitation of DAPI-stained samples would be at 360 nm, but UV excitation light would not pass through optical elements that could fluoresce. LED cube 1201 and filter cube 1210, in one exemplary embodiment, enable just such an optimal option. UV excitation light is incident only on objective lens 1230, which can be selected not to fluoresce. The emitted light returns to the detector through several 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 levels present in UV light.

[0357] 17 shows the relative locations of an imaging illumination subsystem 1600 for wide-field imaging in non-fluorescence modalities. This system can be 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.

[0358] Additional embodiments and components of the imaging system are further described in PCT Patent Application No. 2022 / 120047, "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.

[0359] 18 shows an exemplary wide-field imaging system 1800 without a confocal option, deployed in one exemplary embodiment. The wide-field imaging system includes an imaging subsystem module 1200 visually accessing micro-wells 200 of a microplate 300 located on a carriage 310 housed within a measurement chamber 320, and images of the micro-wells 200 can be captured by a tube lens 1250 and a camera 1560.

[0360] As mentioned above, the microplate 300 can be disposed on a carriage 310 that positions the wells 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 an objective turret 1232. The relative position of the imaging illumination subsystem 1600 is shown in FIG. 15, and the imaging illumination subsystem 1600 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 is provided below in FIGS. 16 and 17, in which some elements of FIG. 15 are omitted for clarity.

[0361] Further details of the imaging subsystem module are described below with respect to Figures 16 and 17. Components of the wide-field imaging system are further described, for example, in U.S. Patent No. 10,072,982, entitled "Universal multidetection system for microplates," which is incorporated herein by reference in its entirety for all purposes.

[0362] 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 these systems and capabilities.

[0363] Figure 19 is a diagram of a non-imaging analysis subsystem according to one embodiment. Referring to Figure 19, a non-imaging analysis subsystem 1300 of a multi-detection system is provided.

[0364] The analytical modalities of the non-imaging analytical subsystem 1300 can be absorbance, top-down fluorescence, and chemiluminescence. Xe flash bulb 13001 emits radiation in the 200 nm to 1000 nm range. Two stages 13002 and 13003 of a dual fluorescence excitation / absorption monochromator select a narrow bandpass of radiation. The radiation is guided toward the sample by fiber optic cables into either the absorbance channel via fiber 13030, the top fluorescence channel via 13005, or the bottom fluorescence channel via 13033. Because only one fiber operates at a time, there is no optical crosstalk between the various analytical modes. Absorbance is measured by silicon detector 13060 via lenses 13040 and 13050.

[0365] Top fluorescence excitation and emission pickup is via lens 13020, which can be moved up and down to accommodate various microplate and fluid levels. Bottom fluorescence is similarly achieved with lens 13055. Both top and bottom emission are directed by fiber optic cables to the first stage of emission dual monochromators 13010 and 13011 and then to photomultiplier tube 13012. Chemiluminescence fiber optic 13021 may be connected directly to the photomultiplier tube to provide measurement for very weak light by bypassing the monochromator.

[0366] The fluid injection subsystem 1100 provides researchers 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.

[0367] FIG. 20 illustrates an injection subsystem according to one embodiment.

[0368] Referring to Figure 20, an optional injection subsystem is provided. Injection subsystem 1100 may be disposed on top of the multi-detection device, as shown in Figure 21, with fluid lines 1112 and 1111 fed through septum accesses in 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 Figure 20, and delivered to the wells via injection needles 1102 and 1101.

[0369] Referring to Figure 19, environmental control can be deployed in a multi-detection system.

[0370] As shown in FIG. 19, a carriage 310 supports a microplate 300 (e.g., a sample carrier) and is positioned within an incubation chamber 320. This ensures that the microplate 300 is maintained at a desired temperature at all positions of the carriage 310 within 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 a chamber will be familiar to those skilled in the art from many multi-detection devices. A typical controlled temperature range can be from room temperature to 65°C.

[0371] FIG. 21 is a diagram illustrating a multi-detection system according to one embodiment.

[0372] For live cells, the temperature is typically 37°C, but additionally, control of the gas around the sample is required. This control is achieved by filling the entire housing 1910 of the device of FIG. 21 with the appropriate gas mixture. This design avoids attempting to contain the gas-controlled environment solely in the measurement chamber or an isolated 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 by using soft gasket materials around the user access door.

[0373] 22A-22C are diagrams illustrating a gas regulation subsystem according to one embodiment.

[0374] 22A-22C, an environmental control subsystem 2000 (e.g., a gas control subsystem) can be located external to the device. The environmental control subsystem 2000 allows a user to set the CO and / or O concentration levels (i.e., CO and / or O) in the chamber to higher CO and lower O levels than normal atmosphere. Gas sampling lines connect the environmental control subsystem 2000 to the interior of the device housing. Based on the composition of the gas sampled or extracted from the device via the sampling lines, a control system can adjust the flow rate of CO or N gas supplied to the device, for example, with the incoming gas dispersed using a small fan. This allows all gas sensors and valves to be located external to the main device, preserving the complexity and reliability of gas control in an external gas controller.

[0375] 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 microplate provides the user with the ability to perform long-term live-cell experiments.

[0376] 21, an external view of the entire device as implemented in the illustrated embodiment and the elements that are subject to user interaction with the device is shown. Carriage 310 presents itself to the user (shown on the right), and microplate 300 is placed on carriage 310, for example by the user or a robotic arm, and then positioned within the multi-detection system. Access to confocal cube 1530, widefield LED cube 1201, and widefield filter cube 1210, confocal disk module, and objective lens 1230 is through door 1905, at the front of the device, thus facilitating user access to more user-interchangeable elements at once.

[0377] According to certain embodiments, the objectives of the present disclosure (eg, objective 1230 or objective 2210) may be fluid immersion objectives.

