Multi-modal system and method for analyzing cells

Abstract

Extended period cell analysis is provided via a device comprising: a chamber configured to receive a sample carrier, the sample carrier comprising a plurality of wells having electrodes in electrical communication with an impedance meter; and a cartridge comprising a compound and at least one delivery port for the compound; a camera configured to acquire an image of the cell culture in each well via an associated window in the well when the sample carrier is positioned in the first position; a fluid processor in communication with the sample carrier and the cartridge when the sample carrier is positioned in the second position, the fluid processor configured to deliver the compound from the cartridge to a given well based on an impedance measurement of the sample in the given well; and a moving stage configured to move the sample carrier between a first position and a second position.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 483,218, filed on February 3, 2023, entitled “Multimodal Systems and Methods for Analyzing Cells,” which is incorporated herein by reference in its entirety. Technical Field

[0003] Aspects and embodiments disclosed herein generally relate to measurements of living cell samples. Background Art

[0004] Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) are key indicators of mitochondrial respiration and glycolysis. These measurements provide a systems-level perspective on cellular metabolic function in cultured cells and isolated samples. In addition, impedance measurements of living cells are widely recognized as a label-free, non-invasive, and quantitative analytical method for assessing cellular status.

[0005] Therefore, there is a need to develop new systems and methods to perform long-term, metabolic and / or impedance-based measurements of living cells and to image these cells in real time. Summary of the Invention

[0006] According to one aspect, a device having an extended period measurement capability is provided, the device comprising: a sensing system comprising an array of sensor cells configured to generate a first signal in response to a first analyte over an extended period and a second signal in response to a second analyte over an extended period, each sensor cell of the array of sensor cells being positioned to correspond to a corresponding well on a sample carrier comprising an array of wells; a stage configured to receive the sample carrier; a motion actuator assembly configured to position the stage and at least one of the sensing system relative to each other on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system that dispenses a substance into at least one well of the sample carrier; a sample control element configured to control a characteristic of a sample within at least one well of the sample carrier to within a predetermined amount of another sample within another well of the sample carrier over an extended period; 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 the environment surrounding the sample carrier over an extended period; and acquire data corresponding to the first signal and the second signal for at least two time points spanning the extended period.

[0007] In some aspects, the extended period measurement is performed in a microchamber having a reduced volume of no greater than 3 microliters, the reduced volume being generated by moving sensor cells of the sensor cell array down predetermined positions into corresponding wells in the sample carrier.

[0008] In some aspects, the extended period measurements are performed in a non-continuous manner between a single modality selected from the group consisting of: flux measurement, impedance measurement, and imaging.

[0009] In some aspects, the extended period measurements are performed in a non-continuous manner between at least two modalities selected from the group consisting of: flux measurement, impedance measurement, and imaging.

[0010] In some aspects, the control element controls the sample environment to maintain environmental parameters at target levels for associated wells in the sample carrier.

[0011] In some aspects, the target level for the environmental parameter is programmatically changed over time measured over an extended period of time.

[0012] In some aspects, the control element controls the sample environment via at least one of direct cellular / intracellular / pericellular / proximity measurement of a sample parameter to achieve a target cellular microenvironment of the biological model in the sample.

[0013] In some aspects, the cellular microenvironment is controlled on a per sample basis.

[0014] In some aspects, the target level of the sample parameter is programmatically changed over the extended period of time measured.

[0015] In some aspects, the device further comprises a ventilation system configured to alter the composition of the headspace gas in the cellular microenvironment.

[0016] In some aspects, the sample control element includes one or both of the following: a sample temperature control element configured to control the temperature of the sample; or a sample environment control element including one or both of the following: a gas control element configured to control the content of one or more gases of O2, CO2 and N2 in the sample, or a humidity control element configured to control the humidity of the environment.

[0017] In some aspects, the sample control element comprises a heater.

[0018] In some aspects, the first signal measures a first analyte that is proportional to the O 2 content in a given well, and the second signal measures a second analyte that is proportional to the pH value in the given well.

[0019] In some aspects, the first signal is measured in parallel with the second signal.

[0020] In some aspects, the extended period 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.

[0021] In some aspects, the device further comprises an image acquisition element configured to image a sample or sample feature within each of a plurality of wells defined in the sample carrier through an opening or window; wherein the image acquisition element is configured to acquire and process at least one image from each well of the sample carrier.

[0022] In some aspects, the sample carrier comprises a plurality of wells configured to hold a predetermined amount of sample, wherein each well of the plurality of wells comprises an opening or window allowing the image capture element to capture at least one image from each well of the sample carrier.

[0023] In some aspects, the device further comprises: an electrode surface comprising a non-conductive carrier positioned at the bottom of the sample carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on a single plane and having substantially the same surface area; and a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures in each of the plurality of holes.

[0024] In some aspects, a plurality of wells configured to hold a predetermined amount of sample are positioned above the plurality of electrode arrays, wherein each well of the plurality of wells includes an opening or window allowing an image capture element to capture at least one image from each well of the sample carrier.

[0025] In some aspects, the device also includes: an impedance measuring device configured to: measure impedance changes caused by sample attachment within each hole of the sample carrier; or stimulate the sample within each hole of the sample carrier through an electrical signal, wherein the electrode surface is located at the bottom of the sample carrier, and wherein the electrode surface includes a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array includes at least two electrode structures positioned on a common plane and having substantially the same surface area; a plurality of connecting pads located on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures; wherein the impedance element detects electrical impedance changes between or among the electrode structures or stimulates the sample through the electrical signal; and wherein the impedance element detects electrical impedance changes between or among the electrode structures, or the excitation output of the sample from the electrical signal.

[0026] In some aspects, a plurality of wells configured to hold a predetermined amount of sample are positioned above the plurality of electrode arrays, wherein each well of the plurality of wells includes an opening or window allowing an image capture element to capture at least one image from each well of the sample carrier.

[0027] According to one aspect, a device having extended period measurement capability is provided, comprising: a sensing system comprising an array of sensor cells, the array of sensor cells 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 the extended period, each sensor cell of the array of sensor cells being positioned to correspond to a respective well on a sample carrier comprising an array of wells; a stage configured to receive the sample carrier; a motion actuator assembly configured to position the stage and the sensing system relative to each other on one or more of an x-axis, a z-axis, and a y-axis; one or both of the system; a liquid handling system that distributes reagents to the sample within each well of the sample carrier; a sample control element that includes one or both of the following: a sample temperature control element that is configured to control the temperature within each well of the sample carrier to within a predetermined temperature of each other; or a sample environment control element that includes one or both of the following: a gas control element that is configured to control the O2, CO2, and N2 content of each well of the sample carrier to within a predetermined ratio of each other; or a humidity control element that is configured to control the humidity within each well of the sample carrier to within a predetermined amount of each other; image a collection element configured to image the sample or a feature of the sample within each hole of the sample carrier through the opening, wherein the image collection element is configured to collect at least one image from each hole of the sample carrier; an impedance element comprising an electrode surface, the electrode surface configured to measure an impedance change caused by sample attachment or to excite the sample by an electrical signal within each hole of the sample carrier, wherein the electrode surface is located at the bottom of the sample carrier, and wherein the electrode surface comprises a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on a common plane and having substantially the same surface area; a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures; and wherein the impedance element detects an electrical impedance change between the electrode structures or excites the sample by an electrical signal; and a signal processing module operably connected to the sensing system, the signal processing module being configured to receive and adjust the first signal and the second signal from the sensor unit, and to process at least one image from the image collection element, and to measure an electrical impedance change from the impedance element between the electrode structures or an excitation output of the sample from the electrical signal.

[0028] According to one aspect, a device having an extended period measurement capability is provided, comprising: a sensing system comprising an array of sensor cells 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 the extended period, each sensor cell of the array of sensor cells being positioned to correspond to a respective well on a sample carrier comprising an array of wells; a stage configured to receive the sample carrier; a motion actuator assembly configured to position the stage and at least one of the sensing system relative to each other on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system configured to dispense a reagent into a sample within each well of the sample carrier; a sample control element comprising one or both of: a sample temperature control element configured to control the temperature within each well of the sample carrier to within a predetermined temperature of each other; or a sample environment control element comprising one or both of: a gas control element configured to control the O2, CO2, and N2 content of each well of the sample carrier to within a predetermined ratio of each other. rate; and a humidity control element configured to control the humidity within each hole of the sample carrier to within a predetermined amount of each other; an impedance element comprising an electrode surface configured to measure an impedance change caused by sample attachment or to excite the sample by an electrical signal within each hole of the sample carrier, wherein the electrode surface is located at a bottom of the sample carrier, and wherein the electrode surface comprises a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on the same plane and having substantially the same surface area; a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures; and wherein the impedance element detects an electrical impedance change between the electrode structures or excites the sample held in the sample carrier by the electrical signal; and a signal processing module operably connected to the sensing system, the signal processing module configured to receive and condition the first signal and the second signal from the sensor unit, and measure the electrical impedance change from the impedance element between the electrode structures or the excitation output of the sample from the electrical signal.

[0029] In some aspects, the apparatus further comprises an image capture element configured to image the sample or a feature of the sample within each well of the sample carrier through the opening, wherein the image capture element is configured to capture at least one image from each well of the sample carrier.

[0030] According to one aspect, a device having an extended period measurement capability is provided, comprising: a sensing system comprising an array of sensor cells 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 the extended period, each sensor cell of the array of sensor cells being positioned to correspond to a respective 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 each other on one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system configured to dispense a reagent into a sample within each well of the sample carrier; a sample control element comprising one or both of the following: a sample temperature control element configured for controlling the temperature within each hole of the sample carrier to be within a predetermined temperature of each other; or a sample environment control element, which includes one or both of the following: a gas control element, configured to control the O2, CO2 and N2 content of each hole of the sample carrier to be within a predetermined ratio of each other; or a humidity control element, configured to control the humidity within each hole of the sample carrier to be within a predetermined amount of each other; an image acquisition element, which is configured to image the sample or a feature of the sample within each hole of the sample carrier through the opening, wherein the image acquisition element is configured to acquire at least one image from each hole of the sample carrier; and a signal processing module, which is operably connected to the sensing system, the signal processing module being configured to receive and adjust the first signal and the second signal from the sensor unit, and process at least one image from the image acquisition element.

[0031] In some aspects, the device further comprises: an impedance element comprising an electrode surface configured to measure impedance changes caused by sample attachment or to excite a sample within each hole of a sample carrier via an electrical signal, wherein the electrode surface is located at a bottom of the sample carrier and wherein the electrode surface comprises a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on the same plane and having substantially the same surface area; a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures; and wherein the impedance element detects electrical impedance changes between the electrode structures or excites the sample via an electrical signal.

[0032] According to one aspect, a device having an extended period measurement capability is provided, comprising: an impedance element comprising an electrode surface configured to perform one or both of: measuring impedance changes caused by sample attachment; or exciting a sample by an electrical signal within each of a plurality of wells defined in a sample carrier, wherein the electrode surface is located at a bottom of the sample carrier, and wherein the electrode surface comprises a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on a single plane and having substantially the same surface area; a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures, wherein the impedance element detects electrical impedance changes between the electrode structures or excites the sample by an electrical signal; an image acquisition element configured to image a sample or a feature of the sample within each well of the sample carrier through an opening, wherein the image acquisition element is configured to acquire at least one image from each well of the sample carrier; and a signal processing module operably connected to the impedance element, the signal processing module configured to measure electrical impedance changes from the impedance element and to process at least one image from the image acquisition element.

[0033] According to one aspect, a sample carrier is provided, comprising: a plurality of wells configured to accommodate a predetermined amount of sample, wherein each well of the plurality of wells comprises an opening allowing an image acquisition element to acquire at least one image from each well of the sample carrier; an electrode surface comprising a non-conductive carrier located on a bottom of the sample carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on a single plane and having substantially the same surface area; a plurality of connecting pads positioned on the sample carrier, wherein each connecting pad is electrically connected to at least one of the electrode structures in each well of the plurality of wells; and a plurality of structures that produce microchambers of reduced volume when matched with sensor cells of the sensor cell array.

[0034] In some aspects, the reduced volume is 3 microliters or less.

[0035] In some aspects, the plurality of structures are brackets, lips, protrusions, or stops configured to control the extent to which the sensor unit can protrude downwardly into the plurality of apertures to a predetermined distance.

[0036] In some aspects, the sample carrier is a microtiter plate, a flow slide, or a 3D tissue or spheroid molding / measurement plate.

[0037] In some aspects, one or more wells of the sample carrier are made of a material that restricts gas diffusion.

[0038] In some aspects, one or more wells of the sample carrier include windows through the electrodes that allow viewing or imaging of the cell sample from the bottom of the well.

[0039] In some aspects, one or more wells of the sample carrier do not include windows through the electrodes, such that the cell sample is viewed or imaged from the top of the well opposite the location where the electrodes are defined.

[0040] In some aspects, the sample carrier comprises a cover.

[0041] In some aspects, the cover includes one or more sensors that measure at least one of O2, pH, and CO2.

[0042] In some aspects, the sample carrier comprises a cartridge.

[0043] In some aspects, the cartridge includes one or more sensor or compound / substance ports.

[0044] According to one aspect, an analytical device is provided, comprising: a sample carrier comprising a plurality of wells, wherein each well of the plurality of wells: is fluidically isolated from each other well of the plurality of wells and comprises an electrode configured to measure impedance of a sample disposed within a given well; and defines a window through which the sample is visible from outside the sample carrier; a flux detector configured to individually address each well of the plurality of wells of the sample carrier and detect a first analyte; an image acquisition element configured to image the sample in a well of the plurality of wells of the sample carrier through the window and to acquire an image from each well of the plurality of wells of the sample carrier; and an environmental control module for maintaining environmental parameters surrounding the sample carrier within a predetermined range for at least six hours at a time.

[0045] In some aspects, the environmental control module maintains a CO 2 concentration, an O 2 concentration, and a N 2 concentration in the atmosphere of the environment surrounding the plurality of wells.

[0046] In some aspects, the apparatus further comprises: a flux box capable of moving relative to the sample carrier on an axis substantially perpendicular to a plane intersecting each of the plurality of wells of the sample carrier, wherein the flux box comprises a plurality of heads, wherein: the flux box is configured such that each of the plurality of wells of the sample carrier is addressable by a head of the plurality of heads; and each of the plurality of heads that addresses a given well comprises a surface proximate the sample carrier that defines a reaction chamber within the well and is configured to limit the volume of liquid or evaporation from the reaction chamber.

[0047] In some aspects, each well of the plurality of wells comprises a volume defining member configured to: limit a range of motion between the sample carrier and the second element of the device; or define a minimum non-zero distance between the sample carrier and the second element of the device.

[0048] In some aspects, the volume defining member comprises at least one of: a bracket; a protrusion; a lip; and a position controller for a motor that moves the sample carrier relative to the sensor array stopped a distance above the bottom of the corresponding well.

[0049] In some aspects, the sample carrier is movable relative to the flux detector and the image acquisition element to allow the flux detector and the imaging element to sequentially address the sample carrier.

[0050] In some aspects, the apparatus further comprises: a liquid handling module configured to deliver liquid to the sample carrier.

[0051] In some aspects, the sample carrier is movable relative to the liquid handling module.

[0052] In some aspects, the liquid handling module is configured to deliver liquid to individual wells of the plurality of wells.

[0053] In some aspects, the flux detector is configured to detect a second analyte.

[0054] According to one aspect, a device is provided, comprising: a chamber configured to receive: a sample carrier comprising a plurality of wells; and a cartridge comprising a compound; a camera disposed in the chamber below a position for receiving the sample carrier into the chamber, the camera configured to capture images of the contents of individual wells of the plurality of wells; a sensor disposed in the chamber, configured to monitor cell growth in individual wells of the plurality of wells; a temperature controller configured to regulate the temperature of samples held in individual wells of the plurality of wells and the sensor; and a fluid processor in communication with the sample carrier and the compound, configured to deliver the compound from the cartridge to a given well of the plurality of wells based on one or more of the pH value of the contents of the given well and the image of the given well captured by the camera.

[0055] In some aspects, the apparatus further includes an environmental controller configured to regulate a temperature of the atmosphere in the chamber, a CO 2 concentration of the atmosphere, and an O 2 concentration of the atmosphere.

[0056] According to one aspect, a device is provided, comprising: a chamber configured to receive: a sample carrier comprising a plurality of wells, each 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 to the first side, the first electrode and the second electrode each being electrically connected to an electrical measurement module on the sample carrier; and a cartridge comprising a compound and at least one delivery port for the compound; a temperature controller configured to adjust the temperature of samples held in the plurality of wells and individual wells of the electrical property measurement module; and a fluid processor connected to the sample carrier and the cartridge, configured to deliver at least one compound from the cartridge to a given well of the plurality of wells, and measure the sample in the given well through the electrical measurement module between the first electrical contact and the second electrical contact.

[0057] In some aspects, the measurement of the sample is a measurement of cell growth and impedance value in a given well, or a measurement of cell excitation.

[0058] In some aspects, the apparatus further includes an environmental controller configured to regulate a temperature of the atmosphere in the chamber, a CO 2 concentration of the atmosphere, and an O 2 concentration of the atmosphere.

[0059] According to one aspect, a device is provided, comprising: a chamber configured to receive: a sample carrier comprising a plurality of wells, wherein each well of the plurality of wells has 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 to the first side, the first electrode and the second electrode being each electrically connected to an impedance meter on the sample carrier; and a cartridge comprising a compound and at least one delivery port for the compound; a camera disposed below the chamber for receiving the sample carrier, configured to capture an image of a cell culture in each individual well of the plurality of wells through an associated window in each individual well when the sample carrier is positioned in a first position in the chamber; a fluid processor, the fluid processor being connected to the sample carrier and the cartridge when the sample carrier is positioned in a second position in the chamber, the fluid processor being configured to deliver the 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 by the impedance meter between the first electrical contact and the second electrical contact; and a motion stage in contact with the sample carrier, configured to move the sample carrier between the first position and the second position.

[0060] In some aspects, the apparatus further comprises: a temperature controller configured to regulate the temperature of a sample held in an individual well of the plurality of wells.

[0061] In some aspects, the apparatus further includes an environmental controller configured to regulate a temperature of the atmosphere in the chamber, a CO 2 concentration of the atmosphere, and an O 2 concentration of the atmosphere.

[0062] According to one aspect, a method of using a device is provided, comprising loading a sample carrier comprising one or more cell samples into the device, each sample being placed within a corresponding well of the sample carrier.

[0063] In some aspects, samples are analyzed over an extended period of time of 6 hours to 72 hours.

[0064] In some aspects, the cell sample comprises living cells.

[0065] According to one aspect, a method of analyzing a cell sample is provided, comprising: providing a device; loading a sample carrier comprising one or more cell samples into the device, each sample being placed in a corresponding well of the sample carrier; and analyzing the cell samples.

[0066] In some aspects, samples are analyzed over an extended period of time of 6 hours to 72 hours.

[0067] In some aspects, the method further includes positioning one or both of the stage and the sensing system relative to each other on one or more of the x-axis, the z-axis, and the y-axis.

[0068] In some aspects, the method further comprises dispensing the compound into the sample within each well of the sample carrier.

[0069] In some aspects, the method further comprises controlling the temperature within each well of the sample carrier to within a predetermined temperature of each other.

[0070] In some aspects, the method further comprises controlling the gas content of each well of the sample carrier to be within a predetermined ratio of one another.

[0071] In some aspects, the method includes 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 an extended period of time.

[0072] In some aspects, the method further includes receiving and conditioning the first signal and the second signal from the sensor unit.

[0073] In some aspects, the method further comprises calculating one or more metabolic flux parameters, including at least one of the following: oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and / or proton efflux rate (PER).

[0074] In some aspects, the method further comprises imaging the sample or a feature of the sample within each well of the sample carrier through the opening.

[0075] In some aspects, the method further includes processing at least one image from the image acquisition component.

[0076] In some aspects, the method further comprises measuring a change in impedance of the sample.

[0077] In some aspects, the sample comprises living cells.

[0078] In some aspects, the sample comprises one or more of loose cells, cell constructs, loose tissue, tissue constructs, organelles, enzymes, cellular products or byproducts, and conditioned medium.

[0079] In some aspects, the sample comprises mammalian cells or tissue.

[0080] In some aspects, the sample comprises stem cells.

[0081] In some aspects, the sample includes cells of the cardiovascular system.

[0082] In some aspects, the sample comprises non-mammalian cells or tissues.

[0083] In some aspects, the sample comprises single-celled organisms.

[0084] In some aspects, wherein the sample comprises whole animal model tissue.

[0085] In some aspects, the sample comprises whole plant model tissue or plant model cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawings will be provided by the Office upon request and payment of the necessary fee.

[0087] The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in different figures is represented by a like reference numeral. For clarity, not every component may be labeled in every figure. In the drawings:

[0088] Figure 1 is a front perspective view of an apparatus for analyzing living cells according to one embodiment;

[0089] Figure 2 is a rear perspective view of an apparatus for analyzing living cells according to one embodiment;

[0090] Figure 3 is a side view of an apparatus for analyzing living cells according to one embodiment;

[0091] Figure 4 is a top view of an apparatus for analyzing living cells according to one embodiment;

[0092] Figure 5 is a side view of selected components of an apparatus for analyzing living cells according to one embodiment;

[0093] Figure 6 is a schematic diagram of a system for analyzing living cells according to one embodiment;

[0094] Figure 7 is a schematic diagram of a system for analyzing living cells according to one embodiment;

[0095] Figure 8 is a schematic diagram of selected components of a system for analyzing living cells according to one embodiment;

[0096] Figure 9 is a schematic diagram of selected components of an apparatus for analyzing living cells according to one embodiment;

[0097] Figures 10A-10Dis a graph showing the oxygen consumption rate (OCR) or extracellular acidification rate (ECAR) of living cells analyzed by the method disclosed herein according to one embodiment, particularly Figures 10A-10B OCR readings from the system disclosed herein and the comparative system, respectively, showing that the system has improved stability performance from 0 to 15 minutes compared to the comparative device;

[0098] Figures 11A-11B is a graph showing the oxygen consumption rate (OCR) of living cells analyzed by the method disclosed herein according to one embodiment, particularly Figures 11A-11B OCR readings from the disclosed system and the comparative system, respectively, showing results with significantly lower variability, especially for metformin-treated cells (lower line);

[0099] Figure 12 is a table showing average evaporation of a substance (e.g., medium) contained in a sample carrier analyzed by the method disclosed herein according to one embodiment;

[0100] Figures 13A-13B It is a comparison picture of a sphere;

[0101] Figure 14 is a block diagram illustrating a multi-detection system according to an embodiment;

[0102] Figure 15 is a block diagram illustrating a multi-detection system according to an embodiment;

[0103] Figure 16 is a block diagram illustrating a multi-detection system according to an embodiment;

[0104] Figure 17 is a block diagram illustrating a multi-detection system according to an embodiment;

[0105] Figure 18 is a block diagram illustrating a multi-detection system according to an embodiment;

[0106] Figure 19 is a diagram of a non-imaging analysis subsystem according to an embodiment;

[0107] Figure 20 is a diagram illustrating an injection subsystem according to an embodiment;

[0108] Figure 21 is a diagram illustrating a multi-detection system according to an embodiment;

[0109] Figure 22A is a perspective view showing an environmental control subsystem according to an embodiment;

[0110] Figure 22B is a rear view showing the environmental control subsystem according to this embodiment;

[0111] Figure 22C is a front view showing the environment control subsystem according to this embodiment;

[0112] Figure 23 is a functional block diagram illustrating modal control of the device according to the embodiment; and

[0113] Figure 24 is a flow chart of a control method of a multi-detection system according to an example embodiment;

[0114] Figure 25A is a first view showing an immersion objective lens according to an embodiment;

[0115] Figure 25B is a second view showing the immersion objective lens according to this embodiment;

[0116] Figure 26 is a diagram showing a fluid pump system according to an embodiment;

[0117] Figure 27 is a diagram illustrating objective lens coupling according to an embodiment;

[0118] Figure 28A is a perspective view showing an immersion objective lens according to a first embodiment;

[0119] Figure 28B is a top view showing the immersion objective lens according to the first embodiment;

[0120] Figure 28C It is along Figure 28B A first sectional view taken along line AA in FIG. 1 shows the immersion objective lens according to the first embodiment in a state where a liquid bubble is provided;

[0121] Figure 28D The immersion objective lens according to the first embodiment is shown along Figure 28B A second cross-sectional view taken along line AA in FIG, wherein a sample carrier (eg, a microplate) is provided on the immersion objective;

[0122] Figure 29A is a top view showing an immersion objective lens according to a second embodiment;

[0123] Figure 29B It is along Figure 29A A first cross-sectional view taken along line BB shows the immersion objective lens according to the second embodiment in a state where a liquid bubble is provided;

[0124] Figure 29C The immersion objective lens according to the second embodiment is shown along Figure 29A In the second cross-sectional view taken along line BB, a sample carrier (e.g., a microplate) is disposed on the immersion objective lens;

[0125] Figure 30A is a top view showing an immersion objective lens according to a third embodiment;

[0126] Figure 30B It is along Figure 30A A first cross-sectional view taken along line CC shows the immersion objective lens according to the third embodiment in a state where a liquid bubble is provided;

[0127] Figure 30C The immersion objective lens according to the third embodiment is shown along Figure 30A In the second cross-sectional view taken along line CC, a sample carrier (e.g., a microplate) is disposed on the immersion objective lens;

[0128] Figure 31A is a top view showing an immersion objective lens according to a fourth embodiment;

[0129] Figure 31B It is along Figure 31A A first sectional view taken along line DD in FIG. 1 shows the immersion objective lens according to the fourth embodiment in a state where a liquid bubble is provided;

[0130] Figure 31C The immersion objective lens according to the fourth embodiment is shown along Figure 31A In the second cross-sectional view taken along line DD, a sample carrier (e.g., a microplate) is provided on the immersion objective lens;

[0131] Figure 32A is a first diagram of a multi-detection system in a laser point scanning confocal modality according to an embodiment;

[0132] Figure 32B is a second diagram of a multi-detection system in widefield or spinning disk confocal modality according to an embodiment;

[0133] Figure 33 is a diagram of an example user interface according to an embodiment;

[0134] Figure 34 is a schematic diagram illustrating a cross-sectional view of a transmission module according to one embodiment;

[0135] Figure 35 is a diagram of a side view of a transport module according to one embodiment;

[0136] Figure 36 including a graph illustrating the accuracy of measurements made by an apparatus having a thermally conductive excitation source according to one embodiment;

[0137] Figures 37A-37C Mitotoxicity is shown to result in negative MTI values;

[0138] Figures 38A-38C Mitotoxicity is shown to result in a positive MTI value;

[0139] Figure 39 Includes an exemplary MTI detection diagram;

[0140] Figure 40 Included are graphs showing kinetic dose-response OCR data for the three test compounds;

[0141] Figure 41 is a graph showing Z' values ​​obtained using a Mito Tox assay and an MTI-based analysis according to one embodiment;

[0142] Figure 42 Showing the light source;

[0143] Figure 43 Relay optics are shown;

[0144] Figure 44 The structure of the excitation-emission separation device is shown;

[0145] Figure 45 showing the holder and associated optical accessory elements;

[0146] Figure 46 A view showing a sample carrier and its imaging;

[0147] Figures 47A-47C Provides a schematic diagram of a device having a two-electrode structure;

[0148] Figure 48 An example of a multimodal analysis device is shown;

[0149] Figure 49 A schematic system diagram illustrating an embodiment of a system for analyzing living cells;

[0150] Figure 50 Show Figure 49 Exploded view of the sample carrier and cartridge;

[0151] Figure 51 and Figure 52 A plan side view of the workpiece stage without a sample carrier is shown in each case.

[0152] Figure 53 and Figure 54 Shown are top and perspective views of the bottom surface of a well of a sample carrier.

[0153] Figure 55 Shown is the feedback mechanism used to control the gas concentration in the sample carrier wells.

[0154] Figures 56A-56C is a graph showing the oxygen consumption rate (OCR) of living cells analyzed by the method disclosed herein according to one embodiment, particularly Figures 56A-56Care three experimental replicates of OCR readings from the analyzer disclosed herein (left) and a comparative analyzer (right), showing that the results from the analyzer have significantly lower variability compared to the comparative instrument, particularly at lower OCR rates when cells are plated at lower densities or after cells are treated with respiratory inhibitors (oligomycin or rotenone + antimycin A);

[0155] Figure 57A-Figure 57B is a graph of the same data, showing the basal oxygen consumption rate (OCR) of living cells analyzed by the method disclosed herein according to one embodiment, particularly Figure 57A-Figure 57B are base OCR readings from an analyzer disclosed herein (left) and a comparative analyzer (right), showing that the results from the analyzer have significantly lower variability compared to the comparative instrument, particularly at lower OCR rates when cells are plated at lower densities;

[0156] Figure 58 is a graph showing the standard deviation of the basal oxygen consumption rate (OCR) of living cells analyzed by the method disclosed herein in three experimental replicates according to one embodiment, particularly Figure 58 is the standard deviation of the underlying OCR readings for the disclosed analyzer (grey) and the comparative analyzer (black), showing that the results from the analyzer have significantly lower variability compared to the comparative instrument. DETAILED DESCRIPTION

[0157] The present disclosure provides, at least in part, systems, methods, and consumables that allow long-term measurements of cell samples. Also disclosed herein are multimodal systems, methods, and consumables that perform bioenergy-based measurements (e.g., measurements of metabolic flux), electronic-based excitation or stimulation, electronic-based signal measurements (e.g., field potential recording, impedance measurement), and imaging. Also disclosed herein are temperature / environmental control of parameters, such as temperature, gas (O2, CO2, N2, etc.), humidity, and atmospheric pressure. Any combination of these components is also disclosed herein.

[0158] The systems (e.g., instruments, devices, and apparatus) and methods (e.g., assays) described herein may include or use one or more components, such as a flux measurement system, an impedance measurement system, an imaging measurement system, or any combination thereof, capable of measuring over extended periods of time. In some embodiments, the flux measurement system may include elements for temperature / environmental control, fluid handling, or both. Conventional systems and methods may not be suitable for long-term measurements, at least in part due to extensive evaporation of the well / sample medium and compound / substance ports. The systems and methods described herein may control the sample environment or microenvironment to allow for long-term measurements. Such control may include, for example, liquid handling, temperature control, gas control, humidity control, or any combination thereof. In some embodiments, evaporation of the compound / substance port may be addressed by injecting the compound through liquid handling, rather than loading the compound / substance port into a cartridge prior to the assay. In some embodiments, calibration may be shortened or even eliminated when a time-based detection method is employed. In some embodiments, the systems or methods described herein may control CO2 levels to allow cell samples to proliferate. In some embodiments, the systems and methods described herein include or use an impedance measurement system suitable for use in conjunction with the flux measurement system described herein.

[0159] Conventional systems and methods for measuring extracellular flux, impedance, and imaging independently may not always 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 cell samples. In long-term extracellular flux assays, evaporation of the compound / substance port volume in the cartridge is a limiting factor, and therefore, assays exceeding six hours may not be possible without increased fluid handling. Imaging may also be affected by interference from electrodes.

[0160] 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 compounds, exchanging cell growth or flow media, and controlling temperature and / or environmental conditions (e.g., gas, humidity) through preloaded cartridges; measuring one or more cell functions simultaneously through attachment / detachment of cell samples by real-time impedance measurement; and facilitating long-term measurements.

[0161] In 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; injecting one or more compounds, exchanging cell growth or flow media, and controlling temperature and / or environmental conditions (e.g., gas, humidity) through an embedded fluid handling device; measuring one or more cell functions simultaneously with attachment / detachment of the cell sample by real-time impedance measurement; and facilitating long-term measurements.

[0162] In 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 time intervals; injecting one or more compounds, exchanging cell growth or flow media, and controlling temperature and / or environmental conditions (e.g., gas, humidity) through preloaded cartridges; measuring one or more cell functions simultaneously with attachment / detachment of the cell sample by real-time impedance measurement; and imaging the cell sample simultaneously by visualization at a specified area at the bottom of the sample carrier without electrodes or impedance conductor gaps.

[0163] In 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 time intervals; injecting one or more compounds, exchanging cell growth or flow media, and controlling temperature and / or environmental conditions (e.g., gas, humidity) through preloaded cartridges; measuring one or more cell functions simultaneously with attachment / detachment of the cell sample by real-time impedance measurement; and imaging the cell sample simultaneously by visualization through a transparent impedance conductor at the bottom of the sample carrier.

[0164] In one aspect, the present disclosure provides a device capable of measuring in real time one or more metabolic parameters of a suspended cell sample flowing from a biological growth chamber or processing unit into a measurement chamber at specified intervals; exposing the sample to one or more compounds, refreshing the sample with growth or flow medium, and controlling temperature and / or environmental conditions (e.g., gas, humidity); simultaneously imaging the cell sample and performing image-based fluorescence measurements; and / or impedance measurements.

[0165] In another aspect, the present disclosure provides a device capable of measuring in real time one or more metabolic parameters of a suspended cell sample flowing from a biological growth chamber or bioprocessing unit into a measurement chamber at specified intervals; exposing the sample to or not exposing the sample to one or more compounds, refreshing the sample with growth or flow medium, and controlling temperature and / or environmental conditions (e.g., gas, humidity); simultaneously imaging the cell sample and performing image-based fluorescence measurements; and returning the cell sample to the biological growth chamber or bioprocessing unit at specified intervals for further cell proliferation, which can be repeated until a user-specified time.

[0166] In 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 compounds, exchanging cell growth or flow medium, and controlling temperature and / or environmental conditions (gas, humidity, etc.) through specific controllers, as well as controlling the medium through an embedded fluid handling device; and simultaneously measuring cell function through attachment / detachment of the cell sample by real-time measurement of impedance.

[0167] In 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 by monitoring an analyte using a barbed lid, wherein an analyte sensor extends in a medium at a specified height above the sample; removing the barbed lid using a robotic / manipulator system and placing a lid or cartridge on the sample carrier; measuring one or more metabolic parameters of a cell sample in a sample carrier in real time at specified intervals by creating a microchamber having a cartridge with an analyte sensor at the distal end; injecting one or more compounds, exchanging cell growth or flow media, and controlling temperature and / or environmental conditions (gas, humidity, etc.) through specific controllers and media, via an embedded fluid handling device or through pre-filled / or user-filled cartridge compound / substance ports, and optionally adding media and / or washing the media in the well plate; removing the cartridge and placing a barbed analyte sensing monitoring lid on top of the sample carrier; and simultaneously measuring one or more cellular functions via attachment / detachment of the cell sample by real-time impedance measurement.

[0168] definition

[0169] Unless defined otherwise, 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.

[0170] As used herein, the articles "a" and "an" refer to one or more of the grammatical object of the article (eg, to at least one).

[0171] As used herein, the terms "about" and "approximately" generally refer to an acceptable degree of error in a measurement given the nature or precision of the measurement. An exemplary degree of error is within 20%, typically within 10%, and more typically within 5% of a given value or range of values.

[0172] As used herein, the term "acquire" means obtaining possession of a physical entity or value, either by "direct acquisition" or "indirect acquisition" of the physical entity or value. "Direct acquisition" refers to performing a process (e.g., performing a synthesis or analytical method) to obtain the physical entity or value. "Indirect acquisition" refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquires the physical entity or value). Direct acquisition of a physical entity includes performing a process that includes a physical change in a physical substance (e.g., a starting material). Exemplary changes include making a physical entity from two or more starting materials, shearing or crushing a substance, separating or purifying a substance, combining two or more separate entities into a mixture, and performing a chemical reaction that includes breaking or forming covalent or non-covalent bonds. Directly obtaining a value includes performing a process that includes a physical change of a sample or another substance, for example, performing an analytical process that includes a physical change of a substance (e.g., a sample, an analyte, or a reagent) (sometimes referred to herein as a "physical analysis"), performing an analytical method, for example, a method that includes one or more of the following: separating or purifying a substance (e.g., an analyte or a fragment or other derivative thereof) from another material; combining an analyte or a fragment or other derivative thereof with another substance (e.g., a buffer, a solvent, or a reactant); or changing the structure of an analyte or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first and a second atom of the analyte; or by changing the structure of a reagent or a fragment or other derivative thereof, for example, by breaking or forming a covalent or non-covalent bond between a first and a second atom of the reagent. In embodiments, directly obtaining includes direct measurement. In embodiments, indirectly obtaining includes inference.

[0173] As used herein, the term "obtaining a sample" refers to obtaining possession of a sample, such as the samples described herein, by "directly obtaining" or "indirectly obtaining" the sample. "Directly obtaining a sample" refers to performing a process (e.g., performing a physical method such as surgery or extraction) to obtain a sample. "Indirectly obtaining a sample" refers to receiving a sample from another party or source (e.g., a third-party laboratory that directly obtains the sample). Directly collecting a sample includes performing a process that involves physical changes to a physical substance, such as a starting material (e.g., tissue, such as tissue from a human patient or tissue previously separated from a patient). Exemplary changes include making a physical entity from a starting material; dissecting or scraping tissue; separating or purifying a substance; combining two or more separate entities into a mixture; or performing a chemical reaction that involves breaking or forming covalent or non-covalent bonds.

[0174] As used herein, the term "ambient temperature" refers to the temperature of the air in or near the environment. Ambient temperature may also be referred to as a baseline temperature or the temperature of a device or object before temperature control is initiated. 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.

[0175] The term "basic mitochondrial ATP productivity" as used herein refers to the rate at which mitochondria in a cell sample produce ATP before the cell sample is contacted with an ATP synthase inhibitor, a mitochondrial uncoupler, and an electron transport chain (ETC) inhibitor to form a reaction mixture. In an embodiment, the basal mitochondrial ATP productivity is calculated by subtracting (basic OCR) from the oxygen consumption rate measurement value (e.g., the last measurement or the average value of multiple measurements) before the cell sample is first contacted with any one of the ATP synthase inhibitor, the mitochondrial uncoupler, or the ETC inhibitor. In an embodiment, the constant (called P / O ratio) * 2 (converting oxygen atoms into oxygen molecules) is multiplied by 2.45 to 2.86.

[0176] As used herein, the term "bioenergetic capacity" refers to the increased level of glycolysis and / or mitochondrial activity that a cell can influence, utilize, and / or induce. In embodiments, bioenergetic capacity is determined in response to increased energy demand and / or in response to inhibition / perturbation of energy production. In embodiments, bioenergetic capacity comprises oxygen consumption (e.g., oxygen consumption rate (OCR)) and proton efflux (e.g., proton efflux rate (PER)). In embodiments, the value of oxygen consumption (e.g., OCR) is a response to mitochondrial uncoupling. In embodiments, the proton efflux value (e.g., PER) is a response to ATPase inhibition. In embodiments, PER is the glycolytic PER (glycoPER), which mathematically removes the contribution of CO2.

[0177] As used herein, the term "bioenergetic balance" refers to the balance between aerobic and glycolytic energy production. In embodiments, bioenergetic balance describes the ratio of ATP produced by glycolysis to that produced by oxidative phosphorylation. In embodiments, bioenergetic balance includes a relationship, such as a ratio, between ATP produced by mitochondria and ATP produced by glycolysis, between ATP produced by mitochondria and total ATP production, between ATP formed by glycolysis and total ATP production, or any combination thereof.

[0178] As used herein, the term "bioenergetic work" refers to the amount of ATP produced by a cell.

[0179] As used herein, the term "cell sample" refers to a sample comprising cells or cell products or by-products. In an embodiment, a cell sample comprises a plurality of cells. In an embodiment, the cells are placed in a medium. A cell sample can be or include one or more of cells, tissues, cell or tissue constructs, organelles, enzymes, and / or conditioned medium.

[0180] As used herein, the term "cellular metabolic function" refers to the ability of an organism to carry out chemical reactions required to sustain life. In an embodiment, the cellular metabolic function of a cell sample can be monitored by measuring OCR and ECAR.

[0181] As used herein, the term "extracellular acidification rate (ECAR)" refers to a measurement of proton extrusion in the extracellular medium over time. The ECAR can be reported as the rate of change in pH units, e.g., millipH / minute (mpH / min) over the assay run time.

[0182] As used herein, the term "glycolysis" or "glycolytic activity" refers to the cellular metabolic function that converts glucose into lactate.

[0183] As used herein, the term "mitochondrial respiration" refers to the metabolic reactions that occur in mitochondria and the processes that require oxygen to convert energy stored in macronutrients into ATP.

[0184] As used herein, the term "mitochondrial toxicity index" (also referred to as "mitochondrial toxicity index" or "MTI") refers to an index value derived from OCR measurements. MTI is a parameter that provides information about the type and extent of mitochondrial toxicity. Positive MTI values ​​(typically between 0 and 1) indicate mitochondrial toxicity due to uncoupling, whereas negative MTI values ​​(typically between 0 and -1) indicate mitochondrial toxicity due to inhibition.

[0185] Unless the context clearly indicates otherwise, "or" is used herein to mean and is interchangeable with the term "and / or." Unless the context clearly indicates otherwise, the use of "and / or" in certain places herein does not mean that "or" is not interchangeable with "and / or."

[0186] As used herein, the term "oxygen consumption rate (OCR)" refers to a quantitative measurement of the amount of oxygen consumed 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 O2 levels, e.g., picomoles / minute (pmol / min) over the assay run time.

[0187] In one embodiment, OCR includes determining the oxygen consumption in a completely sealed system, for example, a system that allows oxygen back diffusion or a large amount of 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 not corrected for oxygen back diffusion into the sample, or determining the oxygen consumption in a sealed system, for example, a system that does not allow oxygen back diffusion or a large amount of oxygen back diffusion into the sample, or the oxygen consumption is equal to or substantially equal to the oxygen depletion in the sample.

[0188] In one embodiment, oxygen consumption is determined directly or indirectly, for example, by inference from a measured oxygen gradient (eg, within a test well or across a capillary tube), or by measuring oxygen at preselected time points.

[0189] In one embodiment, oxygen consumption is reported in units other than rate of change of O2 levels, such as sensor response per unit time (eg, microseconds / minute, relative fluorescence units / minute).

[0190] The term "primary cells" as used herein refers to cells that are separated or harvested directly from a subject, organ or tissue. For example, primary cells can be isolated from the blood of a living subject. Primary cells can be separated or harvested using enzymes or mechanical methods. Once separated or harvested, primary cells can be cultured in a medium containing essential nutrients and growth factors to support proliferation. Primary cells can be suspension cells that do not need attachment to grow (e.g., anchorage-independent cells) or adherent cells that need attachment to grow (e.g., anchorage-dependent cells).

[0191] As used herein, the term "proton efflux rate (PER)" refers to a quantitative measure of extracellular acidification that takes into account medium buffering capacity and plate geometry. PER values ​​can be reported as the rate of change of H+, for example, in picomoles / minute (pmol / min) over the assay run time. + It is a quantifiable analyte that is proportional to pH.

[0192] The term "sample" as used herein refers to a biological sample obtained or derived from a source of interest. In an embodiment, the source of interest includes an organism, such as an animal or a human. The source of the sample can be blood or a blood component; a body fluid; a solid tissue from a fresh, frozen and / or preserved organ, tissue, biopsy, resection, smear or aspirate; or a cell at any time during the subject's pregnancy or development. In an embodiment, the source of the sample is blood or a blood component. In an embodiment, the sample is a raw sample, for example, obtained directly from a source of interest by any appropriate means. In an embodiment, the sample is a preparation obtained by processing (for example, by removing one or more components of the primary sample and / or by adding one or more reagents thereto).

[0193] As used herein, the term "sample carrier" refers to a substrate that can carry a sample. In an embodiment, the sample carrier can include one or more holes. Exemplary sample carriers include, but are not limited to, microplates, microtiter plates, multi-well plates, single-hole plates, microplates, microfluidic chips, microfluidic devices, dishes, slides, flasks, and test tubes. The sample carrier 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 hole of a sample carrier can refer to 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 holes, or any number of holes between or more holes. As used herein, at least two wells of a sample carrier can refer to 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 of wells in between or more.

[0194] Systems with extended-duration capabilities and multi-mode systems

[0195] Without wishing to be bound by theory, it is believed that in some embodiments, the systems, consumables, and methods described herein are particularly well suited for long-term measurements of living cell samples.