[0378] 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 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 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 droplet of fluid (e.g., water or other fluid) is placed on the objective and held in place by the surface tension of the fluid. The objective is then brought close to the sample, and the droplet is sandwiched between the sample and the objective. In this way, light passing to and from the sample to the objective does not pass through air. The refractive index of the fluid, which is higher than that of air, results in an increased numerical aperture, which increases resolution and signal levels. According to an embodiment, the objective can be brought close to the sample, and then a droplet of fluid is placed on the objective.

[0379] 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 example 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.

[0380] With reference to FIGS. 25A and 25B , an immersion objective according to an embodiment of the present disclosure will now be described. 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. Additionally, 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, as shown in FIGS. 25A and 25B , the inlet port and the outlet port can be the same port 31. Referring to FIG. 25B , an excess portion 34 of the droplet of liquid can exit the sleeve 1332 through the port 31. In one exemplary embodiment, the sleeve 1332 can be formed of, for example, anodized aluminum, plastic, or other material.

[0381] According to an embodiment, referring to FIG. 26 , 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). 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. 26 , 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 into 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. 26 . 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 fits over the objective lens 1330 and can direct fluid to the top of the objective lens 1330, helping to hold the droplet 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. 26, the first reservoir 1338 and the second reservoir 1339 are shown as separate source and waste reservoirs, respectively. However, according to embodiments, instead of two separate reservoirs, a single reservoir can be provided and the fluid can be reused. Additionally, the pump can be multipurpose. For example, BioTek's C10 product 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.

[0382] 26, 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.

[0383] 27 , the objective lens coupling portion 1334 may include a kinematic connection portion 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 portion 1334A, and the objective lens turret 1232 may include at least one of the convex portion or the concave portion as a second portion of the kinematic connection portion 1334A corresponding to the first portion. The magnet 1334B may be provided on 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 magnets 1334B corresponding to each other and configured to connect to each other via magnetic force. In other embodiments, only one of the objective lens 1330 and the objective lens turret 1232 may include a magnet 1334B, which may be configured to connect to a magnetic material (e.g., a metal) included in the other of the objective lens 1330 and the objective lens turret 1232.

[0384] According to comparative embodiments, an objective lens can be threaded onto the objective lens turret. However, the use of a sleeve and tube with the objective lens can make it difficult to thread the objective lens onto the objective lens turret, at least in some embodiments. According to embodiments 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.

[0385] 28A-31C, the objective lens 1330 and the sleeve 1332 can have various configurations. According to an embodiment, the sleeve 1332 can also be referred to as a cap.

[0386] 27 is a diagram showing an objective lens coupling portion according to one embodiment; FIG. 28A is a perspective view showing an immersion objective lens according to a first embodiment; FIG. 28B is a top view showing an immersion objective lens according to the first embodiment; FIG. 28C is a first cross-sectional view taken along line AA in FIG. 28B, showing the immersion objective lens according to the first embodiment with spherical liquid provided thereon; FIG. 28D is a second cross-sectional view taken along line AA in FIG. 28B, showing the immersion objective lens according to the first embodiment with a microplate provided thereon; FIG. 29A is a top view showing an immersion objective lens according to a second embodiment; FIG. 29B is a first cross-sectional view taken along line BB in FIG. 29A, showing the immersion objective lens according to the second embodiment with spherical liquid provided thereon; and FIG. 29C is a second cross-sectional view taken along line BB in FIG. 29A, showing the immersion objective lens according to the second embodiment with a microplate provided thereon. Figure 30A shows an immersion objective according to a second embodiment, with a spherical liquid provided thereon; Figure 30B is a first cross-sectional view taken along line CC in Figure 30A, showing an immersion objective according to a third embodiment with a spherical liquid provided thereon; Figure 30C is a second cross-sectional view taken along line CC in Figure 30A, showing an immersion objective according to a third embodiment with a microplate provided thereon; Figure 31A is a top view of an immersion objective according to a fourth embodiment; Figure 31B is a first cross-sectional view taken along line DD in Figure 31A, showing an immersion objective according to a fourth embodiment with a spherical liquid provided thereon; and Figure 31C is a second cross-sectional view taken along line DD in Figure 31A, showing an immersion objective according to a fourth embodiment with a microplate provided thereon.

[0387] In the following description of Figures 28A to 31C, the same or similar features are given the same or similar reference numerals, and for the purpose of clarity, redundant descriptions of the same or similar features may be omitted.

[0388] Referring to Figures 28A to 28D, the upper surface 10A of the sleeve 1332A can be flush, i.e., coplanar, with the upper surface 11A of the objective lens 1330A, and the sleeve 1332A can be configured to clamp to the objective lens 1330A.

[0389] The sleeve 1332A can 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 can be provided separately or integrally with one another 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 can 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 can be provided separately 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 (i.e., the upper portion 50A, the middle portion 60A, and / or the lower portion 70A) can be subdivided into separate bodies and / or comprise additional bodies. According to an embodiment, any number of upper portion 50A, middle portion 60A, and lower portion 70A may be formed from aluminum.

[0390] According to an embodiment, any number of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be formed to exhibit substantial rotational symmetry about a central axis of the objective lens 1330A, which may be, for example, the optical axis of the objective lens 1330A.

[0391] The middle portion 60A may be provided above the lower portion 70A. The middle portion 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. 26 ). The inlet port 62 and the outlet port 63 may be provided on opposite sides of the sleeve 1332A, spaced apart 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 configured by a single port.

[0392] 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 around the central axis of the objective lens 1330A. According to embodiments, 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 embodiments, the tapered portion 64A may alternatively be referred to as a protruding portion.

[0393] According to an embodiment, the inlet port 62 may include a passage extending through the tapered portion 64A 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.

[0394] The upper portion 50A may include a main body. For example, the main body may include a sidewall 52A extending upward from the intermediate portion 60A and a top wall 53A extending radially inward from the sidewall 52A. The sidewall 52A and the top wall 53A may extend substantially at 90 degrees from each other. However, the angle is not limited thereto and may vary depending on the embodiment. The main body, including the sidewall 52A and the top wall 53A, may be formed to exhibit substantial rotational symmetry around the central axis of the objective lens 1330A.

[0395] The groove 84 can be formed by and between the upper portion 50A and the middle portion 60A. For example, the groove 84 can be defined by the inner surface of the top wall 52, the inner surface of the side wall 53, and the outer surface of the tapered portion 64A. According to embodiments, the groove 84 can be formed to exhibit substantial rotational symmetry about the central axis of the objective lens 1330A. The groove 84 can be configured to receive and contain excess liquid. According to embodiments, the groove 84 can be in communication with the outlet port 63 such that excess liquid in the groove 84 can exit the sleeve 1332A via a passageway in the outlet port 63 that is in communication with the groove 84.

[0396] 28C and 28D, at least the upper surface of the upper wall 53A can form an upper surface 10A of the sleeve 1332A that is flush with the upper lens surface 11A of the objective lens 1330A. According to an embodiment, the upper surface of the tapered portion 64 can also be flush with the upper lens surface 11A of the objective lens 1330A.

[0397] According to an embodiment, one or more O-rings 32 may be provided between the sleeve 1332A and the objective lens 1330A. For example, the O-ring 32 may be provided between the intermediate portion 60A and the objective lens 1330A. The O-ring 32 may be configured to seal the bottom side of the space in which liquid is received between the objective lens 1330A and the tapered portion 64A.

[0398] 28D, a microplate 80 holding a sample in at least one well 82 can be provided directly above the sleeve 1332A and objective lens 1330A. A droplet 90 of liquid on the lens of the objective lens can contact the bottom surface of the microplate 80 at a location directly below the well 82. The microplate 80 can correspond to, for example, the microplate 300 described in this disclosure, or other microplates.

[0399] Referring to Figures 29A to 29C, the upper surface 10B of the sleeve 1332B can be above the upper surface 11B of the objective lens 1330B, and the sleeve 1332B can be configured to clamp to the objective lens 1330B.

[0400] Sleeve 1332B can include, for example, upper portion 50B, middle portion 60B, and lower portion 70B.

[0401] The intermediate portion 60B can include a tapered portion 64B, and the upper portion 50B can include a body including a sidewall 52B and a top wall 53B. At least an upper surface of the top wall 53B can form the upper surface 10B of the sleeve 1332B above the upper lens surface 11B of the objective lens 1330B. According to an embodiment, the upper surface of the tapered portion 64B can also be above the upper lens surface 11B of the objective lens 1330B and flush with the upper surface of the top wall 53B.

[0402] Referring to Figures 30A to 30C, the upper surface 10C of the sleeve 1332C can be below the upper surface 11C of the objective lens 1330C, and the sleeve 1332C can be configured to clamp to the objective lens 1330C.

[0403] The sleeve 1332C can include, for example, an upper portion 50C, a middle portion 60C, and a lower portion 70C.

[0404] The intermediate portion 60C can include a tapered portion 64C, and the upper portion 50C can include a body including a sidewall 52C and a top wall 53C. At least an upper surface of the top wall 53C can form an upper surface 10C of the sleeve 1332C below the upper lens surface 11C of the objective lens 1330C. According to an embodiment, the upper surface of the tapered portion 64C can also be below the upper lens surface 11C of the objective lens 1330C and flush with the upper surface of the top wall 53C.

[0405] 33A-33C, an upper surface 10D of the sleeve 1332D can be flush with an upper lens surface 11D of the objective lens 1330D, and the sleeve 1332D can be configured to threadably engage with the objective lens 1330D.

[0406] According to one embodiment, the inner surface of the sleeve 1332D and the outer surface of the objective lens 1330D may include correspondingly engaging threads so 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.

[0407] The sleeve 1332D can include, for example, a first portion 60D and a second portion 50D.

[0408] The first portion 60D can include a tapered portion 64D, and the second portion 50D can include a body including a sidewall 52C and a top wall 53C. At least an upper surface of the top wall 53D can form an upper surface 10D of the sleeve 1332D that is flush with an upper surface 11D of the objective lens 1330D. According to an embodiment, the upper surface of the tapered portion 64D can also be flush with an upper surface 11D of the objective lens 1330D.

[0409] According to an embodiment, the inner surface of the first portion 60D may include threads.

[0410] 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.

[0411] According to embodiments of the present disclosure, various embodiments of confocal microscopy may alternatively or additionally be provided. For example, a laser point-scanning confocal system may be provided. Laser point-scanning confocal microscopy may involve 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 transmitted back through the optical system. This light may then be read point-by-point by a detector, which may be a photomultiplier tube (PMT), but may also be detected using other light measurement sensors. The signal from the sensor may 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 systems and therefore often not suitable for high-throughput applications or live-cell imaging. On the other hand, laser point-scanning confocal systems penetrate deeper into the sample and offer better axial and lateral resolution. Recent 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 motors that drive the system's scan mirrors.