[0196] In one aspect, the present disclosure provides components for measuring cellular bioenergetic parameters in real time, such as metabolic flux parameters, such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and / or proton efflux rate (PER). On the other hand, the present disclosure provides components for measuring cell electrical properties (such as cell impedance) and allows electronic excitation of signals interacting with cell samples. On another aspect, the present disclosure provides optical and / or imaging components, such as a microscope or inverted microscope with a high-definition camera, for bright field imaging and / or fluorescence imaging of cells with or without labels. On the other hand, the present disclosure provides real-time temperature / environmental control, optionally with monitoring / feedback, which is generally to create a measurement / sample environment with more physiological relevance. Such temperature / environmental control can include, for example, temperature, humidity, atmosphere control and gas control (oxygen, carbon dioxide, nitrogen, etc.). On the other hand, the present disclosure provides injection of compounds or fluid control to measure and quantify the interaction between target compounds and cell activity.

[0197] In another aspect, the present disclosure provides a multi-mode system, which includes the above components combined with each other in different pairs. Exemplary components of the multi-mode system are further described below.

[0198] Bioenergetic measurement (e.g. extracellular flux measurement) components

[0199] The bioenergy measurement (e.g., extracellular flux measurement) components of the system can include, for example, the formation of a microchamber, an electro-optical element, and / or a fluorescent analyte sensor that responds to the concentration of the analyte. In some embodiments, an LED can be used to excite the analyte sensor, and a detection device can be used to measure the change in the signal over time. The signal can be measured in any available detection mode, including intensity, DLR, TRF, ratiometry, ToF, etc. In some embodiments, the system can move the z-axis component to form a microchamber between the box consumables and the sample carrier (e.g., a microplate). In some embodiments, a consumable with a microelectrode having an impedance component is used, and these two parameters are measured simultaneously.

[0200] For example, other exemplary extracellular flux components of the system are described in the section herein entitled "Bioenergetic Measurement Module."

[0201] Components for electrical measurements (e.g., electronic excitation and cell impedance measurement)

[0202] The electrical measurement (e.g., electronic excitation and impedance measurement) components of the system can include, for example, consumables with embedded microelectrodes on the cell seeding surface. These microelectrodes can be made of materials that are easy to form and compatible with cell growth. In some embodiments, the microelectrodes are optimized for low impedance characteristics to maximize the accuracy of measurement and excitation pulses. In some embodiments, the microelectrodes are arranged to minimize the space between opposite polarity connections, while increasing the length of the electrodes or connecting electrodes to increase the potential between the microelectrodes. In order to read the impedance measurement value, a low-power signal can be excited at either end of the microelectrode seeding surface. The real and imaginary parts of the impedance can then be measured. In some embodiments, the microelectrodes are excited with a very low alternating current and measured on microelectrodes of opposite polarity. Measuring the current and voltage on the opposite side can provide an opportunity to measure the real and imaginary parts of the impedance of the cell sample compared to the current and voltage measured at the source electrode. Such electronic pulses can also be used to excite cells that respond to electrical pulses.

[0203] For example, other exemplary electronic excitation and cell impedance measurement components of the system are described in the section herein entitled "Electrical Measurement Module."

[0204] Imaging and Optics

[0205] The imaging and optical components of the system can allow for detection modes such as fluorescence intensity, luminescence, fluorescence polarization, time-resolved fluorescence, alpha and UV-visible absorbance, fluorescence, phase contrast, bright field, high contrast bright field, color bright field, and phase contrast, etc. System components can include, for example, filter cubes, image processing elements, cameras such as CMOS or CCD devices, objectives, LEDs, and lasers, as well as programs that allow for optimal image or measurement collection.

[0206] For example, other exemplary imaging components of the system are described in the sections herein entitled "Imaging Module" and "Optical Module."

[0207] Consumable parts

[0208] In some embodiments, the system includes an interface for interacting with consumables. The consumables can be any consumables that accommodate cell samples. Exemplary consumables include, but are not limited to, flow chips, microtiter plates with any number of holes, 2D cell cultures, and 3D tissue or sphere formation / measurement plates. In some embodiments, if impedance measurement or electrical excitation is required, the consumables include microelectrodes. In some embodiments, the consumables can form microchambers to allow flux measurement. In some embodiments, the consumables are made of materials that limit gas diffusion to improve flux sensitivity. In order to image the cell sample, the components that make up the imaging system can be configured to read from below or above the consumables. If imaging from below, the consumables can have an opening or window through the microelectrode to observe the cell sample. The opening or window is made of transparent, light-transmitting plastic or glass that does not contain microelectrodes and can be imaged through the bottom or top surface. If imaging from the top, the consumables can remove any features above the sample (e.g., flux measurement box) to observe the sample.

[0209] Other exemplary consumable components are described in the section herein entitled "Consumables."

[0210] System Features

[0211] The user can set up the system to perform measurements in a specific mode (imaging, flux, impedance, etc.). The user can also configure the measurement length, number of injections, incubation time, imaging settings, etc. The system can be configured to automatically move consumables or consumables within the measurement system.

[0212] Bioenergy measurement module

[0213] The devices and methods described herein provide a comprehensive view of the cellular metabolic function of cultured cell samples and isolated samples over an extended duration, which is longer than previously available devices can provide. Unlike previous devices, which have significant evaporation losses and accumulation of metabolites in the sample (these metabolites do not appear in the body within the corresponding time), the extended period device described controls various characteristics of the sample, including temperature, humidity, atmospheric content, etc., so that the metabolic function in the cultured cell sample and isolated sample can be continuously analyzed for more than 6 hours (e.g., up to 72 hours, 150 hours, etc.) without the need for human intervention. Various analytical systems can be included in the device, including flux, impedance, and imaging systems, for identifying the changing characteristics of the sample over time to researchers and control devices (e.g., computers) to monitor and manage the conditions in the cell, thereby maintaining one or more characteristics within a predefined range over an extended period. Therefore, the device is capable of measuring metabolic parameters in real time to control the injection of various compounds, exchange cell growth or flow medium, and control environmental conditions, thereby facilitating long-term measurements.

[0214] In various embodiments, measurements under "incubator-like" conditions are achieved by controlling the CO2 within the sample chamber, thereby avoiding the need for additional buffers (such as HEPES) that are typically used in long-term measurements. These buffers can be problematic for certain biological models. Controlling CO2 to facilitate long-term metabolic monitoring. Gas control also facilitates metabolic interrogation at lower O2 concentrations (e.g., lower O2 achieved by N2 purging), which can replicate in vivo conditions or be used to simulate specific disease states (e.g., imposing hypoxic insults and / or circulation, such as for ischemia-reperfusion modeling or long-term tumor modeling). Gas levels are controlled by a feedback mechanism that controls the gas purge, which can include a fan to change the CO2 and / or O2 concentrations more quickly at the desired time points.

[0215] Bioenergetic capacity drives cellular biological processes, and cellular metabolism is a core indicator of biological function and cell health. The devices and methods disclosed herein can be used to measure cellular metabolic pathways using high-throughput screening techniques. Therefore, the devices and methods disclosed herein can be used to determine and / or quantify key indicators of healthy cell function by modulating metabolic targets, signaling, and substrates, predict cell performance in in vitro disease models, and discover compounds / substances. The goal is to better understand disease states and thereby gain insight into appropriate therapies to alter disease states, healthy phenotypes, and / or optimize and enhance cell performance.

[0216] The devices and methods disclosed herein can be used to measure two major metabolic pathways (mitochondrial respiration and glycolysis) in living cells in real time to provide functional kinetic measurements of the bioenergetic capacity of the cell.

[0217] The devices and methods disclosed herein can be used to facilitate disease modeling and testing of key cellular processes, including activation, proliferation, differentiation, cell death, cellular homeostasis, and / or disease progression; conduct therapeutic discovery by revealing and validating potential therapeutic compound / substance targets; and optimize the engineering and manufacturing of cell therapies.

[0218] In one embodiment, mitochondrial respiration, glycolytic activity, and / or metabolic balance are temporal measurements of cellular activity, independent of the medium / buffer surrounding the cell. Creating microchambers allows for sensitive measurements of cellular activity. Changes in mitochondrial respiration and / or glycolytic activity in cells result in minute, real-time changes in O₂, CO₂, and lactate in the immediate surroundings of the cell, which are detected by the device through OCR, ECAR, and / or PER measurements.

[0219] In one embodiment, changes in mitochondrial respiration, glycolytic activity, and / or metabolic balance of the cell have a feedback loop that promotes maintenance or transition to a desired metabolic phenotype. This can be achieved, for example, by adding nutrients through an onboard liquid handling device or by changing sample environmental conditions (e.g., by changing O2 concentration). For example, in Figure 55 , a feedback mechanism 5700 is shown in which the oxygen concentration 5710 in a cell growth medium 5720 (in which various cells 5730 are growing) is measured by an oxygen sensor 5754 attached to the distal end of a sensor ridge 5752, which is located in a region 5722 proximate to the oxygen sensor 5754. Although the oxygen sensor 5754 is discussed herein as a non-limiting example, various other sensors for measuring different gas components may be used in addition to or in place of the oxygen sensor 5754. The growth medium 5720 is retained within a well 5760 or other compartment that is sealed or semi-sealed from a measurement chamber 5770 by a cover 5750 (in which the sensor ridge 5752 is defined), which is also sealed or semi-sealed from the external environment. The measuring device 5750 receives signals from the sensor ridges 5752, which are transmitted to a signal processor 5790, which calculates in real time the measured O2 concentration 5710 in the region 5722 to determine whether (and how much) N2 is supplied from the N2 blower 5780 to the chamber 5770 to regulate the amount of O2 or other gas in the chamber 5770 and / or the aperture 5760 (e.g., by exhausting unwanted gases with the inflowing N2).

[0220] In another embodiment, mitochondrial respiration and / or glycolytic activity is a temporal measurement of cellular activity as affected by the medium / buffer surrounding the cells by adding gases, therapeutic drug targets, or agents that affect cellular activity (such as ATP synthase inhibitors, mitochondrial uncouplers, or ETC inhibitors) to the culture medium affecting the cells.

[0221] In particular, the devices and methods disclosed herein can be used to measure oxygen consumption rate (OCR), extracellular acidification rate (ECAR), proton efflux rate (PER), adenosine triphosphate (ATP) production rate and other parameters of multiple cell samples in a porous sample carrier. OCR and ECAR or PER can be used to determine mitochondrial respiration and glycolysis and ATP production rate. The measurements obtained by the devices and methods disclosed herein can provide a comprehensive view of cellular metabolic function in cultured cell samples and in vitro samples.

[0222] It should be noted that cell samples as herein described may include loose cells, cell constructs, loose tissues and tissue construct samples.Cell samples can be or include organelles, enzymes, cell products or by-products and / or conditioned medium.The parameters of each cell sample (each hole) can be measured independently and selectively.In a specific embodiment, living cell samples can be tested, for example, without significantly reducing cell viability.Compared with conventional devices and methods, device and method as herein described can provide lower dissolved oxygen or OCR detection limit, higher accuracy consistency, improved temperature control and improved automation.

[0223] Conventional systems are susceptible to humidity and contamination caused by factors such as the laboratory environment, storage, and manufacturing processes, often exhibit motion errors over time (including inconsistent debris movement / accumulation), and are susceptible to evaporation, edge-hole temperature gradients, and prolonged warm-up caused by ambient heating methods. The devices and methods disclosed herein include components that overcome these shortcomings of conventional systems, thereby improving measurement performance and unexpectedly providing lower O2 detection limits and higher measurement accuracy.

[0224] The combination of hardware and analytical software provided in the devices disclosed herein allows for real-time monitoring of living cells in fields such as immunology and disease using rare, ex vivo, and genetically engineered cells to build better disease models. The enhancements disclosed herein improve measurement performance. These enhancements can often make it easier to identify new compound / substance targets, validate the effects of targets on cell function, optimize disease models, and determine the safety and anti-tumor potential of compounds / drugs for T cell therapies from the research laboratory to biopharmaceutical therapeutic development and toxicity programs.

[0225] The device disclosed herein can provide improved precision at low oxygen consumption rates (OCRs), enabling analysts to confidently interrogate more immune cell types, as well as cell types with impaired bioenergetics.

[0226] The devices and methods disclosed herein provide the ability to analyze living cells over an extended temperature range. For example, temperature control elements and a controlled temperature zone smaller than the headspace of the housing contribute to improvements over previous devices.

[0227] The devices and methods disclosed herein provide greater uniformity in heating temperature control elements, which can improve cell biology at consistent temperatures and sensing with device sensors, reducing system edge effects.

[0228] The devices and methods disclosed herein can provide temperature control with faster start-up times than previous devices.

[0229] The devices and methods disclosed herein include electronic optical panels capable of operating at humidity levels up to 95%. Previous devices often performed suboptimally at 70%-80% humidity. Therefore, the devices can be transported, stored, or used in areas with high humidity, or if it is desirable to control higher humidity levels within the device.

[0230] The devices and methods disclosed herein provide improved performance and detection at lower levels of OCR that previously appeared as noise, which allows analysis of damaged or dysfunctional immune cells, thereby expanding the different types of cells that can be analyzed by the device.

[0231] The two main pathways for energy production (mitochondrial respiration and glycolysis) involve cellular oxygen consumption and proton efflux, respectively. The devices and methods disclosed herein include sensors, such as 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 used to measure analytes outside, inside, and around cells.

[0232] According to certain embodiments, a system, also referred to herein as an apparatus, is disclosed herein. The apparatus may include a stage adapted to support a multi-porous sample carrier, also referred to herein as a sample carrier or a sample carrier cartridge. The apparatus may include a sensor adapted to sense cellular constituents associated with a cell sample in a well of the multi-porous sample carrier. The apparatus may include a dispensing system adapted to introduce a fluid into the well. The apparatus may include a plunger adapted to receive a barrier to generate a reduced volume of a medium within the well comprising at least a portion of the cells, the barrier adapted to be inserted into the well by relative movement of the stage and the plunger.

[0233] In particular, the device can include a plurality of sensors, each adapted to sense cellular constituents in a corresponding well of a multi-porous sample carrier. Thus, the device can include a sensor array. The sensors can independently and selectively sense cellular constituents in each well. The distribution system can include one or more injectors. The distribution system can be configured to independently and selectively introduce a fluid or reagent into each well. A plunger can be adapted to independently and selectively insert into each well.

[0234] The apparatus may include a motion actuator assembly, also referred to herein as a lift mechanism, constructed and arranged to position or orient one or more components along at least one coordinate axis. The motion actuator assembly may include one or more high torque motors configured to drive the system components.

[0235] The motion actuator assembly may include at least one axis actuator assembly. In some embodiments, the motion actuator assembly may include at least one x-axis actuator assembly configured to position the stage relative to the sensor. The x-axis actuator assembly may additionally or alternatively be configured to position the sensor relative to the stage. The x-axis actuator assembly may additionally or alternatively be configured to position the stage relative to the housing. The motion actuator assembly may include at least one z-axis actuator assembly configured to position the sensor and / or dispensing system relative to the stage. The z-axis actuator assembly may additionally or alternatively be configured to position the stage relative to the sensor and / or dispensing system. The motion actuator assembly may include at least one y-axis actuator assembly configured to position the stage relative to the sensor. The y-axis actuator assembly may additionally or alternatively be configured to position the sensor relative to the stage.

[0236] In use, the motion actuator assembly can be configured to align or substantially align the sensor unit and / or injector array with corresponding wells of a porous sample carrier positioned on a stage. In use, the motion actuator assembly can be configured to achieve fluid communication between one or more components (e.g., a sensor unit or injector of a dispensing system) and a sample within a well of the porous sample carrier.

[0237] In an exemplary embodiment, one or more sensors may be adapted to sense changes in the oxygen level and pH (proton concentration) of the cell medium associated with the metabolic activity of the cell sample in the pores of the porous sample carrier. The stage, sensor, and distribution system can be used in conjunction with the sensor to simultaneously measure the basal oxygen consumption rate and basal extracellular acidification rate of the cell sample. Thereafter, the distribution system can be used to sequentially administer one or more reagents to the cell sample. In an exemplary embodiment, the one or more reagents may include a mitochondrial ATP synthase inhibitor (oligomycin A), a mitochondrial uncoupler BAM15, and / or a mixture of mitochondrial compound I and compound III inhibitors (rotenone and antimycin A, respectively). The sensor can optionally measure the oxygen consumption rate and the extracellular acidification rate substantially simultaneously after each distribution of one or more reagents. Before distributing the reagents, additional reagents, such as regulating reagents, may be optionally distributed, or after rotenone / antimycin A is injected into the cells, the extracellular membrane ion carrier monensin may be injected. After each distribution, the same oxygen consumption rate and extracellular acidification rate measurements may be performed.

[0238] Components of the device are further described in, for example, U.S. Patent No. 7,276,351, entitled “Method and Apparatus for Measuring Multiple Physiological Properties of Cells,” and U.S. Patent No. 8,658,349, entitled “Cell Analysis Apparatus and Method,” each of which is incorporated herein by reference in its entirety for all purposes.

[0239] One or more of the following features may be included: The sensor may be configured to analyze the component without disturbing the cell. The well may include a step. The plunger or barrier may be adapted to agitate the medium prior to analyzing the component.

[0240] The sensor can be a photoluminescence-based sensor. The sensor can be, for example, a fluorescence sensor, a luminescence 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 aperture can be adapted to receive the sensor. The reduction in the volume of the medium achieved by the plunger can comprise the sensor, and / or at least a portion of the barrier can comprise the sensor.

[0241] The device may include a light source, such as a fluorescent lamp, a light emitting diode (LED), or a laser, configured to excite the sensor of the sensor unit to generate a signal in response to the target analyte or characteristic being measured. In some embodiments, the light source may be configured to generate a reference signal. By monitoring the reference signal generated by the light source, fluctuations in the intensity of the light source may be proportionally corrected to compensate for drift. The light source may be positioned on a thermally conductive printed circuit assembly that is configured to minimize the drift of the light source. In some embodiments, the thermally conductive printed circuit assembly may be formed of a material configured to minimize the drift generated by the heat-induced fluctuations of the light source by at least 20%, for example, at least 15%, 10%, 5%, or 1%.

[0242] In certain embodiments, one or more sensors may be adapted to analyze (determine the presence or concentration of) extracellular components in the pores, such as CO2, O2, Ca ++ 、H + Analytes that are proportional to O2 content include, for example, CO2, O2. Analytes that are proportional to sample pH include, for example, Ca ++ 、H + More than one analyte may be measured, for example at least one analyte to analyze an extracellular component.

[0243] One or more sensors may be suitable for analyzing a first extracellular component. In some embodiments, one or more sensors may be suitable for analyzing multiple extracellular components, such as 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 separately (e.g., sequentially). The present disclosure generally describes a sensor unit that is configured to analyze a first target analyte, such as 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.

[0244] In certain embodiments, the sensor is an optical sensor. The optical sensor can be a sensor based on fluorescence or phosphorescence. The sensor can alternatively utilize solid-state, nanoparticle, microparticle and / or magnetic sensors, etc. For example, a solid-state sensor can include one or more dots or membranes on a cover, base, protrusion, or a combination thereof, wherein a particle-based sensor can generally be a solution or suspension. Alternatively, in one aspect, a particle-based sensor can be loaded into a cell or coated on a surface. Nevertheless, such sensors can include optical, O2, pH, temperature, CO2, or a combination thereof.

[0245] Furthermore, in one aspect, the sensor can be an electrochemical or potentiometric sensor. Additionally or alternatively, electrodes can also be included in the well to measure electrical properties including impedance. While a sensor is selected, in one aspect, as described above, it should be understood that the well or chamber can also contain one or more reference probes in the form of any of the above-described sensors that generate a signal of a known value for device calibration.

[0246] An exemplary sensor unit is an oxygen-sensitive photoluminescent dye. The photoluminescent dye can be selected from any oxygen-sensitive photoluminescent dye. The appropriate dye can be selected based on the intended use of the probe. A non-exhaustive list of suitable oxygen-sensitive photoluminescent dyes specifically includes, but is not limited to, ruthenium (II)-bipyridine and ruthenium (II)-diphenylphenothiophine complexes, porphyrinones such as platinum (II) / octaethylporphinone, platinum (II) / porphyrins such as tetrakis (pentafluorophenyl) porphine, palladium (II) porphyrins such as palladium (II) porphyrins, tetrakis (pentafluorophenyl) porphyrin, phosphorescent metal complexes of tetrabenzoporphyrin, dichloro, azaporphyrins, and long-decay luminescent complexes of iridium (III) or osmium (II).

[0247] Typically, in these embodiments, the hydrophobic oxygen-sensitive photoluminescent dye can be compounded with a suitable oxygen-permeable and hydrophobic carrier matrix. Suitable oxygen-permeable hydrophobic carrier matrices can be selected based on the properties of the expected biological sample to be tested and the selected dye. A non-exhaustive list of suitable polymers for use as oxygen-permeable hydrophobic carrier matrices specifically includes, but is not limited to, polystyrene, polycarbonate, polysulfone, polyvinyl chloride, and some copolymers. Another approach is to dye oxygen-permeable microbeads with an oxygen-sensitive photoluminescent dye, mix the dyed microbeads with silicone or polyurethane, and apply the mixture as a polymer coating.

[0248] Regardless of the type of solid sensor selected, in one aspect only, the sensor can be embedded in a permeable medium, such as a permeable medium selected from a hydrogel, a silicone resin, and a matrix gel. In some aspects, the sensor is attached to the at least one protrusion by solidifying or removing the medium (e.g., by drying, curing, cooling, evaporation, or other techniques). The solid-state sensor can be applied by dipping or spot-coating the distal end of the at least one protrusion in a fluorescent indicator mixture in the medium.

[0249] However, it should be understood that in some aspects, the sensor can be applied or immersed on all or a portion of one or more protrusions. It should be further understood that in some aspects, the sensor can be removably connected to the body of one or more protrusions of the assembly. It should be further understood that in some aspects, the sensor can be integrally formed with one or more protrusions. The sensor can be integrally formed on one or more protrusions using one or more techniques, such as vapor deposition, chemical coating, spin coating, dipping, and robotic spotting.