[0412] According to embodiments, the confocal subsystem of the present disclosure can include both a laser point-scanning confocal and a spinning disk confocal. A spinning disk confocal system can be used for biological sample imaging and high-throughput applications, while a laser point-scanning confocal system can be used to penetrate deeper into a sample with improved resolution. Similar to using wide-field imaging or other measurement modalities to provide "hits," embodiments of the present disclosure can implement a spinning disk confocal system to rapidly scan a 3D sample and identify the location of several points of interest. A laser point-scanning system can then be used to capture more detailed images of the area of ​​interest. Both laser point-scanning confocal and spinning disk systems are commercially available as two separate devices. However, using two separate devices in this manner presents several challenges. For one, the cost of both the spinning disk and laser confocal microscopes can make implementing the workflow described above impractical. Additionally, there are technical challenges associated with relocating the area of ​​interest to the other microscope. By implementing both a laser point-scanning confocal system and a spinning disk system on the same device, "hits" can be found and the area of ​​interest can be scanned by switching optics without moving the stage. Finally, studying live cells also presents the problem of samples changing over time. Moving a sample to another device takes too long compared to the rate at which biological features change. Moving a sample to another device can result in the "hit" area of ​​interest changing and no longer being of interest.

[0413] Another advantage of having both laser point-scanning and spinning disk confocal systems in the same device is that the laser point-scanning confocal system can be utilized not for imaging but for targeting specific areas of a sample for photobleaching. The laser point-scanning confocal system and the specific control it provides over the XY scanning mirrors allow for very small, specific regions of a sample to be targeted with the laser. This can be a single spot or a block defined by a zigzag scan. Then, once photobleaching occurs, the device 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 F-actin diffusion in primary dendritic cells after a region 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).

[0414] 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.

[0415] FIG. 23 is a functional block diagram illustrating control of modalities in a device according to an embodiment.

[0416] The operation of the modalities may be controlled by a central control unit (e.g., a processor, CPU, microprocessor, etc.), which may also be referred to as a controller (e.g., controller 1000), according to an embodiment.

[0417] 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 90B, the monochromator module 90C, the imager module 90D, the external light source module 932, and the injection module 934.

[0418] The elements of the controlled sample environment 90A can provide temperature control 902 and gas control 904 as described above.

[0419] Sample selection and positioning 90B can be controlled through the use of motors 906 and 908 to position the sample in any X and Y direction.

[0420] The elements of the monochromator module 90C under control 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.

[0421] The elements of the imager module 90D under control can 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, and a spinning disk module and control 926 (e.g., selection and focusing), and a laser scanning confocal module control 927.

[0422] FIG. 24 is a flow diagram of a method for controlling a multi-detection system according to one exemplary embodiment.

[0423] Control of the device may be coordinated through the use of a controller, e.g., as discussed above with respect to Figure 23 and / or Figures 32A and 32B. Input to the device (step S1805) may be achieved 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.

[0424] 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.

[0425] The inputs may be user inputs such as settings and parameters for implementing control of the device.

[0426] In response to receiving input, control of the device can be effected through various elements of the device, e.g., as discussed above with respect to Figure 23 and / or Figures 32A and 32B. For example, in response to receiving user input, the device 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 device (step S1850).

[0427] Although the controls are presented as shown in Figure 24, elements can be controlled individually in any order and control of all elements is not required. Thus, multiple modalities of the device can be controlled in a single assay.

[0428] 24, and other functions described herein that may be performed by the controller, may be implemented through the execution of a processing unit (e.g., a 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 performed by the device.

[0429] Referring to FIG. 33 , 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. 33 shows an example of a user interface when the device 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 complete or the checkbox for element 2302 is deselected. Element 2303 is a drop-down list for EM wavelength selection. While FIG. 33 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. While FIG. 33 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 selection between various modes of instruction. FIG. 33 illustrates selection between modalities, where the system includes spinning disk, laser scanning, and wide-field modalities. According to embodiments, the modalities listed in element 2305 can depend on the modalities present in the system. For example, the system can have any combination of the above-mentioned modalities (and / or additional modalities), or can have only a single modality. If only a single modality is provided, element 2305 can be absent. According to embodiments, elements 2301, 2302, 2303, 2304, and 2305 are not limited to being drop-down menus and selection boxes, and can present options for selection in any manner known to those skilled in the art.

[0430] According to embodiments, the interface may include a display element that allows a user to select multiple modalities to be automatically performed in sequence. For example, a controller may be configured to control a sequence to be automatically performed based on one or more inputs from a user via the interface. The sequence may include any order of modality operations, including those described in this disclosure. For example, an operation using a spinning disk or wide-field imaging system may be followed by an operation using a laser point-scanning confocal system.

[0431] 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.

[0432] For example, according to one embodiment, 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 toward a sample and receives emitted light from the sample, a third optical device that transmits the narrow wavelength band of the emission light, and a detector that converts the narrow wavelength band of the emission light 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 light.

[0433] [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 can include measuring cellular metabolic function, bioenergetic balance, bioenergetic capacity, and bioenergetic work, such as measuring O, CO, and pH using a sensing subsystem. The methods can also include visually observing sample characteristics, such as cell growth, cell health, cell microenvironment, morphological changes, ultrastructural changes, and marker expression, using an optical module with an automated cell imaging reader, such as the 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. This method may be used in conjunction with U.S. Patent 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. Patent Nos. This may include detecting attachment, ultrastructural changes, growth, morphological changes, and cell-cell interactions by impedance measurements using sensing systems or devices such as those described in U.S. Patent Application Publication No. 2018 / 0246019 and WO 2021 / 202264 (each of which is incorporated herein by reference in its entirety for all purposes).

[0434] 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, the brightfield and fluorescence detection optical modules of the xCELLigence eSight are exemplary optical modules that provide live-cell imaging during impedance measurements, as described in U.S. Patent Application Publication No. 2021 / 0301245 (incorporated by reference in its entirety for all purposes).

[0435] It will be appreciated that the successive analyses can be performed by further devices performing different measurements on 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), etc.

[0436] [Application] The systems, consumables, and methods described herein may have a variety of applications. Exemplary applications are described as follows:

[0437] [Cell migration / adhesion] In one aspect, the systems, consumables, and methods described herein are used to analyze cell migration and / or adhesion.

[0438] Cancer cell metastasis and invasion pose a clinical challenge in cancer treatment. Cell migration is typically a bioenergetic-intensive process, and obtaining simultaneous quantitative measurements correlating cell migration / invasion and bioenergetic metabolic activity could open new frontiers for therapeutic development. By combining impedance measurements as a surrogate measure of cell migration with OCR / PER measurements, modulators of cellular metabolism that induce inhibition of cell migration without affecting cell viability can be tested. These measurements can be further combined with fluorescent biosensor imaging of sensors for signaling cascades.

[0439] A similar procedure can be applied to study cell adhesion by coating plates with different extracellular matrices (ECMs) and combining changes in impedance, which represent cell adhesion, with changes in cell metabolism.

[0440] [Stem cell differentiation] In one aspect, the systems, consumables, and methods described herein are used to analyze stem cell differentiation.

[0441] Stem cell differentiation is a lengthy process that can take weeks to months and involves a change in cell phenotype from a proliferative / undifferentiated state to a specialized state. Stem cell differentiation is also accompanied by significant changes in cell morphology and metabolic activity, which must be controlled in order to produce specialized cells with the correct phenotype for direct use as disease models for therapeutic development or as therapeutic agents in cell and gene therapy for various diseases, such as tissue regeneration.

[0442] Simultaneous monitoring of culture environment conditions, changes in cell morphology through impedance measurements, and metabolic activity allows for optimization of cell model development to determine important cellular attributes for stem cell-derived therapies. [Example]

[0443] The embodiments can be further understood with reference to the following examples, which are intended to serve as illustrations and not limitations.

[0444] Example 1: Exemplary Protocol Cells are seeded into assay wells of a multi-well microplate at 50%-90% confluency. To maximize sensitivity, the suspension cells are allowed to adhere to the bottom of the wells. For this exemplary protocol, a 96-well sample carrier constructed and configured to fit the device is used. However, the multi-well sample carrier 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 other numbers of wells. The temperature of the cell suspension is controlled.

[0445] The device lowers a sensor probe into the assay well. The sensor is positioned 200 microns above the bottom of the well, creating an approximately 3 microliter transient microchamber (also referred to herein as a "measurement chamber"). As oxygen and pH levels change, the changes are measured by the sensor. Measurements are typically taken for a predetermined period of time, typically 1 to 5 minutes, e.g., 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.

[0446] The sensor cartridge also contains ports (four per well) that allow injection of modulators (target analytes) into the cell wells during the assay. When specified by the device'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 rate calculations are performed automatically.

[0447] An exemplary protocol was performed 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 system described herein. This testing was also performed on a comparison system with conventional temperature control, signal processing, and motion actuator motor components. The results are presented in the graphs of Figures 10A-10D.

[0448] The graphs in Figure 10A show OCR measurements over assay time measured using a system disclosed herein. The graphs in Figure 10B show OCR measurements over assay time measured using a comparative system. The graphs in Figure 10C show ECAR measurements over assay time measured using a system disclosed herein. The graphs in Figure 10D show ECAR measurements over assay time measured using a comparative system.

[0449] An exemplary protocol was also performed using A549 cells (human lung cancer cells) and 5 mM metformin as a targeting agent. OCR data was measured using the system described herein and a comparative system. The results are presented in the graphs of Figures 11A and 11B.

[0450] The graphs in Figure 11A and 11B show OCR measurements over assay time measured using a system as disclosed herein and a comparative system, respectively.

[0451] Thus, a system 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 comparative systems, 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 system's performance in sensing target analytes and cellular biological characteristics.

[0452] Example 2: Evaporation test protocol using water samples Six assays were performed in the system disclosed herein using known volumes of water in multi-well sample carriers with a modified protocol configured to perform four measurements per hour for six hours. 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 were collected on the test plate immediately after each assay. The standard curve was used to calculate the volume of water in each well of the test plate, and the amount of water volume lost due to evaporation during the six-hour assay was estimated. Results were calculated as a percentage of the total volume lost. The average evaporation for each assay is presented in the table in Figure 12.

[0453] 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. Therefore, the volume of sample fluid lost due to evaporation was small.

[0454] Without wishing to be bound by theory, it is believed that improving the temperature uniformity across the sample within the controlled temperature zones can reduce evaporation of the sample fluid and improve both the system's performance in sensing target analytes and cellular biological characteristics.

[0455] Example 3: Long-term behavior in multimodal analysis When the device is configured for long-term analysis of cell cultures across one or more analysis modes, various environmental and sampling control elements within the device are activated to maintain consistent growth conditions over time and measurement conditions for sensors used to monitor the growth conditions.

[0456] In various embodiments, the extended period can be set by an operator to span at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc., up to 24 hours, 48 ​​hours, 72 hours, etc., in order to analyze cell cultures and other samples for a longer period than provided by previous analytical devices without requiring human intervention to maintain growth conditions for that period.

[0457] The device includes a sensing system and a stage contained within a cavity of the device. The stage is configured to receive a sample carrier having a plurality of wells defined in a first surface thereof. In some embodiments, the stage is connected to a motion actuator assembly that moves the stage relative to the sensing system on one or more of an x-axis, a z-axis, and a y-axis. Additionally or alternatively, the motion actuator assembly can move the sensing system relative to the stage on one or more of an x-axis, a z-axis, and a y-axis, which can include rotation in a yaw, pitch, or roll orientation. In a multimodal analysis device, the motion actuator assembly can also move the stage and / or a sample carrier or substrate carrying a sample between various devices or sensors for sequential analysis or access by those devices or sensors.