[0250] The distribution system can include one or more injectors configured to independently and selectively introduce a fluid or reagent into each well. In some embodiments, the distribution system can include an array of injectors, for example, at least one injector positioned to correspond to each well of the sample carrier. In some embodiments, the distribution system can include one or more movable injectors, each configured to introduce a fluid or reagent into multiple wells of the sample carrier.

[0251] In certain embodiments, to actuate movement of one or more injectors (e.g., across a plurality of apertures), the apparatus may include an injector motion actuator assembly positioned to drive at least one injector. The injector motion actuator assembly may drive the one or more injectors through a row of apertures, a column of apertures, or through any configuration of apertures in a preselected pattern.

[0252] Thus, the device may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 movable injectors positioned to be driven over a plurality of holes, a row of holes, or a column of holes. Alternatively, the distribution system may have one or more arrays of injectors, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 injectors, fixedly positioned to correspond to each hole. The device may have a hole to injector ratio of 1:1 to 1:384, such as 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 may have an injector to orifice ratio of 1:1 to 1:384, such as 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.

[0253] The components and processes according to example aspects of the present disclosure can be well adapted for measuring components in all different types of samples (e.g., biological samples). For example, in one aspect, the systems and processes according to example aspects of the present disclosure can be used to measure one or more components in a cell material or parameters associated with the component. The one or more components can be contained in the culture medium surrounding the cell or in the cell itself. In some embodiments, the biological sample being tested may contain cellular material derived from the cell, such as organelles, mitochondria, cell extracts, cell products or byproducts, or conditioned culture medium. The measurement can be performed in a label-free manner.

[0254] An example system is Figure 1-Figure 4 As shown. Figure 1-Figure 4 As shown, the device or apparatus 100 includes a housing 10 having an opening in a side wall of the housing 10. The opening can be optionally closed by a door 12. A stage 20 is disposed within the housing 10 and is adapted to receive a porous sample carrier 30. The stage 20 can be moved through the opening into or outside the housing 10 by an x-axis actuator assembly. When the stage 20 is placed within the housing 10 for testing, the door 12 can be closed. The housing can include one or more electronic ports 14 that can be connected to a computer and / or a power source.

[0255] 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. Figure 1-Figure 4The device shown includes an electronics port 14, however, it should be noted that the device can be connected to an external computer by any means known in the art, such as wireless fidelity (WiFi), ultra-high frequency radio waves (also known as ) or any other data transmission connection. In an embodiment, the device can be connected to an external computer via the cloud.

[0256] Figure 5 An exemplary assembly 110 is shown in FIG. Component 110 may be included in Figure 1-Figure 4 10 is shown within the housing 10. Assembly 110 includes components of a sensing system 40 (e.g., optical fiber) and a dispensing system 50. Sensing system 40 includes an array of sensor units, and dispensing system 50 includes an array of injectors disposed on a manifold. In some embodiments, the manifold includes apertures through which pressurized air can be forced through injection ports on a sensor cartridge having ports corresponding to the apertures in the manifold and "sealed" by a gasket and force applied to the manifold. One or more components of the manifold can be independently moved 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 can be controlled by a manifold temperature controller 52. Assembly 110 includes a stage 20 adapted to receive a multi-porous sample carrier 30 (with the cartridge shown at the top). The temperature of the sample within the multi-porous sample carrier 30 can be controlled by a sample temperature control element 22. Stage 20 can be moved along the x-axis, as indicated by an x-axis actuator assembly 24 of the motion actuator assembly. The motion actuator assembly further includes a y-axis actuator assembly 26 configured to move the stage 20 along the y-axis.

[0257] The device may include an automatic measurement system. The device may also include or be connectable to a computer, and the automatic measurement system is in electrical communication with the computer. In certain embodiments, the device may also include a controller for adding one or more fluids or reagents to one or more wells of the microplate. The controller can operate the sensor to achieve sensing of one or more components in one or more wells of the microplate. The system can communicate with the controller and the sensor through a graphical user interface residing on the computer. The graphical user interface can be configured to receive instructions for designing a multi-well experiment according to the method disclosed herein, instruct the controller to perform the multi-well experiment, and receive data acquired by the sensor in response to the execution of the multi-well experiment.

[0258] In certain embodiments, the graphical user interface may include multiple display areas, each area being assigned to one of the wells. The graphical user interface may be configured to receive instructions written in each area assigned to one of the wells to design a multi-well experiment, and to receive data acquired by the sensor in response to the execution of the multi-well experiment for display in each area assigned to one of the wells. Thus, a method executable by the controller can be independently and selectively applied to one or more wells via instructions from the graphical user interface.

[0259] Figure 6 An exemplary system is shown, including a system (laboratory device) that can be connected to a cloud-based computing network and a computer connected via the cloud-based network. The system includes a detector or sensor unit and other electronic components (such as a signal processing module and a motion actuator). The detector and electronic equipment 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 distribution system. The 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, laptop computer and / or tablet computer or other mobile device. The web browser software platform can provide cloud-based data processing, cloud-based storage and / or cloud-based connection between the computer and the system. Other mechanisms for connecting to the cloud can be used, such as desktop software or driver software. A data storage module can also be included in the system, such as a local memory storage device (e.g., a server, an external drive, a portable drive) and / or a cloud-based memory storage device. The data storage module can store historical data, protocols, data processing algorithms and / or controller executable instructions.

[0260] Figure 7-Figure 8 is a schematic diagram of the system disclosed herein and showing the electronic components in greater detail. Figure 7 is a schematic diagram of a system operably connected to a central control computer. The baseboard includes a microcontroller or system controller that is operably connected to temperature control elements for the manifold and tray (i.e., sample temperature control elements) and a distribution system or injection unit. Another microcontroller (also referred to herein as a "motion controller") is shown as being operably connected to the controller and motion actuator assemblies, including a z-motor that operates the z-axis actuator assembly and an x-motor that operates the x-axis and optional y-axis actuator assemblies. The devices described herein can include stepper motors with higher torque, which improve measurement accuracy over the service life of the device and reduce the need to provide maintenance and / or replace motor components.

[0261] A proximity and / or encoder sensor is also provided as part of a motion actuator assembly configured to sense the relative positioning of a stage or multi-well sample carrier and other device components, such as a sensor unit and a dispensing system injector. The proximity sensor can be configured to generate a notification signal, and optionally pause a protocol, if one component is positioned within a predetermined distance from another component, such as a sensor unit within a predetermined distance from a corresponding well of a sample carrier. Additionally or alternatively, the proximity sensor can be configured to generate a notification signal, and optionally pause a protocol, if an opening in a side wall of the housing is ajar and / or external light is detected within the housing.

[0262] The system may also include a stall sensing module programmed to generate a notification signal and optionally pause the protocol (e.g., stop motor movement) if the predetermined protocol steps are not completed within a predetermined time interval. The stall sensing module may be configured to detect stalls using an encoder. For example, the encoder may operate by looking for a timing-related delay in the encoder travel and flagging a stall.

[0263] Figure 7 The schematic diagram also includes sensing units for O2 and pH analytes, which are operably connected to a signal processing module comprising an amplifier and a microcontroller configured to receive and amplify signals from the sensor units. The signal processing module is also operably connected to a system controller and a central control computer. The system also includes a barcode scanner configured to scan barcode-encoded information that is operably transmitted to the central control computer.

[0264] Figure 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 the barcode and transmit the information to the computer. The system controller is operably connected to a tray heater or sample temperature control element, which is configured to control the temperature of the consumables or samples within the porous sample carrier. The system controller is also operably connected to a transmission amplifier or a 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, which is configured to control the temperature of the injection manifold or distribution system. Optionally, a separate system controller operably connected to the manifold heater or manifold temperature control element can be provided.

[0265] Figure 9An exemplary sensor unit 41 is shown disposed within the well 31. The exemplary sensor unit 41 is a fluorescence sensor. A fluorophore having a fluorescence property that depends on at least one of the presence and concentration of a component in the well 31 can be disposed on the surface of the well 31. The sensor unit 41 can include a housing for receiving a waveguide for at least one of exciting the fluorophore and receiving fluorescence emissions from the fluorophore.

[0266] The present disclosure provides a method, apparatus and measurement system for adding a test compound to a hole and measuring the composition of the hole with a sensor. The method can be performed as a high-throughput assay by adding one or more test compounds to one or more holes, or adding a plurality of identical or different test compounds to a plurality of holes of a microplate. In certain embodiments, the test compound is introduced while the sensor probe is kept in balance with the liquid contained in each hole (e.g., kept immersed therein). In these embodiments, since the sensor probe remains immersed in the compound delivery process, the equilibrium time can be shortened. Therefore, a system and method for storing and distributing a single preselected test compound or a compound of a preselected concentration per hole is provided.

[0267] In certain embodiments, the apparatus and method store and deliver one or more test compounds or target agents per well. Test compounds can be delivered using a compressed gas supply from a remote source to initiate compound delivery. In certain embodiments, both the sensor probe and the test compound delivery mechanism are combined within a single disposable cartridge. Also described is a pneumatic multiplexer that, when temporarily connected to the cartridge, allows a single actuator to initiate test compound delivery from multiple ports using a compressed gas supply from a remote source.

[0268] In one aspect, a cartridge adapted to cooperate with a porous sample carrier having a plurality of wells is provided. The cartridge can include a substantially planar element having a plurality of regions having the same number of corresponding openings as the wells in the porous sample carrier. At least one port can be formed in at least one region of the cartridge, adapted to deliver a test fluid, such as an aqueous solution of a candidate compound / substance compound or other reagent, to the corresponding 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) a well adapted to receive a sensor located in a subregion of at least one region of the cartridge.

[0269] For example, U.S. Patent No. 9,170,255, entitled "Cellular Analysis Apparatus and Method," which is incorporated herein by reference in its entirety for all purposes, further describes components and features of the cartridge.

[0270] The device may include a lifting mechanism suitable for moving the box relative to the stage or plate to place the sensor in the hole, typically placing multiple sensors in multiple holes at the same time. A pressure source suitable for cooperating with the cartridge fluid can be provided to deliver the test fluid from the port in the cartridge to the hole. The device may also include a multiplexer arranged between the pressure source and the cartridge, the multiplexer being suitable for being in fluid communication with multiple ports formed in the cartridge. The multiplexer can selectively be in fluid communication with a dedicated port group formed in the box. A controller may be provided to control the lifting mechanism, the multiplexer and / or the pressure source so that when the relevant sensor is arranged in the hole, the test fluid can be delivered from a given port or a group of ports to the corresponding hole or a group of holes. As previously mentioned, the controller can communicate with a computer or a graphical interface.

[0271] In certain exemplary embodiments, a well of a cartridge adapted to receive a sensor can include a sensor sleeve structure having a surface proximate to a well of a porous sample carrier. Disposed on the surface can be a fluorophore having a fluorescent property that depends 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 exciting the fluorophore and receiving a fluorescent emission from the fluorophore.

[0272] The sensor array corresponding to the well array may be integrated with the cartridge, but may also be a separate element that cooperates with and is disposed within the wells formed in the cartridge.The sensor array may be flexibly mounted relative to the sample carrier.

[0273] Methods for analyzing cells using the devices disclosed herein are provided. These methods can be used to measure cells disposed in a medium in a porous sample carrier. The methods can include one or more of the following: disposing at least a portion of a sensor in a medium in a pore of the porous sample carrier, analyzing components associated with the cells in the medium in the pores, delivering a test fluid to the pores while the sensor remains disposed in the medium in the pores, and further analyzing the components to determine any changes therein. In certain embodiments, one or more components can be analyzed substantially simultaneously. In particular, the rate of change of one or more components can be measured over a measurement time, for example, to determine metabolic or other activity of a cell sample.

[0274] The analyzing step may include analyzing each component associated with each cell in the medium in each well. The each component may be the same component. The delivering step may include delivering the corresponding test fluid or target reagent to the corresponding well while the corresponding sensor remains disposed in the medium in the corresponding well. The each test fluid or reagent may include the same test fluid or agent.

[0275] The analyzing step can include analyzing the various components associated with the various cells in the medium in each well to determine any corresponding changes therein. The delivering step and further analyzing step can be repeated. Between two measurements, different test fluids or reagents or additional aliquots of the same test fluid or reagent can be delivered. The method can include maintaining a substantial 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.

[0276] Described method can include before, during and / or after the analysis step, control the temperature and / or environment of cell sample.In certain embodiments, described method can include in the execution process of whole analysis method, control the temperature and / or environment of cell sample.Controlled environment can include, for example, control the composition (such as N2, O2 and / or CO2 concentration) of relative humidity (RH) and / or ambient gas.For example, in certain embodiments, controlled environment can include by using N2 gas purge air to induce anoxic environment.

[0277] The method may further comprise imaging or scanning the sample during the analyzing step, during the delivering step, and / or after the analyzing step and / or delivering step.

[0278] 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 living cell samples. The sample can include or exist in the form of one or more of loose cells, cell constructs, loose tissues, tissue constructs, organelles, enzymes, cell products or by-products, and conditioned medium. 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 (e.g., seeds, pods, or other plant materials). The sample can include single-cell organisms, such as microorganisms. In certain exemplary embodiments, the sample can include whole plants or animal model tissues (e.g., zebrafish, Caenorhabditis elegans, fruit flies).

[0279] The biological material being analyzed may include cellular material. The biological material may contain living cells, including bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, insect cells, etc. These cells may be animal cells, human cells, immune cells, or immortalized cells.

[0280] Exemplary cells include human T cells (CD4+, Pan-CD3+, CD8+, PBMCs, such as naive, activated, effector, and memory), mouse T cells (splenic-derived CD8 naive and activated), immortalized mouse myoblasts (e.g., C2C12), Jurkat cells, lung cancer cell models (A549, PC9, H1373), leukemia cancer cell models (THP-1), human hepatoma cells (e.g., HepG2), human epidermoid carcinoma cells (e.g., A431), and analysis of whole organisms, such as zebrafish, Caenorhabditis elegans, and Drosophila. Certain aspects of the devices and methods disclosed herein enable analysis of living cells that require a temperature of 28°C–40°C without the need to place the device in a temperature-controlled chamber.

[0281] The devices and methods disclosed herein can be used to facilitate research in areas such as cancer, immunology, toxicology, compound / substance discovery, and immunotherapy.

[0282] In one aspect, cell sample is obtained or derived from a subject (such as humans or non-human animals). In one aspect, the subject is a mouse, and in one aspect, it suffers from a disease or has the risk of suffering from a disease. However, in one aspect, cell sample can include primary cells, cells directly separated or harvested from living tissues or organs, cultured cells and / or immortalized cells. However, in one aspect, cell sample can include primary cells, cells directly separated or harvested from living tissues or organs, cultured cells and / or immortalized cells. In one aspect, cell sample includes modified cells (such as genetically engineered to heterologously express a gene of interest, and / or genetically engineered to inhibit gene expression, such as cells from knockout mice or CRISPR KO libraries). However, in one aspect, cell sample includes stem cells or cells derived from stem cells. However, no matter what kind of cell is used, in one aspect, cell sample includes culture medium, such as culture medium or growth medium, wherein cells can be placed in culture medium. In addition, as will be understood, in one aspect, cell sample includes multiple cells (such as multiple cells as described herein).

[0283] The cells tested may include any suitable cell samples, 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, bird cells, insect cells, reptile cells, amphibian cells, etc. The cells tested may also include monolayer cells, two-dimensional cell samples, three-dimensional cell samples (such as tissue samples, cell spheroids, organoids, biopsy samples, cell scaffolds, organs on chips, etc.). Examples of parameters that can be measured and related to the above-mentioned battery function include carbon dioxide concentration, oxygen concentration or oxygen partial pressure, calcium ions, hydrogen ions, etc. However, in one aspect, the parameter measured is oxygen concentration (e.g., oxygen consumption). Through these tests, people can understand the factors that drive cell phenotype and function and / or the accurate situation of the cell environment or microenvironment.

[0284] The assembly and method according to the exemplary aspects of the present disclosure can be used to measure the (micro) environmental conditions of living cells or any living cells. For example, the cell material tested can include bacterial cells, fungal cells, yeast cells, prokaryotic cells, eukaryotic cells, etc. The cells that can be tested include mammalian cells (including animal cells and human cells). The specific cells that can be tested include cancer cells, immune cells, immortalized cells, primary cells, induced pluripotent stem cells, cells infected with viruses or bacterial pathogens, etc.

[0285] For example, in one aspect, the assemblies and methods according to the exemplary aspects of the present disclosure can be used to assist in immunotherapy. Immunotherapy is a treatment method that enhances a patient's immune system to fight cancer, infection, and other diseases. For example, the immunotherapy process can include adoptive cell-based therapies, such as the production of T cells, natural killer (NK) cells, monocytes, macrophages, combinations thereof, and the like. For example, during T cell therapy, T cells are removed from the patient's blood. The T cells are then sent to a bioreactor for expansion or culture. In addition, T cells can be modified to have specific proteins called receptors. The receptors on the T cells are designed to recognize and target unwanted cells in the body, such as cancer cells. The modified T cells are cultured in a bioreactor to reach a certain cell density and then supplied to the patient's body for use in fighting cancer or other diseases. T cell therapy can also be referred to as adoptive T cell therapy or T cell transfer therapy, an example of which is chimeric antigen receptor (CAR) T cell therapy. Due to the great success in fighting blood diseases, T cells have recently been widely used for adoptive T cell therapy or T cell transfer therapy. In some embodiments, aspects of the present disclosure 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 present disclosure can be used to monitor T cell activation, T cell exhaustion, T cell metabolism (including starting materials and modified products, etc.).

[0286] NK cells are a type of cytotoxic lymphocyte that seeks out and destroys infected cells in the body. They can mount very rapid immune responses, and their application in cancer therapy has therefore attracted significant interest and attention. However, the number of NK cells in mammalian blood is limited, requiring them to be grown to relatively high cell densities within bioreactors.

[0287] The cultivation of cells (such as T cells, NK cells or other mammalian cells) generally requires a slightly complex process from inoculation to use in patients. The assembly and method disclosed herein can be used to monitor cell metabolism at any point in the culture process to ensure that the cells are healthy and / or have a desired metabolic phenotype and that the culture medium in which the cells are grown contains nutrients at optimized levels. For example, the system and process can be used to make adjustments to ensure the metabolic adaptability of the cells during growth.

[0288] In addition to immune cells, the metabolism of cancer cells can also be monitored to understand which nutrients fuel the cancer cells. For example, assemblies and methods according to exemplary aspects of the present disclosure can reveal mechanisms or components that affect cancer cell metabolism to inhibit growth. Assemblies and processes according to exemplary aspects of the present disclosure can also be used to determine the rate at which cancer cells proliferate. The systems and methods of the present disclosure are also very suitable for use in toxicology. For example, the methods and assemblies of the present disclosure can be used to detect mitochondrial liability in potential therapies. For example, the risk of mitochondrial toxicity can be assessed with high specificity and sensitivity. In this way, the mechanism of action of some mitochondrial poisons can be determined.

[0289] Electrical measurement module

[0290] According to certain embodiments, the system further comprises an electrical measurement module configured to measure various electrical properties of the 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 sample held in each well and provides an electrical signal of the measured property to the control module to track changes in the electrical property over an extended period of time (e.g., between 6-72 hours). In other embodiments, the electrical measurement module excites the sample held in the wells of the sample carrier and measures the electrical signals of the excited cells.

[0291] Figure 47A4900 is a schematic diagram of a consumable 4900 having two electrode structures having the same or similar area and deposited on a substrate (e.g., a sample carrier) in which one or more holes are formed. The first electrode structure has electrode elements 4910a-4910c, and the second electrode structure has electrode elements 4910d-4910f (commonly or collectively referred to as electrode elements 4910). The electrode elements within the electrode structure are connected to each other by an arc-shaped connecting electrode bus 4925. Like the electrode element 4910, this connecting bus 4925 is also made of a conductive material (e.g., a gold film on a gold film, a platinum film, a chromium film, or a titanium film). These conductive connection paths or connecting buses 4925 may have an insulating coating. The electrode element 4910 includes an electrode wire with connected circles added thereto. The total area of ​​the electrode elements 4910 and the gaps between the electrode elements 4910 can correspond to the bottom of the well (e.g., a cylindrical well, a conical well, or a cubic well), or can be slightly larger or slightly smaller than the bottom of the well (e.g., a commonly used 24-well sample carrier, a 96-well sample carrier, or a 384-well sample carrier). The entire surface of the well can be covered with electrodes to ensure that molecular interactions occurring on the bottom surface of the well will result in impedance changes. The advantage of this arrangement is that the non-uniform molecular interactions occurring on the bottom surfaces of these wells will result in only small changes in the impedance measured between the electrode elements 4910. Although three electrode elements 4910 extending from each connection bus 4925 are shown, in various embodiments, more or fewer electrode elements 4910 of different lengths, widths, and surface features may be used.

[0292] 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.

[0293] One or more gaps or windows 4920 are defined between the electrode elements 4910 to allow imaging of the various contents of the well in which the consumable 4900 is disposed. In various embodiments, the window 4920 can be located in the center of the consumable 4900 to correspond to the center of the well, but various sub-windows 4920 can also be defined so that the space is not occupied by the electrode structure 4910, the connection bus 4925, or the connection pad 4950. These sub-windows 4920 can be aligned with other sub-divisions defined within the microwell or well, or various features of the sample to be imaged.

[0294] Figure 47B is a schematic diagram of consumable 4900 having two electrode structures of similar area deposited on a substrate. Figure 47B As shown, electrode elements 4910a-4910f are rectangular wires and together form an interdigitated electrode structure unit, although other shapes and sizes may be used in various embodiments. Figure 47A Similarly, the electrode elements 4910 within each electrode structure are connected by arcuate conductive paths or electrode buses 4925. The connection pads 4950 are connected to the electrode structures by electrical connection traces 4930. One or more gaps or windows 4920 are defined between the electrode elements 4910 to allow imaging of the various contents of the well in which the consumable 4900 is disposed. In various embodiments, the window 4920 can be located in the center of the consumable 4900 to correspond to the center of the well, but various sub-windows 4920 can also be defined so that the electrode structure 4910, the connection bus 4925, or the connection pads 4950 do not occupy this space. These sub-windows 4920 can be aligned with other sub-divisions defined within the microwell or well, or various features of the sample to be imaged.