[0458] For example, the movement actuator assembly can move the sample to a first position for access by a flux detector, a second position for access by an imaging module, a third position for access by an electrical measurement module, etc. In various embodiments, the first position is provided for access by an image capture element and the second position is provided for access by an impedance element. In various embodiments, for example, as shown in FIG. 48 , a first position is provided in the device 5000 for access by an image capture element 5080 and an impedance element 5050 (coupled to the sample carrier 5040 via an electrical interface 5051), and a second position is provided for access by a flux detector 5070. In various embodiments, the fluid handler 5030 can be accessed in the third position, or in one or both of the first and second positions.

[0459] 48, various accessory elements for the image capture element 5080 and the flux detector 5070 are provided in alignment at first and second positions, respectively. For example, excitation sources 5090a and 5090b, and optical conditioning elements 5095a-5095f (e.g., mirrors, lenses, filters, light paths, etc.) are provided between the image capture element 5080 and the flux detector 5070 and the sample carrier 5040 (if in their respective positions) to optically communicate the wells with their respective measurement elements. Additionally, the flux cartridge 5060 can be pre-loaded with various chemical compounds, growth media, and other compounds that are injected into or exchanged with the wells in the sample carrier 5040.

[0460] In various embodiments, the flux cartridge 5060 is movable relative to the sample carrier 5040 (or vice versa) on an axis that is substantially perpendicular to an axis along which the sample carrier 5040 moves between a first position and a second position, or to a plane that intersects each of the multiple wells on the sample carrier 5040. The flux cartridge 5060 comprises a plurality of heads, each corresponding to a given well in the sample carrier 5040, the heads comprising a surface proximal to a surface of the sample carrier 5040 in which the wells are defined, such that when placed in contact with the sample carrier 5040, the heads define closed reaction chambers. The closed reaction chambers are configured to maintain a seal that limits the volume of liquid contained in each sample and / or reduces the rate of volumetric evaporation from the reaction chamber over a duration.

[0461] The sensing system comprises an array of sensor units configured to generate an electrical signal proportional to an observed analyte in a sample well. For example, a first sensor in the sensor array can monitor a first analyte proportional to the gaseous O content in a given well over time to generate a first signal, and a second sensor in the sensor array can monitor a second analyte proportional to the pH value in a given well over time to generate a second signal. The sensor units in the array of sensor units are positioned (via a movement actuator assembly) to correspond to corresponding wells on the sample carrier and analyze the contents thereof for control and monitoring of the samples held in the wells.

[0462] A liquid handling system is included in the device for dispensing various substances into the samples in each well of the sample carrier. In various embodiments, the liquid handling system is provided via a cartridge that is insertable into (and removable from) the device without affecting the atmosphere of the cavity. The cartridge can include reservoirs for various substances to be provided to the wells, including water, aqueous solutions (e.g., aqueous solutions of candidate compounds / substances or other agents), dyes, cell growth media, cell culture media, N2, O2, CO2, etc. Exemplary components and features that may be used in the cartridge are further described, for example, in U.S. Pat. No. 9,170,255, entitled "Cell analysis device and method," which is incorporated herein by reference in its entirety for all purposes.

[0463] Sampling control elements are included in the device to control one or more properties measured by the sensing system for samples in each well of the sample carrier over time. The sampling control elements control the properties of any given sample so that the properties are within a predetermined amount of any other sample in another well of the sample carrier. For example, the sampling control elements can include a sample temperature environmental control element configured to control the temperature of the sample (e.g., within + / - X degrees), a gas control element configured to control the gas content of at least one of the O2, CO2, and N2 content of the sample (e.g., within + / - X parts per million (ppm)), and a humidity control element configured to control the humidity of the sample (e.g., within + / - X% relative humidity). In various embodiments, the sample temperature environmental control element is a heater.

[0464] In addition to controlling relative properties between various wells in parallel (e.g., a first well versus a second well at a first time), the sampling control element can also control the properties of a given well over time (e.g., a first well at a first time and a second time) so that the property being monitored in the well remains within a controlled range of values ​​over the analysis period. Various consumables provided by the fluid handler and / or cartridge allow for the addition of material to compensate for lost material (e.g., due to evaporation, sublimation, consumption by the sample (e.g., cellular respiration), or diffusion to the environment external to the well). Material sources (e.g., cartridges) can be replaced throughout time to account for material consumption and provide a reserve of unused material to supply to wells as needed to maintain properties within the wells, or to supply additional material for analysis (e.g., cell growth compounds, pharmaceuticals under test, etc.). Thus, the described devices can maintain consistent growth conditions across multiple samples held in corresponding multiple wells simultaneously in parallel and chronologically (e.g., a first well through an nth well at each of a first through nth time).

[0465] At different times during an extended analysis period, the device can observe different properties of the sample under observation. In various embodiments, the device includes one or both of an image capture element, such as an optical module described herein, and an impedance element, such as an electrical measurement module described herein, which can operate at different locations within the device's cavity where a motion actuator assembly positions the sample carrier at different times. Each of the various observation modules can be located within a different cavity or sub-cavity within the device. For example, as shown in FIG. 48 , an image capture element 5080 and a flux detector 5070 are both disposed within the cavity 5010 of the device 5000, but are separated into different sub-cavities by a divider 5020. The divider 5020 can include various vents and atmosphere or environmental controls to provide different temperature, humidity, or airflow characteristics to different portions of the cavity 5010 at different times.

[0466] The image capture element comprises one or more cameras and various camera attachment devices (e.g., mirrors, lenses, light sources) to enable it to capture images of the samples (or sample features) in each well of the sample carrier. These images are captured from the underside of the sample carrier (e.g., opposite the surface in which the wells are defined) through a transparent or semi-transparent window. If the wells comprise electrodes (e.g., for electrical stimulation of the sample and / or impedance measurements of the sample), the electrodes are positioned to leave a gap of a predetermined size between them that defines a window through which imaging is performed. In various embodiments, the image capture element can be configured to capture and process images from each well of the sample carrier individually (allowing the operator to select specific wells or in a specific order, or to forgo imaging) or batches of some or all of the wells in parallel.