[0295] Figure 47C FIG4 is a schematic diagram of a consumable 4900 having electrode structures 4930a-4930d deposited on a substrate and having similar areas. The electrode structures 4930a-4930d include a plurality of interconnected electrode elements 4910a-4910h. The electrode elements 4910 are rectangular wires and together form an interdigitated electrode structure unit, although other shapes and sizes may be used in various embodiments. Figure 47A and Figure 47B In contrast, an electrode structure having electrode elements 4910a-4910c and 4920a-4920d is connected to a connection pad 4950. One or more gaps or windows 4920 are defined between the electrode elements 4910 to allow imaging of the various contents of the well in which the consumable 4900 is disposed. In various embodiments, the window 4920 can be located in the center of the consumable 4900 to correspond to the center of the well, but various sub-windows 4920 can also be defined so that the space is not occupied by the electrode structure 4910, the connection bus 4925, or the connection pad 4950. These sub-windows 4920 can be aligned with other sub-divisions defined within the microwell or well, or various features of the sample to be imaged.

[0296] Examples of electrical measurement modules are further described in, for example, US Patent No. 7,470,533, entitled "Impedance-Based Devices and Methods for Use in Assays," the entire contents of which are incorporated herein by reference for all purposes.

[0297] Temperature control

[0298] Device as described herein includes one or more temperature control elements, which are used to reduce the temperature gradient between the outer pore (such as boundary hole) and the inner pore of the porous sample carrier. Sample temperature control element and manifold temperature control element are described herein. Temperature control element can be designed to control temperature independently of each other. Temperature control element is usually formed by heat-conducting material, and this material is optionally positioned to be in close proximity or direct contact with one or more parts (such as porous sample carrier, sensor unit and / or injector). For example, the size of sample temperature control element can be designed to be suitable for porous sample carrier. The size of manifold temperature control element can be suitable for sensor, injector and / or cartridge, and optionally, when cartridge is positioned to cooperate with porous sample carrier, for example, when sensor unit and / or injector are in communication with the hole fluid of porous sample carrier, cover porous sample carrier. In certain embodiments, a microenvironment including manifold and heater and a heating component around sensor cartridge and a tray heater in direct contact with sample carrier is formed, which allows this temperature to be maintained for a period of extended time. Manifold temperature control element can be configured to cooperate with sample temperature control element, to cover porous sample carrier.

[0299] The design of the temperature control element forms a controlled temperature zone or microenvironment within the device. The controlled temperature zone typically includes the array of holes of the sample carrier. In particular, the controlled temperature zone does not include the head space of the housing, nor does it include a large part of the head space, for example, the temperature control does not extend to the entire inner cavity of the device, so that the temperature of the components outside the controlled temperature zone will not be substantially changed (e.g., increased or decreased) by activation of the temperature control element. In some embodiments, the volume of the controlled temperature zone does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times the volume of the sample carrier. In some embodiments, the volume of the controlled temperature zone does not exceed 10% of the volume of the housing, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0300] Surprisingly, the design of the temperature control element allows the device to operate at lower temperatures than expected, for example, at 8°C or lower, compared to the typical low-end operating temperature of 12°C. The lower limit of the operating temperature is sometimes limited by the heat generated by system components (such as motors or motor control components, power supplies, circuit boards, and light sources). The lower operating temperature allows the device to be used to examine sample types that were previously impossible to examine with such devices, such as zebrafish, whole-cell organisms, or non-mammalian cells. Therefore, in some embodiments, the temperature control element can control the sample temperature in each well to less than 12°C, for example, less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1°C.

[0301] The creation of a controlled temperature zone or microenvironment typically allows the device to maintain the sample temperature within each well of the sample carrier within a predetermined range of the target temperature within approximately 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.

[0302] Furthermore, the design of the temperature control element enables the device to achieve temperature uniformity and a wider operating temperature range than previous designs. This wider operating temperature range allows the device to be used with a wider variety of cell types, such as non-mammalian cells that may require lower or higher temperatures than previously possible, thereby improving viability during the assay. Higher operating temperatures can increase the sensitivity of the sensing element, for example, allowing the device to achieve a lower OCR detection limit than previous devices. In some embodiments, this improves the uniformity and / or accuracy of measurements.

[0303] The manifold temperature control element can be configured to control the temperature of a target reagent and / or sensor unit to within 3° C., e.g., 2° C., 1° C., 0.6° C., 0.5° C., 0.4° C., 0.3° C., 0.2° C., or 0.1° C., of another injector and / or sensor unit. In certain embodiments, the manifold temperature control element can be configured to control the temperature of a target reagent and / or sensor unit, and the sample temperature control element can be configured to control the temperature of samples within the well array of a sample carrier to within 3° C., e.g., 2° C., 1° C., 0.6° C., 0.5° C., 0.4° C., 0.3° C., 0.2° C., or 0.1° C., of each other. Thus, the temperature control elements disclosed herein can generally maintain temperature uniformity between different samples in a sample carrier, e.g., interior and boundary samples of a sample carrier, and / or between test component components of a sample carrier and their corresponding samples.

[0304] In some embodiments, the sample temperature control element is configured to control the sample temperature in each well of the sample carrier to within a predetermined range. Exemplary predetermined ranges include 0°C–70°C above ambient temperature, for example, 8°C–20°C above ambient temperature, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, or 70°C above ambient temperature. In some embodiments, the sample temperature control element is configured to control the temperature of the sample (e.g., two identical or substantially identical samples) in each well of the sample carrier so that the sensor signal generated or consumed in response to the level of the target analyte does not differ by more than a predetermined amount between the two identical or substantially identical samples, for example, when the samples are analyzed under identical or substantially identical conditions, the difference between the two samples does not exceed 10%, for example, 5%, 3%, 1%, or 0.1%. In particular, the temperature control element can be configured to reduce or suppress fluctuations in sensor readings, such as photoluminescence sensor readings, cellular metabolism and other functions, and / or analyte concentrations that may arise due to temperature differences.

[0305] The design of the temperature control element reduces sample evaporation during protocol execution. When evaporation is severe enough to change the concentration of analytes in the culture medium, evaporation can affect cell function. The temperature uniformity achieved by the sample temperature control element and / or the manifold temperature control element compared to conventional devices indicates reduced sample evaporation. In some embodiments, the temperature control element can be configured to control evaporation of the sample within the well array to less than 25%, such as less than 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. For long-term assays, such as 6-hour assays, 8-hour assays, 10-hour assays or longer, evaporation can be controlled by these percentages. In addition, the porous sample carrier can be designed to reduce evaporation during cell culture and incubation.

[0306] Surprisingly, the design of the temperature control element provides a lower O2 detection limit and improved measurement accuracy. For example, the system disclosed herein can have an OCR detection range of 2000 pmol / min to 0.01 pmol / min, such as 700 pmol / min to 0.01 pmol / min, such as 50 pmol / min to 0.01 pmol / min. In some embodiments, the system can have an improved lower OCR detection limit of less than 50 pmol / min, such as less than 40 pmol / min, 30 pmol / min, 20 pmol / min, 10 pmol / min, 5 pmol / min, 3 pmol / min, 1 pmol / min, 0.1 pmol / min, or 0.01 pmol / min.

[0307] In addition, the temperature control element can be designed to reduce, limit or inhibit differential (gradient) diffusion of gases within the sample carrier, cartridge and / or the internal environment of the sample carrier or controlled temperature zone. The temperature control element can be configured to control, for example, reduce, limit or inhibit the diffusion of gases within the controlled temperature zone, cartridge, sample carrier so that the gas composition of the environment does not change significantly during the assay, for example, does not change by more than 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or 20% during the assay.

[0308] Environmental Control

[0309] Device as herein described may include or be associated with one or more environmental control modules, and the environmental control module is designed to control the environment around the porous sample carrier. The environmental control module can be designed to control the ambient gas and / or relative humidity (RH) of the sample surrounding environment. For example, the environmental control module can be configured to control one or more of the N2, O2 and CO2 concentrations of the gas around the sample. RH can be increased or decreased by the environmental control module. For example, RH can be reduced to less than 75%, 65%, 55%, 45% or 35%, or RH can be increased to greater than 65%, 75%, 85% or 95%. The environmental control module can make the device be used for ischemia / reperfusion modeling and other controlled gas experiments.

[0310] The environmental control module can include a gas source, such as one or more of N2, O2, and CO2, fluidly connected to the sample carrier. The environmental control module can form a controlled environment zone that includes the array of holes in the sample carrier. The controlled environment zone can be open or closed to the surrounding environment. The environmental control module can include a pump or fan configured to direct gas to the sample carrier or purge gas from the sample carrier.

[0311] In certain embodiments, an environmental control module is incorporated into the device. The controlled environment zone can be formed in an airtight container, such as an airtight container. The environment can be formed by moving a heating element to surround, cover, or seal a heated sample carrier. The heating element can be made of a heat-conducting material, such as metal, aluminum, steel, etc. These heat-conducting materials can be anodized to reduce / eliminate conductivity. The heat-conducting heating element can also block stray light (ambient light). In some embodiments, the container is substantially enclosed so that the air flow is minimized. The container can accommodate a sample carrier, such as a stage for accommodating a sample carrier. In some embodiments, the container can accommodate a cartridge with a sample carrier. In order to form a controlled environment zone, the sealed container can be connected to a gas source fluid and purged with one or more selected gases accordingly.

[0312] In some embodiments, an environmental control module is associated with the device. For example, in some embodiments, the device can be placed in a gas-controlled incubator or hypoxic chamber. Thus, the device can be configured for use in a gas-controlled environment, for example, by being formed from materials suitable for use in a gas-controlled environment, such as materials with low gas solubility.

[0313] The environmental control module can be integrated with the system software, for example, operatively connected to the controller and / or system processor. The software can be programmed to cycle the environmental control module according to a selected schedule.

[0314] The environmental control module can be integrated with the system software, for example, operably connected to a controller and / or system processor, to take input from measurements of the cell microenvironment (e.g., intracellular O2, pericellular O2, or O2 measurements near a cell sample), thereby allowing environmental control to deliver a target cell microenvironment. The software can be programmed to cycle the environmental control module to deliver the target microenvironment according to a selected protocol.

[0315] Imaging module

[0316] According to certain embodiments, the system further comprises an imaging module configured to capture and process images of a sample held in a sample carrier. An example imaging module comprises an image capture element (e.g., a camera or camera array) and associated auxiliary optical components that assist the image capture element in imaging the sample or sample features within each well of the sample carrier through windows on opposite sides of the sample carrier, from which the plurality of wells are defined. The image capture element or the sample carrier can be moved relative to each other by a motion stage to position the camera or camera array in alignment with the windows in the wells so that the contents can be imaged.

[0317] In various embodiments, a light source is associated with the imaging module to illuminate the sample, fluorescent labels, etc. The light source can be placed on the same side of the sample carrier as the image acquisition element (e.g., below the sample carrier, as a flash or direct light), on the opposite side of the sample carrier (e.g., as a backlight), or in another part of the cavity (e.g., for use as ambient light). In addition, the light source can be configured to generate light within the visible spectrum, infrared spectrum, ultraviolet spectrum, and combinations thereof, and the image acquisition element is configured to detect such light. In various embodiments, the image acquisition element or controller can color-shift portions of the image acquired outside the visible spectrum into the visible spectrum, apply grayscale scaling, color correction, etc. Example light sources are further described, for example, in U.S. Patent No. 10,072,982, entitled "Universal Multi-Detection System for Microplates," the entire contents of which are incorporated herein by reference for all purposes.

[0318] Figure 42A light source 4400 is shown according to an exemplary embodiment of the present disclosure. In some embodiments, the light source 4400 includes two light generating devices: a xenon flash lamp 4410 and a tungsten lamp 4420. In other embodiments, the light source 4400 may include a xenon continuous wave lamp, a light emitting diode (LED), a laser, or any other light generating device.

[0319] Tungsten sources are very stable, and their radiation extends from the blue of the visible spectrum into the far infrared, with a peak around 1 μm. They are best suited for measurements in the visible and infrared regions of the spectrum. In contrast, xenon flash sources provide most of their radiation in the deep ultraviolet, ultraviolet, and short visible spectral ranges. Furthermore, xenon flash sources provide very fast bursts of light, lasting a few microseconds and decaying quickly, making them suitable for time-resolved measurements in modern multi-detector systems.

[0320] The xenon flash lamp 4410 has a parabolic reflector 4411 positioned so that the arc 4412 of the lamp 4410 is located near the focus of the reflector 4411, thereby providing a substantially collimated light beam from the reflector 4411. The tungsten lamp 4420 has a parabolic reflector 4421 positioned so that the filament 4422 of the lamp 4420 is located near the focus of the reflector 4421, thereby providing a substantially collimated light beam from the reflector 4421. Figure 42 As shown, lens 4423 can be used to focus the light beam from reflector 4421 onto the exit port 4430 of light source 4400. Figure 43 As shown, according to an embodiment of the present disclosure, a relay optical device can be used to focus the light beam onto the entrance of the optical fiber. Alternatively, the lens 4423 can focus the light beam from the reflector 4421 directly onto the entrance of the optical fiber in the excitation spectroscopy device 4500.

[0321] In various embodiments, the excitation spectroscopy device 4500 has two spectral selection devices that differ in the physical techniques they use to separate light of different wavelengths. The first device is a filter selection device 4520 that has a variety of user-changeable filters 4521. The second device is a double monochromator 4530.

[0322] The first path directs light through one of the filters 4521 in the filter selection device 4520, which transmits narrowband light. The light then propagates through optical fiber 4522 to the exit port 4540. The second path bypasses filter 4521 by directing light through an aperture 4523 in the filter selection device 4520. The light then continues through optical fiber 4531, which accepts the circular image formed by the arc or filament spot from the light source 4400 at the entrance 4510 and shapes the spot into a slit shape to match the input slit of the double monochromator 4530. Monochromator 4530 selects the narrowband light, and filter 4521 then changes the shape of the light from the exit slit of monochromator 4580 to a circular shape similar to the aperture in the sample carrier 4700.

[0323] The optical path selector 4550 can be moved relative to the filter selection device 4520, providing the ability to direct light to the output port 4540 spectrally selected by the filter 4521 or the monochromator 4530.

[0324] The movable off-axis parabolic reflector 4440 has two working positions. In the first position, as Figure 42 As shown by the solid line in , reflector 4440 reflects and focuses the light from reflector 4411. Figure 42 In the second position, shown in dashed lines, reflector 4440 blocks light from reflector 4421. This arrangement allows light from either lamp to be focused on the same location. Additionally, fan 4417 directs air through fins 4415 of the cooling extrusion of xenon source 4410 and onto tungsten source 4420. This arrangement allows both sources to share a single cooling system.

[0325] Two light sources are arranged close together with their optical axes offset (preferably at an angle of approximately 90 degrees to each other), creating a very compact illumination system with a shared cooling system. Using parabolic reflectors around the light sources, combined with off-axis parabolic reflectors, allows for efficient coupling of arc and filament light into the system. The final focus of both light sources is the same. This system allows for a more compact arrangement than systems utilizing separate light source compartments, each with a separate exit point, thus requiring mechanical movement of an optical relay system to switch between the light sources.

[0326] Figure 44 The structure of an excitation-emission separation device 4600 according to an exemplary embodiment of the present invention is shown. The general purpose of the excitation-emission separation device 4600 is to illuminate a sample with excitation light and / or collect emission light from the sample. The excitation-emission separation device 4600 can be located above or below the sample carrier 4550 relative to the surface defined by the well, or one can be located above the sample carrier 4450 and the other below.

[0327] The sample carrier 4450 includes a substrate having several wells defined therein for accommodating samples for analysis. Each well includes a volume defining member configured to limit the range of motion between the substrate and a second component of the system and / or to define a minimum non-zero distance between the substrate and the second component of the system (e.g., to prevent the second component from contacting a sample held in the well). In various embodiments, these volume defining members include supports, protrusions, and stops located at a point above the bottom of the corresponding well that define the volume and shape of the well, as well as the spacing between the well, other wells in the sample carrier 4450, and other components of the system that operate with the sample carrier 4450.

[0328] In some embodiments, several optical paths can be used, depending on the measurement technique. For absorbance measurements, the excitation light and the emission light are preferably collinear. Figure 44 As shown, absorbance measurements are made in block 4640 with full illumination with excitation light from below at point G. This excitation light may come from monochromator 4530 or filter selection device 4520. Detector 4650 is placed on the opposite side of the well to collect the emitted light that has passed through the sample.

[0329] For luminescence measurements, no excitation light is required, and only the emission light is collected from the sample by the excitation-emission separation device 4600. In block 4630, a single fiber optic bundle 4735 is used to maximize the light gathering capability of the system, thereby improving the signal.

[0330] For fluorescence measurements, two optical paths can be used to illuminate the sample with excitation light and collect the sample's emission light. These paths can be optimized to further improve overall system performance.

[0331] Block 4620 depicts the first optical path for fluorescence measurement, which can use a partially reflecting mirror or a dichroic mirror to make the excitation light and the emission light collinear when entering and leaving the sample, respectively. The light is transmitted to block 4620 via optical fiber 4532. A movable aperture 4601 has multiple openings, preferably with diameters ranging from approximately 1.5 mm to 4 mm, and is positioned in front of the guide optical fiber 4522. Images of the openings positioned in front of the optical fiber 4522 are formed in the aperture 4555 by lenses 4621 and 4622. The opening size of the movable aperture 4601 is selected to fill the aperture with light as completely as possible while preventing light from entering adjacent apertures and causing crosstalk.

[0332] The light is reflected by a partially transmissive mirror 4623 on a movable holder 4627. Multiple mirrors can be placed on the holder 4627. Some of the mirrors can be dichroic mirrors to improve the signal because all the excitation light is reflected toward the aperture and all the emission light is transmitted toward the exit fiber. The dichroic mirrors can also improve the signal-to-noise ratio of the measurement system because the remaining excitation light that reaches the aperture and is reflected by the meniscus lens is blocked from reaching the exit fiber. The emission light from the aperture is focused onto the fiber bundle 4731 by lenses 4621, 4622, and 4670. The focusing lens 4670 in front of the fiber bundle 4731 ensures that the emission light from the full depth of the aperture is collected, thereby increasing the system signal.

[0333] The high energy collection characteristics of the system ensure low detection limits and allow acceptable results for a wide range of fluid levels without the need to refocus the optical system based on the fluid volume. This contrasts with confocal measurements, such as those described 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.

[0334] In some embodiments, linear polarizers 4624 and 4625 are included in holder 4627, and the same motion that positions the appropriate mirror in the optical path can also be used to select the polarizer used for fluorescence polarization measurement. This eliminates the need for a separate mechanism to switch polarizers, thereby improving the reliability of the system.

[0335] Box 4610 depicts a second optical path for fluorescence measurements, which uses a tilted V-shaped optical arrangement for direct aperture illumination and light collection. This allows the system to direct the entire amount of light from fiber 4532 into aperture 4555. To this end, the numerical apertures of optical elements 4611 and 4612 are matched to those of fiber 4532. The excitation light cone enters the aperture and excites the contents of the aperture through the first branch of the V. The emitted light is collected by the second branch of the V. The numerical apertures of lenses 4614 and 4613 are matched to those of exit fiber 4732. The V is tilted relative to the vertical plane to direct excitation light specularly reflected from the aperture surface away from the light collection branch of the V. Thus, in addition to spectral separation, this arrangement introduces spatial separation of the emitted and excitation light, significantly improving the signal-to-noise ratio. This tilted V-shaped arrangement can also be used to perform fluorescence polarization measurements.

[0336] Inlets A and B of the excitation-emission separation device 4600 receive fiber bundles from the excitation spectroscopy device 4500. The optical fiber can be positioned to guide the light spectrally separated by the filter in the excitation spectroscopy device 4500 to input B of block 4620. The optical fiber can also be positioned to guide the light spectrally separated by the monochromator in the excitation spectroscopy device 4500 to input A of block 4610. Alternatively, the inputs can be reconfigured by switching optical fibers 4522 and 4532. This switching can be done manually. The emitted light is gathered from ports C and D by optical fibers 4731 and 4732. The arrangement of optical fibers 4731 and 4732 in the outlet ports C and D determines the source of the emitted light in the optical fibers.

[0337] Figure 45 A holder 4627 with associated dichroic mirrors 4623, 4628, and 4629 and linear polarizers 4624, 4625, and 4626 is shown according to an exemplary embodiment of the present invention. The holder 4627 is fixed to a slider 4650, which slides along a track 4651 due to the force applied by a motor 4652 via a belt 4653. The holder 4627 moves in a direction perpendicular to the plane defined by the optical axes of the excitation light and the emission light. Although the two different optical fibers 4522 and 4532 may occupy Figure 45 The optical fiber positions are shown in , but for clarity, only optical fiber 4522 is shown.

[0338] In the depicted design, there are five possible positions for the retainer 4627 relative to the optical fiber 4522 that transmits the excitation light. Figure 45 The first position shown represents a situation where the center of mirror 4628 is aligned with the optical axis of optical fiber 4522. In this position, fluorescence polarization-based measurements cannot be performed. If holder 4627 is moved to the left by a distance equal to the distance between the centers of mirrors 4628 and 4629, holder 46227 will be in the second position. In the second position, mirror 4629 plays an active role, and fluorescence polarization-based measurements cannot be performed.

[0339] The other three positions of the holder 4627 correspond to three different situations. First, when the right third of the reflector 4623 is in front of the optical fiber 4522, analysis based on fluorescence polarization cannot be performed. Second, when the middle third of the reflector 4623 is in front of the optical fiber 4522, the linear polarizer 4624 is in the optical path of the excitation light, and the 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 the reflector 4623 is in front of the optical fiber 4522, the linear polarizer 4624 is still 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 the holder 4627 can not only select which mirror to place in the optical path, but also perform fluorescence polarization measurement.

[0340] like Figure 45 As shown, linear polarizers 4625 and 4626 have parallel surface orientations and perpendicular polarization axis orientations. Their active areas are equal in size, with each dimension comparable to the cross-sectional size 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 that of linear polarizer 4625. The area of ​​mirror 4623 is at least three times that of linear polarizer 4625. Mirror 4623 is partially reflective and partially transparent.