[0467] The impedance element includes an electrode surface configured to measure impedance changes resulting from the deposition of a sample in each well of the sample carrier, stimulate the sample in each well of the sample carrier with an electrical signal, or both. In various embodiments, the electrode surface is located at the base of the sample carrier (e.g., opposite the side on which the wells are defined), and the electrode surface includes a non-conductive carrier. When positioned to interact with the sample carrier, the impedance element includes multiple electrode arrays positioned in contact with the sample carrier. Each of these electrode arrays includes at least two electrode structures positioned on the same plane (e.g., a shared plane) and having substantially the same surface area as each other. The electrode structures interface (e.g., are in electrical communication) with corresponding members of multiple connection pads located on the sample carrier. Thus, when in electrical communication with the sample carrier, the impedance meter, impedance analyzer, or impedance measurement circuitry of the impedance element can detect changes in electrical impedance between the electrode structures resulting from changes in the sample. Similarly, when in electrical communication with the sample carrier, the impedance element's voltage or current source can stimulate the sample with an electrical signal.

[0468] The operation of each of the elements and modules of the device is coordinated by a controller, which can be any type of computing device (e.g., FPGA array, microcontroller, processor and associated memory, ASIC, etc.) operably connected to the various elements and modules. The controller is configured to control one or more of the temperature, humidity, and gas content of each well of the sample carrier over time via sampling control elements, acquire data corresponding to first and second signals for each analysis over at least two time points spanning the time via various sensing systems (including image capture elements and impedance elements), and condition the first and second signals. In various embodiments, conditioning the first and second signals includes at least one of amplifying, filtering, time-shifting, frequency-shifting, and digitizing one or more of the signals at one or more times.

[0469] FIG. 49 shows a schematic system diagram of one embodiment of a system 5100 for analyzing live cells, particularly for measuring extracellular flux, impedance, and imaging for long-term measurements of a cell sample, including simultaneous measurement of one or more combinations of extracellular flux, impedance, and imaging. The system incorporates a sample carrier 300 configured to interface with a sensor cartridge 5102. The sample carrier 300, sometimes referred to as a well plate, comprises an array of wells 200 for individually holding cell samples and an impedance and / or stimulator component or array thereof. One or more of the wells comprises an electrode array for acquiring impedance measurements across the well bottom and a window in the well bottom for light transmission and imaging through the well bottom via an optical subsystem 5120, such as, for example, an inverted microscope. The optical subsystem can incorporate the functionality of any number of optical configurations and / or imaging modalities, such as wide-field fluorescence microscopy, wide-field bright-field microscopy, or confocal fluorescence microscopy, as well as any of the imaging systems described above. One exemplary configuration of a windowed sample carrier having impedance and optical measurement capabilities is described, for example, in PCT Patent Application No. 2021 / 202264, entitled "SYSTEMS AND METHODS FOR ELECTRONICALLY AND OPTICALLY MONITORING BIOLOGICAL SAMPLES," which is incorporated herein by reference in its entirety for all purposes.

[0470] The sample carrier 300 can be positioned on a carriage 310 and heated stage 360 ​​that can move to interact with a fluid handling system 5130, which can include a fluid injection manifold 5130 and a heater / cooler 5140 for a fluid temperature controller (see FIG. 8). In one embodiment, the manifold is configured to grip the cartridge and move the stage up and down. A substance / media source 5170 is coupled to the manifold 5130 for delivery of fluid to the sample carrier 300.

[0471] Optical components such as optical filters 5160a, 5160b, focusing optics 5162a, 5162b, excitation source(s) 5166, and detector 5164 are positioned above the sample carrier 300 and cartridge 5102. The light manifold 5166 optics can be a fiber optic array, or a glass or plastic slug, with each fiber to an excitation LED or multiplexing one excitation source to many fibers, and can be scanned by the optical components described above.

[0472] Figure 50 shows an exploded view of the sample carrier 300 and cartridge 5102, which comprises a plurality of spines 5104, each having an analyte sensor 5106 at its distal tip, the spines sized and shaped to interface with the wells 200 to form individual microchambers. An electrode interface / impedance reader 5108 is electrically coupled to the sample carrier for receiving and delivering electrical signals from and to the impedance electrodes in the wells 200 (see Figure 53).

[0473] Figure 49 shows a schematic system diagram of one embodiment of a system 5100 for analyzing live cells, Figure 50 shows an exploded view of the sample carrier 300 and cartridge 5102, and Figures 51 and 52 show top and side views, respectively, of the stage 360 ​​without the sample carrier 300. The stage 360 ​​may include a heater / cooler and may include peripheral features 5112 for holding and aligning the sample carrier 300. One or more environmental property sensors 5114 (e.g., temperature, gas, humidity, etc.) may be included in the stage.

[0474] Figure 53 shows a top perspective view of the bottom of the well 200 of the sample carrier 300. The well bottom can include standoffs which can be in the form of ridges 5118, ledges, or other engagement features that interface with the spine 5104 of the sensor cartridge, thereby forming a stop to allow the spine to rest on the ridge at a specified distance to define the microchamber. Figure 54 shows a window 5112 positioned between electrode elements 5124 on the surface of the well, which allows imaging of the well bottom.

[0475] Example 4: 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 compound / substance candidates and post-market compound / substance withdrawal (Wallace, KB, 2008, "Mitochondrial off targets of drug therapy", Trends Pharmacol. Sci. 29, 361-366). Among the methods used to assess compound / substance-induced mitochondrial toxicity in compound / substance discovery and preclinical safety, direct measurement of mitochondrial oxygen consumption using Agilent's Seahorse XF technology has been 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, pp. 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", a,c Toxicology in Vitro 52 (2018) 374-383).

[0476] Thus, disclosed herein is a standardized XF solution that enables the evaluation 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, these improvements are utilized to detect compound / substance-induced mitochondrial dysfunction using OCR measurements.

[0477] 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.

[0478] Based on the response of test compounds in basal oligomycin and / or FCCP OCR compared to appropriate controls, the XF Mito Tox assay can dist...

Claims

1. a sensing system comprising an array of sensor units configured to generate a first signal in response to a first analyte over time and to generate a second signal in response to a second analyte over said time, each sensor unit of the array of sensor units 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 at least one of the stage and the sensing system relative to one another along one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system for dispensing a substance into at least one well of said sample carrier; a sample control element configured to control a characteristic of a sample in at least one well of said sample carrier to be within a predetermined amount of another sample in another well of said sample carrier over said extended period of time; a controller operably connected to the sensing system and the sample control element, the controller being configured to control one or more of a temperature, humidity, and gas content of an environment surrounding the sample carrier over the extended period of time, and to acquire data corresponding to the first signal and the second signal for at least two points across the extended period of time; A device having long-term measurement capability.

2. The device of claim 1, wherein the long-term measurement is performed in a microchamber having a reduced volume of 3 microliters or less, which is created by moving the sensor units of the array of sensor units down a predetermined position into corresponding wells in the sample carrier.

3. The device of claim 1 , wherein the long-term measurements are performed discontinuously between a single modality selected from the group consisting of flux measurements, impedance measurements, and imaging.

4. The device of claim 1 , wherein the long-term measurements are performed non-continuously between at least two modalities selected from the group consisting of flux measurements, impedance measurements, and imaging.

5. 10. The device of claim 1, wherein the control element controls a sample environment to maintain an environmental parameter at a target level in an associated well within the sample carrier.

6. The device of claim 5 , wherein the target level for the environmental parameter is programmatically changed over the time of the long-term measurement.

7. 10. The device of claim 1, wherein the control element controls the sample environment to achieve a target cellular microenvironment for the biological model in the sample via at least one of direct cellular, intracellular, pericellular, and proximal measurements of sample parameters.

8. The device of claim 7 , wherein the cellular microenvironment is controlled on a sample-by-sample basis.

9. The device of claim 5 , wherein the target level for the sample parameter is programmably varied over the time period of the long-term measurement.

10. 10. The device of claim 1, further comprising a ventilation system configured to alter the headspace gas composition in the cell microenvironment.

11. The sample control element comprises: a sample temperature control element configured to control the temperature of the sample; A sample environment control element, ２ , CO ２ , and N ２ a sample environment control element comprising one or both of a gas control element configured to control the gas content of one or more of the contents, or a humidity control element configured to control the humidity of the environment; The device of claim 1 .

12. The device of claim 11 , wherein the sample control element comprises a heater.

13. The first signal is a signal representing the O ２ 10. The device of claim 1, wherein the first signal is a measurement of the first analyte proportional to its content and the second signal is a measurement of the second analyte proportional to the pH value in the given well.

14. The device of claim 1 , wherein the first signal is measured in parallel with the second signal.

15. 10. The device of claim 1, wherein the extended period of time is between 6 hours and 72 hours, between 6 hours and 170 hours, between 6 hours and 168 hours, between 12 hours and 60 hours, between 24 hours and 48 hours, between 12 hours and 36 hours, between 24 hours and 48 hours, between 36 hours and 60 hours, between 6 hours and 60 hours, between 6 hours and 48 hours, between 6 hours and 36 hours, between 6 hours and 24 hours, between 6 hours and 12 hours, between 60 hours and 72 hours, between 48 hours and 72 hours, between 36 hours and 72 hours, between 24 hours and 72 hours, between 12 hours and 72 hours, between 12 hours and 24 hours, between 24 hours and 36 hours, between 36 hours and 48 hours, or between 48 hours and 60 hours.

16. an image capture element configured to image a sample or a feature of the sample in each well of a plurality of wells defined in the sample carrier through an opening or window; The device of claim 1 , wherein the image capture element is configured to capture and process at least one image from each well of the sample carrier.

17. The sample carrier comprises:

2. The device of claim 1, comprising a plurality of wells configured to hold a predetermined amount of sample, each well of the plurality of wells comprising the opening or window that allows an image capture element to capture at least one image from each well of the sample carrier.

18. an electrode surface comprising a non-conductive carrier on the base of the sample carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a single plane and having substantially the same surface area; a plurality of connection pads located on the sample carrier, each connection pad in electrical communication with at least one of the electrode structures in each well of the plurality of wells; The device of claim 1 further comprising:

19. 2. The device of claim 1, wherein a plurality of wells configured to hold a predetermined amount of sample are positioned above the plurality of electrode arrays, and each well of the plurality of wells comprises the opening or window that enables the image capture element to capture at least one image from each well of the sample carrier.

20. an impedance measuring device configured to measure impedance changes due to deposition of a sample in each well of the sample carrier or to stimulate the sample in each well of the sample carrier with an electrical signal, the electrode surface being at the base of the sample carrier and comprising a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, each electrode array comprising at least two electrode structures positioned on a common plane and having substantially the same surface area; a plurality of connection pads located on the sample carrier, each connection pad being in electrical communication with at least one of the electrode structures; Further provided with the impedance element detects a change in electrical impedance between the electrode structures or stimulates the sample with an electrical signal; The impedance element detects a change in electrical impedance between the electrode structures or a stimulation output of the sample with an electrical signal. The device of claim 1 .

21. 21. The device of claim 20, wherein a plurality of wells configured to hold a predetermined amount of sample are positioned above the plurality of electrode arrays, each well of the plurality of wells comprising the opening or window that enables the image capture element to capture at least one image from each well of the sample carrier.

Images