[0341] Figure 46 A view of the sample carrier 4550 from above along a vertical axis toward the frame 4610 of the excitation-emission separation device 4600 is shown. Points A and B' are the input ports of the excitation-emission separation device 4600. Lenses 4611, 4612, 4663, and 4664 focus the excitation light onto the aperture 4455 in the sample carrier 4550. Lenses 4613, 4614, 4673, and 4674 collect and focus the emission light onto points C and D', which are the exit ports of the excitation-emission separation device 4600. The optical axes of lenses 4611, 4612, 4663, 4664, 4613, 4614, 4673, and 4674 are oriented diagonally along the aperture 4555 defined in the sample carrier 4550. Using this arrangement, the same aperture 4555 can be read simultaneously by either a filter-based or a monochromator-based spectroscopy system. Since the excitation light from point A is reflected to point B' and vice versa, very little excitation light is reflected toward exit ports C and D'. Thus, the emission light is spatially separated from the excitation light.

[0342] Optical modules

[0343] The device may further comprise an optical module positioned to image or scan the sample within the porous sample carrier. The optical module may be placed within the housing. The optical module may be operably connected to the controller. The optical module may be controlled or operated via a graphical user interface. In addition, the images or scans obtained by the optical module may be viewed and / or recorded 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 sample in real time.

[0344] Cell-based assays, particularly those involving live cells, are becoming increasingly popular in life science research. Microplates are increasingly being used as a container for qualitative and quantitative investigation of cell growth processes. Typically, researchers utilize multiple specialized devices for cell-based research.

[0345] Photoluminescence, such as fluorescence and / or phosphorescence, is read using an instrument with a beam diameter large enough to obtain a representative measurement of total well fluorescence, or a beam size measurement to perform area scanning and mapping of the signal across the well, which can be accomplished with a dedicated traditional fluorescence reader or a multi-detection reader. Most devices provide for plate incubation, fluid injection, and also allow for gas control (CO2 and / or O2) options similar to tissue culture incubators.

[0346] Widefield imaging allows for more information to be obtained from cells than can be obtained from the fluorescence signal level of the well. Laboratory microscopes are commonly used for brightfield and phase contrast imaging of unstained cells, as well as fluorescence imaging of stained cells. Some systems allow for culture chamber and environmental control. For clearer imaging or sectioning of 3D cell clusters, such as spheroids, confocal microscopy is used as a third option.

[0347] Typically, these devices are purchased from a variety of vendors, and users may be forced to physically transfer containers (e.g., microplates) from one device to another as needed, and track the entire sample analysis process, collating and combining data from several devices to obtain a complete overall analysis of the cell sample. Without robotics, it may be almost impossible to properly perform long and complex experiments or analyses. The use of robotic technology further increases the cost and complexity of analysis. The combination of non-imaging analysis modes (fluorescence, absorbance, and chemiluminescence), wide-field fluorescence imaging at the cellular level, confocal fluorescence imaging, environmental control, and reagent injection will provide a complete overall analysis solution in a single device and free users from tedious microplate handling, microplate tracking, and data transmission. This article describes a combined system solution in which data obtained from a single device can be stored, collated, and analyzed.

[0348] Consumables

[0349] The present disclosure provides consumables that can be used to analyze cell samples according to the systems and methods described herein.

[0350] In some embodiments, the system includes an interface for interacting with consumables. Consumables can be any consumables that accommodate cell samples. Exemplary consumables include, but are not limited to, flow chips, microtiter plates with any number of holes, 2D sample and 3D tissue or spheroid formation / measurement plates. For example, a microtiter plate can have 6, 12, 24, 48, 96, 384 or more holes. In some embodiments, if impedance measurement or electrical excitation is required, the consumables include microelectrodes. In some embodiments, the consumables can form microchambers to allow flux measurement. In some embodiments, the consumables are made of materials that limit gas diffusion to increase flux sensitivity. In order to image the cell sample, the components that make up the imaging system can be configured to read from below or above the consumables. If imaging from below, the consumables can have a window through the microelectrode to observe the cell sample. If imaging from the top, the consumables can remove any features above the sample (e.g., flux measurement box) to observe the sample.

[0351] Consumables include, but are not limited to, sample carriers (e.g., cell culture plates) with and without impedance electrodes, lids, and cartridges. In some embodiments, the lid can have one or more sensors, such as O2 / pH / CO2 sensors. In some embodiments, the cartridge can have one or more sensors and / or compound / substance ports. Consumables can be shuttled between different steps of the automated workflow.

[0352] In some embodiments, the sample carrier is a cell culture plate. In some embodiments, the sample carrier includes a plurality of wells. In certain embodiments, the wells in the plurality of wells include impedance electrodes. In other embodiments, the wells in the plurality of wells do not include impedance electrodes. For example, the impedance electrodes can be wired to detect real and imaginary impedance components of the cell sample during growth and / or measurement. In some embodiments, the wells are fabricated with uniform electrodes on the bottom. In some embodiments, the wells are fabricated with windows for imaging cells at the bottom. If the wells are fabricated with windows for imaging cells at the bottom, normalization can be performed and applied to the measurement. In some embodiments, the wells include protrusions to facilitate the formation of microchambers that do not interfere with the impedance electrodes. During measurement, a cartridge can be loaded into the well and placed on the protrusions. The microchambers can then be refreshed by removing the cartridge from the protrusions.

[0353] Sample Control Module

[0354] 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 comprises one or more of: a sample environment temperature control element (such as the 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 at least one of the O2, CO2, and N2 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 / promote evaporation); and a measuring device control element configured to control the temperature of a sensor interfaced with the sample and / or well to determine various characteristics thereof.

[0355] In addition, the sampling control module can operate in conjunction with the fluid processor or cartridge to control the temperature of various compounds / substances (e.g., reagents, test agents, other media) within certain predetermined ranges. By controlling the temperature of the compounds / substances added to the wells, the controller can reduce the impact of temperature shock on any sample of the material introduced into these wells and store the compounds / substances at a temperature different from the compound / substance delivery temperature (e.g., freezing the compound to extend shelf life, heating the compound to reduce viscosity). In various embodiments, the controller can maintain the compound / substance at a standby temperature that is different from the rest of the device while waiting to introduce the compound / substance into the wells. The controller can additionally or alternatively adjust the temperature of the compound / substance based on the ambient temperature (or standby temperature) before introducing the compound / substance into the wells. For example, a compound can be stored at X degrees as a standby temperature (in an environment with 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.

[0356] In various embodiments, the sample temperature environment control element and / or the measurement device control element is a heater that generates heat through electrical resistance in response to electrical current passing through the various heating elements.

[0357] In various embodiments, a gas control element is in communication with one or more gas tanks containing gas to control the atmosphere of a single sample well, or the atmosphere of a cavity within a system into which a sample carrier is inserted. The gas control element can include various sensors that detect the balance of gas content in the well and / or cavity and / or the pressure of the gas therein. Based on the sensor readings, the gas control element can vent, apply negative pressure, or otherwise remove a portion of the gaseous atmosphere from the well and / or cavity and replace the removed portion with a desired composition of at least one of O2, CO2, and N2 at a desired pressure to maintain the desired atmosphere composition. Additionally or alternatively, the gas control element can inject at least one of O2, CO2, and N2 at a desired pressure to adjust the existing atmosphere without venting, pumping, or otherwise removing a portion of the existing atmosphere.

[0358] In various embodiments, the humidity control element includes a dehumidification element to remove moisture from the atmosphere of the wells and / or cavities into which the sample carrier is inserted and / or connected to a water source to inject additional water into the sample wells or their atmosphere (e.g., via a sprayer or humidifier element).

[0359] In various embodiments, the gas control element and the humidity control element release unwanted atmosphere by opening and closing the cover of one or more wells in the sample carrier and are connected to a liquid handling element or flux / consumables box, which includes various consumable growth gas supplies for adjusting or re-establishing the desired atmosphere composition in a given well, and also includes various growth media, stimulants, regulators, etc. supplied to the samples held in the wells.

[0360] Signal processing module

[0361] High-impedance transimpedance amplifiers are susceptible to parasitic current paths. These can be caused by surface contamination from flux residue or surface cleaners during soldering and manufacturing. These paths can also be exacerbated by high humidity environments and moisture absorption in the dielectric materials used to insulate the conductive paths.

[0362] The device disclosed herein is 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%. It was unexpectedly discovered that the performance of the signal processing module at high relative humidity allows for longer analyses and experiments to be performed on the device. Thus, real-time cellular data can be collected from cell samples, and assays can be performed for periods exceeding 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, without negatively impacting the sensitivity of the sensor unit.

[0363] The signal processing module is a processor operably connected to the sensor unit array and configured to receive and amplify signals from the sensor units. The signal processing module can receive and amplify multiple signals from the sensor unit array simultaneously or individually (e.g., sequentially). In some embodiments, the signal processing module is capable of adjusting the amplification of the signal to collect data at a faster or slower rate, for example, reducing the amplification to increase the acquisition speed. In some embodiments, the signal processing module is configured to operate under reduced parasitic currents (e.g., reduced interference, dark current, or noise) associated with the detection and / or amplification of the signals from the sensor units.

[0364] The signal processing module can be configured to detect the signal using time-based detection or intensity-based detection. In short, the radiation emitted by the excitation probe can be measured in units of intensity and / or lifetime / time domain (including, for example, decay rate, phase shift or anisotropy detection). Intensity-based detection can include detecting and / or processing ratio measurements. In short, the measurement may include a signal measurement that is sensitive to the analyte and a reference measurement that is insensitive to the analyte or substantially insensitive to the analyte. Ratios between references can be combined to facilitate ratio assessment of analyte flux or concentration.

[0365] In some embodiments, the signal processing module includes a printed circuit assembly formed of an insulating material with a high dielectric constant. In some embodiments, the signal processing module includes a printed circuit assembly having a transimpedance amplifier including a ground guard trace. In some embodiments, the signal processing module may include one or more photosensitive components, such as semiconductor diodes, photomultiplier tubes, avalanche photodiodes, CMOS sensors, CCDs, etc. In some embodiments, these photosensitive components may be connected to the transimpedance amplifier. In some embodiments, the signal processing module includes a printed circuit assembly formed of surface mount components, such as a high-gain component substantially free of secondary hand soldering. In some embodiments, the signal processing module includes a printed circuit assembly including a thermally conductive excitation source, optionally wherein the thermally conductive excitation source is thermally connected to a heat sink, such as in thermal contact. The thermally conductive excitation source can be any excitation source that changes intensity relative to temperature, such as a laser diode or a light-emitting diode (LED). In some embodiments, the signal processing module includes a printed circuit assembly having an integrator design. In some embodiments, the signal processing module includes a printed circuit assembly having an operational amplifier design.

[0366] Surprisingly, the design of the thermally conductive excitation source significantly reduces thermal drift, so less reference calibration is generally required, which can reduce calibration errors and thus improve measurement accuracy ( Figure 36 ). Figure 36The data shown in the graph of indicates that thermal drift is reduced after the addition of a thermally conductive excitation source. In some embodiments, the improved design of the thermally conductive excitation source can alleviate (or eliminate) the need to include a reference signal detector, reducing the complexity of 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 may not have a reference signal detector.

[0367] The components and features of the signal processing module are further described in "Section 5: High Impedance Sensors" by Kester et al., and are incorporated herein by reference in their entirety for all purposes.

[0368] Transmission Module

[0369] According to some embodiments, the system further comprises a transmission module configured to transmit the optical signal from the sensor unit array to the signal processing module. For example, the transmission module can transmit one or more of the excitation, reference and emission optical signals.

[0370] The transmission module may be formed from multiplexed fiber optic material. Figure 34-35 is a diagram showing several views of an exemplary transport module 60, including a side view of the transport module 60 ( Figure 35 ) and sectional views ( Figure 34 In an embodiment, the transmission module 60 may include an array of fiber optic bundles, each fiber optic bundle communicating with a corresponding sensor unit of the sensor unit array. The fiber optic bundles may be positioned and arranged to interface directly with one or more sensor units. Each fiber optic bundle may be formed by an array of fiber optic cables contained within a fiber optic probe housing, such as a metal fiber and / or a plastic probe housing, such as Figure 34 As shown in the cross-sectional view.

[0371] In some embodiments, the transport module can be in the form of a homogenized fiber optic waveguide that optically connects the sensor units to the transport module, e.g., each sensor unit is connected to a corresponding fiber optic bundle of the transport module. The homogenized fiber optic waveguide can be configured to evenly distribute light to one or more sensor units. The homogenizer can improve mechanical and optical shuffling.

[0372] Combination of devices

[0373] In certain embodiments, cells can be continuously analyzed by continuously measuring the same cell sample. In no particular order, samples can be analyzed to measure the bioenergetic work of the cell, such as O2, CO2, pH. The data can be stored in a cloud-based storage and optionally analyzed on a cloud-based data processing and visualization system. Electrochemical measurements (e.g., impedance measurements) can be used to analyze the same cell sample, different samples, or samples from the same cell line. The data can be stored on a cloud-based system. The cell growth and morphology of the same sample, different samples, or samples from the same cell line can be visually observed. The data can be stored on a cloud-based system. By marking the sample, such as by a barcode or other digital identification system, the data obtained from the independent measurement can be associated with the corresponding sample / measurement result. The data can be collected and organized in a cloud-based storage and optionally processed in a cloud-based data processing and visualization system. The organized data from analyzing the same cell sample can be queried to obtain patterns and information.

[0374] Each measurement may be performed within a device described herein or a combination of devices, each operatively connected to a data storage and processing system, such as a cloud-based system or a computer.

[0375] In certain embodiments, samples can be analyzed in parallel by taking one or more aliquots from the original cell sample or a sample from the same cell line to generate multiple substantially identical cell samples for each measurement, for example, to generate three or more corresponding substantially identical samples. The samples can be analyzed simultaneously or substantially simultaneously. As previously described, the data can be collected and organized in a cloud-based storage system. As previously described, the organized data can be queried for patterns and information.

[0376] In certain embodiments, a sample or aliquot of a sample can be analyzed by measuring parameters such as O2, CO2, pH, or other metabolism-related parameters to measure the bioenergetic work of the cells, and visually observing cell growth and morphology simultaneously (e.g., simultaneously, substantially simultaneously, or after an extended period of time). In some embodiments, a sample or aliquot of a sample can be analyzed by measuring parameters such as O2, CO2, pH, or other metabolism-related parameters, and performing electrochemical measurements (e.g., impedance) simultaneously (e.g., simultaneously, substantially simultaneously, or after an extended period of time) to measure the bioenergetic work of the cells. In some embodiments, cell growth and morphology of a sample or aliquot of a sample can be visually observed and electrochemical measurements, such as impedance measurements, can be analyzed simultaneously, i.e., simultaneously, substantially simultaneously, or after an extended period of time, while visually observing cell growth and morphology, e.g., simultaneously, substantially simultaneously, or after an extended period of time.

[0377] It is understood that the cell sample is moved between modalities throughout the extended time period while the cell sample is normalized between modalities. The sample is not measured continuously in one modality throughout the extended time period, but rather is measured in one modality, allowing normalization and re-measurement in the same modality. In one embodiment, the sample is measured in a first modality and normalized before being measured again at multiple different time points in the first modality over an extended investigation duration, for example, before a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or Nth measurement of extracellular flux, the sample is measured for 72 hours with a measurement time of 3 minutes and a recovery and normalization time of 5, 10, 15, 30, 60 minutes or more. Additionally or alternatively, the sample is measured in one modality and then moved to a second or third modality for a second or third measurement, or any variant thereof. In one embodiment, a sample is measured over an extended period of time starting with a measurement in a first modality (e.g., extracellular flux in response to an analyte (e.g., measuring O2 consumption or changes in pH), and the sample can be moved to a second modality (e.g., impedance measurement) and / or a third modality (e.g., imaging) before returning the sample to the first modality (e.g., flux measurement).

[0378] In one embodiment, each of the multiple cell samples is monitored separately by each modality, for example, one sample of the same cell line is monitored for bioenergetic metabolism, another sample of the same cell line is monitored for impedance, and another sample of the same cell line is monitored for cell growth visually. In another embodiment, the same cell sample can be analyzed simultaneously or substantially simultaneously, for example, the bioenergetic metabolism of the cell sample can be monitored simultaneously or substantially simultaneously by imaging of the cell sample. In another embodiment, the same cell sample can be analyzed simultaneously or substantially simultaneously, for example, the bioenergetic metabolism of the cell sample can be monitored simultaneously or substantially simultaneously by impedance measurement. In another embodiment, the same cell sample can be analyzed simultaneously or substantially simultaneously, for example, the bioenergetic metabolism of the cell sample can be monitored simultaneously or substantially simultaneously by impedance measurement and imaging.

[0379] 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 by imaging during a first time period, and the same cell sample can be analyzed for bioenergetic metabolism and imaging after a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and longer time periods, for example, between 6 hours and 72 hours, for example, between 12 hours and 60 hours, between 24 hours and 48 hours, between 12 hours and 36 hours. 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 a week (e.g., 168 or 170 hours).

[0380] 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 by impedance during a first time period, and the bioenergetic metabolism and impedance analysis can be performed on the same cell sample after a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and longer time periods, for example, between 6 hours and 72 hours, for example, between 12 hours and 60 hours, between 24 hours and 48 hours, between 12 hours and 36 hours. 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 a week (e.g., 168 or 170 hours).

[0381] 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 simultaneously by impedance measurement and imaging during a first time period. The same cell sample can be subjected to bioenergetic metabolism, impedance, and imaging analysis after a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, and longer time periods, for example, between 6 hours and 72 hours, for example, 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 72 ... or 168 to 170 hours.

[0382] It should be noted that although the present disclosure generally relates to measuring metabolism, similar methods can be used to measure or detect cell microenvironmental characteristics, such as the environmental conditions experienced by a sample. Conditions may be manipulated through environmental control to promote the achievement of desired microenvironmental conditions. These conditions can be manipulated to correlate with cellular responses. As an exemplary embodiment, impedance, specific imaging cell parameters, parameters of fluorescence measurements (such as cell metabolism) can be controlled, and these parameters vary based on cell oxygenation, oxygen or pH to achieve a model that describes the effect of tumor microenvironmental conditions on cell function. As another example, these characteristics can be controlled to analyze the heart rate and / or metabolism of cardiomyocytes as a function of reduced oxygen and / or nutrient availability, as controlled by drugs or by the use of electrical pacing through a device to control heart rate.

[0383] The embodiments described herein overcome the above disadvantages and other disadvantages not described above. In addition, the embodiments are not required to overcome the above disadvantages, and the exemplary embodiments may not overcome any of the problems described above.

[0384] According to one aspect of an example embodiment, there is provided an apparatus for analyzing one or more samples, the apparatus comprising a support for a container accommodating the sample; an imaging subsystem to image the sample; and an analysis subsystem to analyze the sample.

[0385] According to one aspect of an exemplary embodiment, a sample analysis method is provided, comprising selecting at least one subsystem from a plurality of subsystems of a sample analysis device for examining one or more samples, the plurality of subsystems comprising an imaging subsystem for imaging the one or more samples and an analysis subsystem for analyzing the one or more samples; and controlling the selected at least one subsystem to examine the one or more samples, the examination comprising an imaging operation of the imaging subsystem and an analysis operation of the analysis subsystem.

[0386] According to one aspect of an example embodiment, a non-transitory computer-readable medium is provided, having a program embodied thereon, which, when executed by a computer, causes the computer to perform a sample inspection method, the method comprising selecting at least one subsystem from a plurality of subsystems of a sample analysis device for inspecting one or more samples, the plurality of subsystems comprising an imaging subsystem for imaging the one or more samples and an analysis subsystem for analyzing the one or more samples; and controlling the selected at least one subsystem to inspect the one or more samples, the inspection comprising an imaging operation of the imaging subsystem and an analysis operation of the analysis subsystem.

[0387] According to one aspect of the example embodiments, a device for analyzing a sample is provided. The device may include: a container support configured to support a microplate, the microplate including microplate wells configured to accommodate the sample, also referred to herein as a multi-porous sample carrier, plate, or sample carrier. In one embodiment, the sample is analyzed using, for example, an automated cell imaging reader (e.g., Cytation TM 5. Cytation TM 7) is used to image the sample, as disclosed in U.S. Patent No. 10,072,982, which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the sample is imaged using a confocal imaging apparatus, the apparatus comprising: a container support configured to support a microplate, the microplate comprising a microplate well configured to accommodate the sample; an objective lens configured to image the sample; a laser point scanning confocal system configured to image the sample via the objective lens; and a spinning disk and / or widefield imaging system configured to image the sample through the objective lens, wherein at least a portion of the laser point scanning confocal system and the spinning disk and / or widefield imaging system is movable so that the laser point scanning confocal system and the spinning disk and / or widefield imaging system are configured to be selectively aligned with the objective lens to image the sample.

[0388] It will be appreciated that the cell sample may be observed using any type of imaging modality capable of visually inspecting the cells.

[0389] In certain embodiments, cell samples can be observed using phosphorescence lifetime imaging microscopy (PLIM) and / or fluorescence lifetime imaging microscopy, including two-photon excitation imaging.

[0390] In certain embodiments, an imaging modality known as confocal imaging may be well suited for imaging cellular samples, such as 3D cellular structures such as spheroids. In confocal imaging, the sample can be illuminated one point or portion at a time. For example, light can pass through a small hole, such as a pinhole, located in an optically conjugate plane. Point illumination substantially eliminates out-of-focus light and background light, thereby improving the optical resolution and contrast of the image. The complete image, constructed or stitched together point by point by the scanning function, is very sharp, with well-defined features. The scanning function can be performed with a spinning disk, also called a scanning disk or Nipkow disk.

[0391] Confocal imaging is a particularly well-suited imaging method for spheroids. It allows spheroids to be sliced ​​layer by layer and a 3D model created in a computer for accurate cell counting and 3D image processing, allowing for observation of the spheroid from different angles.

[0392] Figures 13A-13B This is a comparison chart of a sphere. Figure 13A Spheroids are shown imaged at twenty times (20X) magnification using widefield imaging. Figure 13B The spheroids were imaged at twenty times (20X) magnification using confocal imaging. Although the size of the spheroids can be determined using Figure 13A images were evaluated, but single cells and spheroid structures were only Figure 13B can be seen in confocal imaging.

[0393] Figure 13B The resolution advantage of confocal imaging comes at the expense of reduced light intensity due to the confocal aperture, so Figure 13A Longer exposure times are usually required compared to widefield imaging.

[0394] Adding confocal fluorescence imaging to a device that also includes non-imaging analysis modes (fluorescence, absorbance, chemiluminescence, etc.) and widefield fluorescence imaging at the cellular level, combined with a controlled live cell environment, will provide the modern researcher with the most versatile single device for analyzing microplate-based assay formats, including those targeting 3D spheroid studies.

[0395] In an example, there may be a workflow where widefield imaging is performed for faster screening, while confocal imaging is performed for publication images associated with O2, CO2, pH measurements obtained from a sample.

[0396] HCS-based assays can be imaged using widefield imaging, which provides faster throughput and statistically robust image analysis. Confocal imaging can then be used to obtain representative wells of "hits" and "controls" for publication or presentation purposes.

[0397] In an example, there could be a workflow where widefield imaging is performed to more quickly perform an initial screening of spheroids based on size. Then, confocal imaging is used to more deeply assess the size of each "hit" based on nuclear counts, which is more accurate using confocal imaging.

[0398] Typically, widefield imaging cannot "see" 3D spheroids well enough to reliably count individual nuclei, however, widefield can still identify "hits" based on the overall size of the spheroid. Once the wells of "hits" are identified using widefield imaging, the identified wells can be imaged using confocal imaging to obtain improved image analysis for counting the total number of nuclei in the spheroid, which is not possible with widefield imaging alone.

[0399] In this example, there might be a proliferation assay (3D endothelial cell spheroid assay) to identify wound healing compound / substance candidates. A primary compound / substance screen could be performed in a microplate format, where small endothelial spheroids are treated with a library of unknown compounds to determine which compounds cause an increase in cell growth / proliferation. Compounds that result in increased growth are likely candidates for further wound healing studies.

[0400] In the analytical workflow, a plate reader can be used to quickly screen microplates using GFP fluorescence intensity to identify wells with increased spheroid size. Wells that reach a GFP intensity threshold (the threshold is statistically determined during assay development) are considered "hits" and are selected for further imaging. Control wells are also always imaged further as reference wells for comparison with hit wells. Confocal imaging of 3D spheroids can be performed to acquire a dual-channel z-stack image set (Hoescht33342 nuclear marker and GFP marker) of the entire spheroid sample. During image processing and analysis of the maximum projection of the Z stack, the cell count of the spheroid is determined to quantify the spheroid size. The distribution of the nuclear mask in the image is visually inspected to determine whether there is cell death within the spheroid. Furthermore, the results of the hit well image analysis are compared with the control group to determine the percentage of growth relative to the control group.

[0401] In the example workflow, a 3D Tumor-like Cytotoxicity and Immune Response Assay (3D Tumor-like Material from Surgical Samples Assay to Determine Immune and Cytotoxic Therapy Response) is performed. This assay involves culturing tumor-like material obtained from animal models or patient surgical samples. Because these tumor-like materials are derived from animals / patients, in vitro tumor-derived immune cell responses can be assessed, allowing analysis of tumor responses to various therapies. This assay can be used to evaluate the effectiveness of new therapies in a microplate format using heterogeneous multicellular tumor models.

[0402] For example, tumors can be stained for nuclear counts (e.g., blue) and immune cell markers (e.g., red). A microplate reader can be used to assess: wells with high cytotoxicity appear as low blue signals; wells with high immune responses appear as high red signals. 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 also always imaged further for comparison with hit wells. Confocal imaging of 3D tumors is performed to acquire a two-channel z-stack image set (Hoescht 33342 nuclear marker and CY5 marker) of the entire tumor sample. Maximum projections of the z-stack are imaged and analyzed, and tumor-like cell counts are quantified. Red blood cell counts are determined for immune responses. The results of the image analysis of the hit wells are compared with the control wells to determine the percentage of cytotoxicity or immune response compared to the control wells.

[0403] Several of the examples above leverage the power of a single device to run analyses as “hit picking.” The first rapid read typically uses a rapid read method to identify samples of particular interest, which can be a fluorescent non-imaging read or a fluorescent or brightfield widefield imaging read performed at lower magnification. Once wells of interest, called hits, are identified, a second, more time-consuming mode is deployed to identify results of particular interest. This type of processing is particularly important if the end result is high-resolution confocal imaging, where large amounts of data storage are required and collecting extensive information on only a few samples of interest can significantly save data storage space. This processing also saves processing time during data acquisition and data review, as most samples are not “hits” and are discarded in the first analysis step. A single, unified device that performs the various processing steps can streamline the analysis.

[0404] The ability of a single device to perform diverse functions opens up other potential applications for studying spheroids. Spheroids are typically grown in round-bottomed wells. Typically, in the final imaging step, the spheroids are transferred to a flat-bottomed plate to prevent the round well bottom from acting like a lens during imaging, thereby unnecessarily introducing optical aberrations and negatively impacting the final 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 position of the spheroid in the well is no longer known. In a preferred embodiment, widefield imaging with a lower magnification but larger field of view can be performed to image the well to locate the spheroid (region of interest). The well is then positioned so that the located spheroid position (region of interest) is aligned with the optical axis, and the spheroid is imaged in confocal mode using a higher magnification objective with a smaller field of view. Z-stacks are performed 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 by performing a fluorescence readout area scan using the device's non-imaging analysis modality and selecting the region of maximum fluorescence signal during imaging.

[0405] Figure 14 is a block diagram illustrating a multi-detection system according to an embodiment.

[0406] like Figure 14 As shown, 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 holder 310, a culture chamber 320 for culturing 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.

[0407] The sample is placed into the well 200 (e.g., microwell) of the microplate 300. The microplate 300 is transported to and from the measurement and incubation chamber 320 by a carriage 310. When positioned to be exposed to the external environment of the multi-detection system, the microplate 300 can be accessed from outside the incubation chamber 320 and / or the housing 1900 for access by a technician or a robotic arm. When the microplate 300 is positioned within the chamber, various supported imaging and non-imaging analysis modalities can be performed.

[0408] The carriage 310 is part of a microplate transport subsystem for positional manipulation of the microplate 300 and may include any suitable combination of a belt, a platform, a microplate holder, a motor, and positioning software executed under hardware control for positional manipulation. When the microplate 300 is placed in the culture chamber 320, the entire microplate 300 remains in culture. The culture system and the culture chamber 320 will be described in detail later.

[0409] The non-imaging analysis subsystem 1300 can be based on illumination via a flash lamp, dual excitation and emission monochromators, a photomultiplier tube (PMT), and a silicon detector. The non-imaging analysis subsystem 1300 supports absorbance, fluorescence, and chemiluminescence analysis modes for detecting the corresponding properties of the sample in the well 200. The non-imaging analysis subsystem 1300 can be implemented as a filter-based subsystem, or as a hybrid of any or all of the above subsystems.

[0410] The imaging subsystem includes widefield 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 widefield imaging and can provide illumination for brightfield, color brightfield, and phase contrast imaging modalities.

[0411] The external subsystem 2100 may be an external confocal illumination subsystem for confocal imaging, which may be modularly connected to or disconnected from the imaging subsystem within the housing 1900 via optical fibers to increase the flexibility of the physical placement of the external subsystem 2100 relative to the device. Alternatively, the confocal imaging illumination subsystem may be provided integrated within the housing 1900.

[0412] If required by the assay, the fluid injection subsystem 1100 delivers reagents to the wells 200. The fluid injection subsystem 1100 may include any combination of pumps, reservoirs, lines or tubing, pipettes and tips, and software executing under hardware control for delivering fluids to the wells and, when necessary, aspirating fluids from the wells.

[0413] The environmental control subsystem 2000, shown as being externally located relative to the housing 1900, may include a gas control module that provides control over the atmospheric conditions within the housing 1900. Other control modules may include modules for controlling temperature, humidity, and other conditions, which may be controlled within the housing 1900 under the control of the environmental control subsystem 2000. The environmental control subsystem may include any combination of pumps, reservoirs, lines or tubing, fans, heating and cooling elements, and the like, for controlling all conditions within the housing 1900. The housing 1900 houses most of the subsystems and defines a physical space within which a gaseous environment conducive to living cells may be effectively maintained and controlled by the environmental control subsystem 2000.

[0414] The controller 1000 can control all operations of the multi-detection system. The controller 1000 can communicate with each subsystem in the multi-detection subsystem via wired or wireless means. The controller 1000 can include any combination of hardware (e.g., CPU, memory, cables, connectors, etc.) and software for executing the hardware to control the operations of the multi-detection system.

[0415] Figure 15 is a block diagram illustrating a multi-detection system according to an embodiment.

[0416] The multi-detection system enables a variety of imaging methods. In addition to confocal imaging mode, fluorescence, bright field and phase contrast widefield imaging can also be performed. The optical components of the confocal imaging system and the widefield imaging system are as follows: Figure 15 shown.

[0417] The microplate 300 can be placed on a carrier 310 (e.g., a carrier for a sample carrier), which positions the well 200 of interest in alignment with the imaging optical axis of the objective lens 1230. The objective lens can be selected from several objectives of various magnifications placed on the objective turret 1232. The relative position of the imaging illumination subsystem 1600 is shown in FIG. Figure 15 As shown, the imaging illumination subsystem 1600 can be used to perform bright field, color bright field, and phase contrast imaging of the sample. Many optical components are shared between the wide-angle and confocal systems and will be discussed below. Figure 16-17 A more detailed description of these parts is provided in Figure 15 some components.

[0418] Figure 16 is a block diagram illustrating a multi-detection system according to an embodiment.

[0419] Confocal imaging Figure 16 The widefield imaging subsystem components (e.g., LED cube 1201 and filter cube 1210) are automatically removed from the light path of the sample. Figure 15 The system shown is converted to Figure 16 The confocal optical system is shown to understand the confocal light path.

[0420] A spinning disk confocal system is deployed as an example embodiment of a confocal imaging system. The system is based on utilizing a spinning disk ( Figure 18) in the optical path of the sample. The disk is placed in an intermediate image plane that is conjugate to the sample and the detection plane. The disk is therefore in both the excitation and emission paths. In an exemplary embodiment, the disk is typically about 2 mm thick and made of glass or quartz. The disk can be coated to be opaque or to have a given transparency or opacity, except for transparent areas where a pattern of pinholes or slits is left. Ideally, the disk surface does not reflect oncoming light. The sample to be imaged is illuminated by the excitation light transmitted through the pinholes. Only the radiation emitted by the sample (generated by these illumination points on the sample) passes through the pinholes of the disk to the detector. The pinholes or slits, although numerous, are spaced far apart from each other and act optically independently. The energy from adjacent pinholes does not ideally affect the sample point illuminated by a given pinhole. The disk pattern is usually arranged in several spirals, such as Figure 18 As shown in Figure 1, the disk can be controlled to rotate continuously, thereby scanning the sample. As the disk rotates, the sample is illuminated one point at a time, and the complete sample image is detected on the detector to reconstruct a complete image of the sample.

[0421] Back to Figure 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 laser or a semiconductor-based laser (laser diode). In an exemplary embodiment, the output tip of the optical fiber can be a light (radiation) source. Radiation is an example because the excitation spectrum may be outside the 380-630nm range commonly referred to as light. However, the term "light source" is more commonly used in imaging, and the term light will be used interchangeably with radiation in this article. The input end of the optical fiber can be illuminated from a light source module outside the device to flexibly select the light source that best suits the sample imaging requirements. The optical fiber also allows the flexibility of bifurcating inputs from multiple external light sources. The output tip of the optical fiber is imaged by the condenser 1522 to or near the intermediate sample image plane where the rotating disk 1504 is located. The light from the optical fiber can be sent through the excitation filter 1531, then reflected by the dichroic mirror 1533 and focused by the tube lens 1520 onto the rotating disk 1504. As will be understood by those skilled in the art, the term "lens" herein and throughout this description can refer to a single lens or a group of lenses, depending on the embodiment and function. As previously described, the disk has a spiral-shaped slit aperture. Field lens 1519 minimizes light loss and directs light exiting the disk to be focused by tube lens 1250. Tube lens 1250 directs the excitation radiation via mirror 1220 into objective lens 1230. Objective lens 1230 illuminates a small spot on the sample near the bottom of the aperture. Sample components have been dyed with a dye corresponding to the excitation wavelength. These components are excited by the incoming radiation and emit radiation, typically at a longer wavelength. The emitted light is directed to a detector as follows.

[0422] Light emitted by the sample is collimated by objective lens 1230, reflected by mirror 1220, and focused by tube lens 1250 and field lens 1519 onto spinning disk 1504. An intermediate image of the sample in the emitted light is formed on the surface of spinning disk 1504. Tube lens 1520 and lens 1521 invert the image and form a sample image at detector 1560. Detector 1560 is typically a pixelated digital camera, such as a charge-coupled device (CCD) camera or a complementary metal oxide semiconductor (CMOS) camera. The sample image is captured by the camera and can be stored in the memory of a multi-detection system or an external computing system, and can be enhanced and analyzed for various properties and / or presented to the user on a visual display.

[0423] A confocal cube 1530 (e.g., a confocal excitation / dichroic / emission cube) is shown between tube lens 1520 and lens 1521, an arrangement used for fluorescence microscopy. The filters and dichroic mirror can be thin-film coatings on glass. Excitation filter 1531 forms a bandpass for excitation, emission filter 1532 forms a bandpass for emission, and dichroic mirror 1533 separates excitation and emission to maximize utilization of available energy and suppress the amplitude of excitation light that reflects from various optical surfaces as it propagates toward the sample (including the disk surface) and reaches the detector. Lens 1521 (e.g., an emission filter) provides the majority of excitation light suppression, but dichroic mirror 1533 also contributes. Another arrangement of the cube could be multiple filter wheels housing the excitation filters, emission filters, and dichroic mirrors. In exemplary embodiments, a cube is one way to arrange these components, allowing for easy replacement as imaging needs change. Multiple filter cubes (e.g., confocal cube 1530) can be arranged on a motorized slider and can be identified by user settings in the software, or electronically or optically marked with a code to be automatically read by a barcode or some other automatically available method.

[0424] The surface of the spinning disk is imaged along with the sample on the detector. Therefore, any dust particles adhering to the disk surface may appear as artifacts in the image, such as bright streaks caused by the disk's rotation. Small particles can easily adhere to the disk surface with sufficient force to resist centrifugal forces. 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 cleanroom, and sealed from the surrounding environment to prevent dust particles from settling on the disks. Windows 1551 and 1550 in the module allow light to pass through but prevent dust from entering. Ideally, these dust-proof windows should be as far away from the intermediate image plane as possible so that dust that may have landed on the window panes does not create artifacts in the image. The disks are fully contained within the disk modules 1502 and 1553. Therefore, users should not open the modules to avoid introducing dust particles onto the disks.

[0425] Figure 16 Two disk modules 1553 and 1502 are shown installed in a multi-detection device. The disks can be moved to position one or the other into the optical path. Alternatively, both disks can be moved out of the optical path and spaced 1501 along the optical axis. This allows for widefield imaging modalities such as fluorescence imaging, brightfield imaging, or phase contrast imaging.

[0426] A significant benefit of allowing users to use both confocal and widefield imaging options in the same setup is the ability to overlay images from various imaging modes, such as the same image from widefield and confocal imaging modes. Alternatively, a brightfield image can be used to locate a region of interest, which can then be imaged confocally. To properly acquire images, the magnifications of the two modalities must be exactly matched; otherwise, the images will not overlay correctly. The light in the section between tube lenses 1520 and 1250 is non-parallel. In confocal mode, several flat windows are located in the optical path of this section: the confocal disk and the dust window. These windows are not required in widefield mode. However, to match the optical path lengths in the non-parallel light paths, glass 1505 is added to the space 1501 between the confocal disks, through which widefield imaging is performed. This ensures that the specimen remains in focus at a fixed objective lens position when the imaging modality is changed. This ensures that the magnifications of the confocal and widefield imaging modes match. The thickness of glass 1505 should match the sum of the planar windows of the disk used in confocal imaging (window 1551, spinning disk 1504, and window 1550). Glass 1505 should be placed as far away from the intermediate image plane as possible so that dust that may fall on the glass does not create artifacts in the image.

[0427] The pinhole size on the confocal disk is ideally selected based on the parameters of the imaging objective 1230. In an embodiment, the size of the disk pinhole image formed 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 disk pinhole size given in the Zeiss "Introduction to Spinning Disk Microscopy" is

[0428] Disk pinhole diameter = 1.2 * objective lens magnification * emission wavelength / objective lens numerical aperture.

[0429] The numerical aperture (NA) and magnification of the objective are both part of the equation. If the pinhole is too small, too much light will be lost and the time to capture the image will increase. If the pinhole is too large, the confocal effect may be reduced or completely lost. Most commercial spinning disk microscopes have non-interchangeable spinning disks with pinholes ranging from 50-70µm. This compromises fairly well with the range of high-magnification objectives typically deployed in confocal microscopes. However, it is best to have a disk with the appropriate pinhole that matches the objective being used.

[0430] Some spinning disk implementations do not feature a spiral pattern of circular holes, but instead employ slit apertures. Slit apertures can provide relatively brighter specimen illumination and a more intense emission signal, while pinhole apertures can provide higher axial resolution. Therefore, for some imaging applications, including biofluorescence, slits may be preferred to reduce image acquisition time, another reason to change disks even with fixed objectives.

[0431] Multiple disks can be deployed in the imaging device so that the user or a multi-detection system can automatically select from the disks.

[0432] Figure 16 An example of two disk modules 1502 and 1553 used in a multi-detection device is shown. All disk modules are user-replaceable. These modules can be identified through user-controlled software settings or electronically or optically marked with a code that can be automatically read using a barcode or other available method to enable automatic configuration of the multi-detection system.

[0433] Another advantage of a modular disk module is that a 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 protection from dust.

[0434] Module recognition enables automated software setup and automatically resets and calibrates the module's axial position in the optical path. In a spinning disk confocal imager, the disk surface plane, the detector sensitive element plane, and the sample plane should be conjugate to one another. This means that if the sample's emission rays are followed, 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 chip plane of detector 1560 is fixed by the camera design. The objective lens 1230 can be moved along the focusing axis to sharpen the sample image on the detector. Ideally, the disk should be positioned at an intermediate plane that joins the detector and intermediate sample image planes, so that all three planes are conjugate. In the proposed embodiment, the disk module design maintains the disk axial position very close to the ideal conjugate position, but the final disk surface position can be automatically adjusted by observing the disk pattern on the detector and bringing it into sharp focus. Various image-based focusing methods are available and well-known in the industry. Once the optimal disk surface position is found, it can be stored in the software and memory and associated with the disk module. If the disk module is removed and reinstalled, the correct disk position can be automatically restored by the software. If a new disk module is introduced, the system will alternately initiate the disk focusing routine and select the optimal axial position for the new disk module, thus freeing the user from having to keep track of which disk modules are deployed in the unit and their various positioning.

[0435] Alternatively, if only a few disk modules are envisioned for use, the user may set up the disk modules through a setup screen in the calibration portion of the user interface of the software included with the multi-detection system.

[0436] The two concepts of user-replaceable disk modules and automated axial disk positioning are best used together, but can also be implemented separately. If automated axial disk positioning is not available, the disk modules can be configured to be interchangeable with respect to the disk position and some data on the module ensures correct placement in the device. The concept of easily replaceable disk modules, which do not need to be opened by the user and are therefore protected from environmental influences, still applies and offers benefits to users who want the flexibility to use multiple disks that best suit the deployment of their imaging targets and samples.

[0437] Even if the number of disk modules in an installation is limited to one or two, automatic axial adjustment can be used to alleviate the need to strictly control the position of the sensitive surface of the detector image sensor in the detector 1560 (e.g., a camera). In this case, the user has maximum flexibility in camera selection and also allows for camera upgrades within a multi-detection system. If the sensor surface moves after changing cameras, the disk surface can be automatically repositioned so that it is conjugate to the sensor surface through an image-based autofocus routine.

[0438] Figure 17is a block diagram illustrating a multi-detection system according to an embodiment.

[0439] exist Figure 17 , shows widefield imaging deployed in an example embodiment. As described above, the optical section (with elements labeled 15xx) does allow for both confocal imaging (with rotating disks 1504 or 1503 in the light path) and widefield imaging (through the space 1501 between the disks). However, for widefield modalities that researchers may want to deploy in a single, multifunctional device, using such optics with a confocal light source 1540 and confocal cube 1530 may have drawbacks. For confocal imaging, the excitation radiation should be directed onto the disk through multiple optical elements located before the disk surface (e.g., dichroic mirror 1533, tube lens 1520, window 1551). After the disk, the excitation radiation is directed to the sample via more optical elements (e.g., window 1550, field lens 1519, tube lens 1250, mirror 1220, objective lens 1230). For confocal imaging, there is no alternative. However, some excitation light is reflected back from every surface encountered. Good design relies on careful ray tracing to ensure that reflected light is directed as far away from the detector as possible, and on emission filters 1532 to suppress unwanted reflected light. Optical elements before the disk surface such as the tube lens 1520 and window 1551, as well as the surface of the spinning disk 1504, are exposed to very strong levels of excitation radiation that is partially reflected. In addition, any dust particles may become excited and fluoresce. Despite the designer's best intentions, some light does pass through the detector, reducing the signal-to-noise ratio. Therefore, a non-fluorescent sample that appears very dark on the image may not appear dark. This may be due to a significant background signal caused by the reflected light, an effect that tends to be uniform across the image. For samples using the above Figure 16 In widefield microscopy with confocal cross-section excitation elements, image quality and system capabilities will be significantly affected.

[0440] In one example embodiment, an alternative subsystem for widefield fluorescence imaging is provided in the same apparatus. The confocal cube 1530 of the confocal subsystem is placed aside and the spinning disk module is placed in space 1501 for widefield imaging. Figure 15 The configuration is converted to Figure 17 The dedicated widefield section components are the LED cube 1201 and the widefield 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 is typically matched to the LED cube 1201 for optimal signal-to-noise performance. Several cube pairs are available on the slider, corresponding to the specific chemistry being studied.

[0441] This design has several advantages.

[0442] First, the LED excitation optics are placed closer to the sample, so the excitation light encounters fewer optical surfaces on its way to the sample. Consequently, reflections from these surfaces that reach the detector are significantly reduced, improving the signal-to-noise ratio in the image.

[0443] Second, there are a wide variety of LEDs used in the commercially available LED cubes 1201, which may not be powerful enough for the confocal optical channel but may provide sufficient excitation if placed closer to the sample, e.g. Figure 17 shown.

[0444] Third, if the sample must be excited in the UV range, this is particularly important. Some objectives are rated as UV-compatible, transmitting UV light and exhibiting very low fluorescence when excited. However, commercially available optical components in the rest of the optical path, such as the tube lens, are generally not guaranteed to be free of fluorescence under UV illumination. If widefield images of a sample stained with the common DAPI nuclear stain are desired, a common approach in the confocal optical path is to use a wavelength around 400 nm to avoid strong excitation of optical components outside the sample. However, shifting the excitation wavelength from 360 nm, the ideal wavelength for DAPI stain excitation, toward 400 nm can significantly reduce emitted light. This would require researchers to place higher dye concentrations in the sample or increase detector gain, which would reduce the signal-to-noise ratio of the imaging. Ideally, excitation of a DAPI-stained sample would occur at 360 nm, but without the UV excitation light passing through optical components that could fluoresce. In exemplary embodiments, LED cube 1201 and filter cube 1210 allow for this optimal option. The UV excitation enters only objective lens 1230, which can be selected to not fluoresce. The emitted light does pass back to the detector through multiple optical elements common to confocal and widefield, but because the emitted light is in the visible spectrum, the optical elements it encounters generally do not fluoresce in the same way that UV light does.

[0445] Figure 17 The relative position of the imaging illumination subsystem 1600 for non-fluorescence widefield imaging is shown. This can be brightfield, color brightfield with three LEDs that can be switched one at a time, or a phase contrast illumination system with an annular aperture to match the phase contrast objective.

[0446] Other embodiments and components of the imaging system are further described in PCT Patent Application No. WO2022120047A1, "Universal Multi-Detection System for Microplates with Confocal Imaging," which is incorporated herein by reference in its entirety. These components include, for example, a laser point scanning confocal (LSC) modality, a spinning disk confocal system, and widefield functionality in a single device.

[0447] Figure 18 An exemplary widefield imaging system 1800 without a confocal option as deployed in an example embodiment is shown. The widefield imaging system includes an imaging subsystem module 1200 that provides direct access to microwells 200 of a microplate 300 located on a carrier 310 housed in a measurement chamber 320, wherein images of the microwells 200 can be imaged via a tube lens 1250 and a camera 1560.

[0448] As described above, the microplate 300 can be placed on the bracket 310, which positions the well 200 of interest in alignment with the imaging optical axis of the objective lens 1230. The objective lens can be selected from several objectives of various magnifications placed on the objective turret 1232. The relative position of the imaging illumination subsystem 1600 is as shown in FIG. Figure 15 As shown, the imaging illumination subsystem 1600 can be used to perform bright field, color bright field, and phase contrast imaging of the sample. Many optical components are shared between the wide-angle and confocal systems and will be discussed below. Figure 16-17 A more detailed description of these parts is provided in Figure 15 some components.

[0449] Will refer to it later Figure 16 and Figure 17 The imaging subsystem modules are discussed in further detail. Components of the widefield imaging system are further described in, for example, US Patent 10,072,982, entitled "Microplate Universal Multi-Detection System," the entire contents of which are incorporated herein by reference for all purposes.

[0450] Configurations according to embodiments of the present disclosure may also combine or include a laser point scanning confocal system, a spinning disk confocal system, and widefield functionality in a single device. However, embodiments of the present disclosure may include any combination of the above systems and functionality.

[0451] Figure 19 is a schematic diagram of a non-imaging analysis subsystem according to an embodiment. Figure 19 , a non-imaging analysis subsystem 1300 of a multi-detection system is provided.

[0452] The analysis modes of the non-imaging analysis subsystem 1300 can be absorbance, fluorescence from the top and bottom, and chemiluminescence. The xenon flash bulb 13001 emits radiation in the range of 200-1000nm. The two stages 13002 and 13003 of the fluorescence excitation / absorption double monochromator select narrowband radiation. The radiation is guided toward the sample through a fiber optic cable, reaching the absorption channel through fiber 13030, the top fluorescence channel through 13005, or the bottom fluorescence channel through 13033. Only one fiber is active at a time, so there is no optical crosstalk between the various analysis modes. The silicon detector 13060 measures the absorbance through lenses 13040 and 13050.

[0453] Top fluorescence excitation and emission pickup are performed by lens 13020, which can be moved up and down to accommodate various microplates and liquid levels. Bottom fluorescence is performed in a similar manner to lens 13055. Both top and bottom emission are guided by fiber optic cables to the first stage of emission dual monochromators 13010 and 13011, and then to photomultiplier tube 13012. Chemiluminescence fiber 13021 can be connected directly to the photomultiplier tube, providing measurement of very low light levels by bypassing the monochromator.

[0454] The fluid injection subsystem 1100 can provide researchers with the ability to inject reagents via fluid lines 1112 and 1111 and quickly measure the injection results through the analysis subsystem, further expanding the range of tests that can be performed in the device.

[0455] Figure 20 is a diagram illustrating an injection subsystem according to an embodiment.

[0456] refer to Figure 20 , provides an optional injection subsystem. The injection subsystem 1100 can be placed on top of the multi-detection device, and the fluid lines 1112 and 1111 are supplied through the bulkhead inlet on the top of the housing, such as Figure 21 The reagent is delivered to the micropores by the pump in the fluid injection subsystem 1100 through the fluid lines 1111 and 1112 (which can be PTFE lines), and is delivered into the pores through the injection needles 1102 and 1101, as shown. Figure 20 shown.

[0457] refer to Figure 19 ,Environmental control can be deployed in a multi-detection system.

[0458] The bracket 310 supports the microplate 300 (eg, a sample carrier) and is located in the culture chamber 320, as shown in FIG. Figure 19As shown. This ensures that the microplate 300 is maintained at the desired temperature at all locations within the incubation chamber 320, within the carrier 310. The incubation chamber 320 can be constructed of a material well-suited to maintaining a constant temperature, such as a continuous sheet of aluminum, while still providing access to the optical components through a small opening. The incubation chamber 320 is typically insulated. This chamber design is well known to those skilled in the art and is well known to many multi-detection devices. A typical controlled temperature range can be from room temperature to 65°C.

[0459] Figure 21 is a diagram illustrating a multi-detection system according to an embodiment.

[0460] For living cells, the temperature is usually 37°C, but additionally the atmosphere surrounding the sample needs to be controlled. This is done by filling the sample with an appropriate gas mixture. Figure 21 Control is achieved through the entire housing 1910 of the device. This design avoids attempts to confine the gas control environment to a measurement chamber or a separate bulkhead. The goal is to equalize the atmosphere within housing 1910. Therefore, housing 1910 is designed to be as airtight as possible by avoiding gaps in the housing and using soft gasket material around the user access door.

[0461] Figures 22A-22C is a diagram illustrating a gas control subsystem according to an embodiment.

[0462] Reference Figures 22A-22C , the environmental control subsystem 2000 (e.g., a gas control subsystem) can be located outside the device. The environmental control subsystem 2000 allows the user to set the CO2 and / or O2 concentration levels in the room to be different from the normal atmosphere: higher CO2 and lower O2. A gas sampling line connects the environmental control subsystem 2000 to the interior of the device housing. Based on the gas composition sampled or extracted from the device through the sampling line, the control system can adjust the flow of CO2 or N2 gas fed into the device, for example by dispersing the input gas with a small fan. This allows all gas sensors and valves to be placed outside the main device and maintain the complexity and reliability of gas control within the external gas controller.

[0463] The combination of a culture chamber surrounding the XY carrier travel area and gas control within the housing and around the microplates enables users to perform long-term live cell experiments.

[0464] refer to Figure 21, shows an external view of the entire device and the elements with which the user interacts with the device, as implemented in an example embodiment. A carrier 310 is presented to the user (as shown in the right image), and the microplate 300 is placed on carrier 310, for example, by the user or a robotic arm, and then placed within the multi-detection system. The confocal cube 1530, widefield LED cube 1201, widefield filter cube 1210, confocal disk module, and objective lens 1230 are accessed through the front of the device via door 1905. This allows the user to easily access more replaceable components at once.

[0465] According to some embodiments, the objective lens of the present disclosure (eg, objective lens 1230 or objective lens 2210 ) may be a fluid immersion objective lens.

[0466] One way to improve the optical performance of a microscope is to use a fluid immersion objective. In an optical microscope, a fluid immersion objective is a specially designed objective lens used to improve the resolution of the microscope. According to an embodiment of the present disclosure, the optical system is an inverted microscope, which means that the objective lens is located below the sample and observes the sample from below. In the inverted microscope device of the present disclosure, when fluid immersion is performed, a drop of fluid (such as water or other fluid) is placed on the objective lens and is held in place by the surface tension of the fluid. The objective lens is then brought to the sample, with the droplet sandwiched between the sample and the objective lens. In this way, the light traveling between the sample and the objective lens does not pass through air. The higher the refractive index of the fluid relative to air, the larger the numerical aperture. This improves resolution and increases signal level. According to an embodiment, the objective lens can be brought to the sample and then the droplet is placed on the objective lens.

[0467] In addition to water immersion objectives, the objectives of the present disclosure can also be equipped with other types of fluids to increase the numerical aperture. Some examples of fluids include, for example, oil and glycerin. In embodiments of the present disclosure, the fluid can be water, oil, glycerin, or some other type of fluid that increases the refractive index.

[0468] Reference Figures 25A-25B , the following describes an immersion objective lens according to an embodiment of the present disclosure. According to an embodiment, the objective lens 1330 can be provided with a sleeve 1332 that is sleeved on the objective lens 1330. The sleeve 1332 can be configured to provide a fluid path in and out of the sleeve 1332. In addition, the sleeve 1332 helps to keep the droplet 33 in place. According to an embodiment, the sleeve 1332 has a port for pumping in fluid and a port for pumping out fluid. According to an embodiment, as Figures 25A-25B As shown, the inlet and outlet can be the same port 31. Figure 25B , excess droplets 34 can exit the sleeve 1332 through the port 31. In an example embodiment, the sleeve 1332 can be formed of, for example, anodized aluminum, plastic, or other materials.

[0469] According to the embodiment, referring to Figure 26 , a fluid pump system may be provided. The fluid pump system may include a first pump 1336, a second pump 1337, a first reservoir 1338 (source reservoir) and a second reservoir 1339 (waste reservoir). The fluid may be pumped from the first reservoir 1331 to the head of the objective lens 1330 by the first pump 1336. Figure 26 As shown, the first pump 1336 can be a syringe pump. The fluid is then removed from the objective lens 1330 by a second pump 1337 that pumps the fluid to a second reservoir 1339. The second pump 1337 can be called a waste pump or a syringe pump, such as Figure 26 As shown. The first pump 1336 and the second pump 1337 can be other types of pumps that perform the same or similar functions. The sleeve 1332 can be assembled onto the objective lens 1330 to guide the fluid to the top of the objective lens 1330 and help keep the droplet in place. The sleeve 1332 can also have a drain port, and the fluid can be configured to be discharged from the sleeve 1332. The objective lens 1330 can be a specially designed objective lens optimized for fluid (e.g., water) immersion applications. Figure 26 In the figure, the first reservoir 1338 and the second reservoir 1339 are shown as separate source reservoirs and waste reservoirs, respectively. However, according to embodiments, a single reservoir can be provided instead of two separate reservoirs, in which the fluid can be reused. In addition, these pumps can be multi-purpose. For example, the BioTek C10 product has a fluid distribution module that can be used to distribute reagents into samples. This same distribution module can be configured to have additional purposes (including the purposes of the first pump 1336 and / or the second pump 1337) to reduce costs.

[0470] Further references Figure 26 , the objective lens 1330 can be attached to the objective lens turret 1232 via the objective lens coupler 1334. Figure 27 A description of objective lens coupler 1334 is provided.

[0471] like Figure 27As shown, the objective lens coupler 1334 may include a kinematic connection 1334A and a magnet 1334B, which are configured to couple the objective lens 1330 and the objective lens turret 1232 together. For example, the objective lens 1330 may be provided with at least one of a protrusion or a recess as a first portion of the kinematic connection 1334A, and the objective lens turret 1232 may include at least one of the other of the protrusion or the recess as a second portion of the kinematic connection 1334A corresponding to the first portion. The magnet 1334B may be provided with one or more of the objective lens 1330 and the objective lens turret 1232. According to an embodiment, the objective lens 1330 and the objective lens turret 1232 may both be provided with magnets 1334B corresponding to each other and configured to be connected to each other by magnetic force. In other embodiments, only one of the objective lens 1330 and the objective lens turret 1232 may be provided with the magnet 1334B, and the magnet 1334B may be configured to be connected to a magnetic material (eg, metal) provided on the other of the objective lens 1330 and the objective lens turret 1232 .

[0472] According to a comparative embodiment, the objective lens can be screwed into the objective turret. However, in at least some embodiments, using a sleeve and tube with the objective lens can make screwing the objective lens into the objective turret difficult. According to an embodiment of the present disclosure, the objective lens coupler 1334 including a kinematic connector 1334A and a magnet 1334B can easily be installed with the sleeve and tube objective lens.

[0473] According to the embodiment, referring to Figures 28A-31C The objective lens 1330 and the sleeve 1332 may have various configurations. Depending on the embodiment, the sleeve 1332 may also be referred to as a cover.

[0474] Figure 27 is a diagram illustrating objective lens coupling according to an embodiment; Figure 28A is a perspective view showing an immersion objective lens according to a first embodiment; Figure 28B is a top view showing the immersion objective lens according to the first embodiment; Figure 28C It is along Figure 28B A first sectional view taken along line AA in FIG. 1 shows the immersion objective lens according to the first embodiment in a state where a liquid bubble is provided; Figure 28D The immersion objective lens according to the first embodiment is shown along Figure 28B A second cross-sectional view taken along line AA in FIG, wherein a microplate is provided on the immersion objective lens; Figure 29A is a top view showing an immersion objective lens according to a second embodiment; Figure 29B It is along Figure 29A A first cross-sectional view taken along line BB shows the immersion objective lens according to the second embodiment in a state where a liquid bubble is provided; Figure 29C The immersion objective lens according to the second embodiment is shown along Figure 29AThe second cross-sectional view taken along line BB shows a microplate disposed on the immersion objective lens; Figure 30A is a top view showing an immersion objective lens according to a third embodiment; Figure 30B It is along Figure 30A A first cross-sectional view taken along line CC shows the immersion objective lens according to the third embodiment in a state where a liquid bubble is provided; Figure 30C The immersion objective lens according to the third embodiment is shown along Figure 30A The second cross-sectional view taken along line CC shows a microplate disposed on the immersion objective lens; Figure 31A is a top view showing an immersion objective lens according to a fourth embodiment; Figure 31B It is along Figure 31A A first sectional view taken along line DD in FIG. 1 shows the immersion objective lens according to the fourth embodiment in a state where a liquid bubble is provided; and Figure 31C FIG. 1 is a diagram showing an immersion objective lens according to a fourth embodiment. Figure 31A In the second cross-sectional view taken along line DD, a microplate is provided on the immersion objective lens.

[0475] In the following Figures 28A-31C In the description of the present invention, the same or similar features are given the same or similar reference numerals. For the sake of clarity, redundant descriptions of the same or similar features may be omitted.

[0476] Reference Figures 28A-28D , a top surface 10A of the sleeve 1332A can be flush with a top surface 11A of a lens of the objective 1330A, and the sleeve 1332A can be configured to be clamped to the objective 1330A.

[0477] Sleeve 1332A may include, for example, an upper portion 50A, a middle portion 60A, and a lower portion 70A. Depending on the embodiment, the upper portion 50A, the middle portion 60A, and the lower portion 70A may be separated from each other or integrally provided to constitute a single body or multiple bodies. Depending on the embodiment, two of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be integrally provided to constitute a single body, while the other of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be individually provided as an independent body that is configured to be attached to the other two. Depending on the embodiment, the upper portion 50A, the middle portion 60A, and / or the lower portion 70A may be subdivided into independent bodies, and / or additional bodies may be provided. Depending on the embodiment, any number of the upper portion 50A, the middle portion 60A, and the lower portion 70A may be formed from aluminum.

[0478] According to an embodiment, any number of upper portions 50A, middle portions 60A, and lower portions 70A may be formed to substantially exhibit rotational symmetry about a central axis of the objective lens 1330A. The central axis may be, for example, an optical axis of the objective lens 1330A.

[0479] The middle portion 60A may be disposed above the lower portion 70A. The middle portion 60A may include an inlet 62 and an outlet 63. The fluid may be pumped through a fluid pump system (e.g., see Figure 26 ) is pumped into the sleeve 1332A via the inlet 62 and is pumped out of the sleeve 1332A via the outlet 63. The inlet 62 and outlet 63 can be separately arranged on opposite sides of the sleeve 1332A. However, the positions of the inlet 62 and outlet 63 are not limited to this configuration and can be variously modified. According to an embodiment, the inlet 62 and outlet 63 can be composed of a single port.

[0480] The middle 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 the exterior of the middle portion 60A. The tapered portion 64A may be formed to substantially exhibit rotational symmetry about the central axis of the objective lens 1330A. Depending on the embodiment, the tapered portion 64A may have a shape other than a cone, as 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) enables the droplet 90 to have a desired shape on the objective lens 1330A for immersion. Depending on the embodiment, the tapered portion 64A may alternatively be referred to as a protrusion.

[0481] According to an embodiment, the inlet port 62 may include a channel extending through the tapered portion 64A to the inside of the tapered portion 64A, eg, configured to supply the liquid of the droplet 90 into the space between the objective lens 1330A and the tapered portion 64A.

[0482] The upper portion 50A may include a main body. For example, the main body may include a sidewall 52A extending upward from the middle 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 to each other. However, the angle is not limited thereto and various modifications may be made according to the embodiment. The main body including the sidewall 52A and the top wall 53A may be formed to substantially present rotational symmetry about the central axis of the objective lens 1330A.

[0483] Groove 84 can be formed by top 50A and middle portion 60A, and is positioned between top 50A and middle portion 60A.For example, groove 84 can be limited by the inner surface of top wall 52, the inner surface of sidewall 53 and the outer surface of tapered portion 64A.According to an embodiment, groove 84 can be formed to present the rotational symmetry of the central axis around objective lens 1330A basically.Groove 84 can be configured to receive and hold excess liquid.According to an embodiment, groove 84 can be communicated with outlet 63, so that the excess liquid in groove 84 leaves sleeve 1332A via the passage of outlet 63 communicated with groove 84.

[0484] Reference Figure 28C-Figure 28DAt least one upper surface of the top wall 53A may constitute the top surface 10A of the sleeve 1332A, which is flush with the top surface 11A of the lens of the objective lens 1330A. Depending on the embodiment, the top surface of the tapered portion 64 may also be flush with the top surface 11A of the lens of the objective lens 1330A.

[0485] Depending on the embodiment, one or more O-rings 32 may be disposed between the sleeve 1332A and the objective lens 1330A. For example, an O-ring 32 may be disposed between the middle portion 60A and the objective lens 1330A. The O-ring 32 may be configured to seal the bottom side of the space containing the liquid between the objective lens 1330A and the tapered portion 64A.

[0486] Reference Figure 28D A microplate 80 can be disposed directly above the sleeve 1332A and the objective lens 1330A, the microplate 80 holding a sample in at least one well 82. A droplet 90 on the objective lens can contact the bottom surface of the microplate 80 at a position directly below the well 82. The microplate 80 can correspond to, for example, the microplate 300 or other microplates described in the present disclosure.

[0487] Reference Figures 29A-29C , a top surface 10B of the sleeve 1332B can be positioned above a top surface 11B of a lens of the objective 1330B, and the sleeve 1332B can be configured to be clamped to the objective 1330B.

[0488] Sleeve 1332B may include, for example, an upper portion 50B, a middle portion 60B, and a lower portion 70B.

[0489] The middle portion 60B may include a tapered portion 64B, and the upper portion 50B may include a body including sidewalls 52B and a top wall 53B. At least one upper surface of the top wall 53B may constitute the top surface 10B of the sleeve 1332B, which is located above the top surface 11B of the lens of the objective lens 1330B. Depending on the embodiment, the top surface of the tapered portion 64B may also be located above the top surface 11B of the lens of the objective lens 1330B and flush with the top surface of the top wall 53B.

[0490] Reference Figures 30A-30C , a top surface 10C of the sleeve 1332C can be positioned below a top surface 11C of a lens of the objective 1330C, and the sleeve 1332C can be configured to be clamped to the objective 1330C.

[0491] Sleeve 1332C may include, for example, an upper portion 50C, a middle portion 60C, and a lower portion 70C.

[0492] The middle portion 60C may include a tapered portion 64C, and the upper portion 50C may include a body including sidewalls 52C and a top wall 53C. At least one upper surface of the top wall 53C may constitute the top surface 10C of the sleeve 1332C, which is located below the top surface 11C of the lens of the objective lens 1330C. Depending on the embodiment, the top surface of the tapered portion 64C may also be located below the top surface 11C of the lens of the objective lens 1330C and flush with the top surface of the top wall 53C.

[0493] Reference Figure 33 A- Figure 33 C. The top surface 10D of the sleeve 1332D may be flush with the top surface 11D of the lens of the objective lens 1330D, and the sleeve 1332D may be configured to be screwed onto the objective lens 1330D.

[0494] According to an embodiment, the inner surface of the sleeve 1332D and the outer surface of the objective lens 1330D may include threads corresponding to and engaging with each other, 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 or the objective lens 1330D.

[0495] The sleeve 1332D may include, for example, a first portion 60D and a second portion 50D.

[0496] The first portion 60D may include a tapered portion 64D, and the second portion 50D may include a body including sidewalls 52C and a top wall 53C. At least one upper surface of the top wall 53D may constitute the top surface 10D of the sleeve 1332D, which is flush with the top surface 11D of the lens of the objective lens 1330D. Depending on the embodiment, the top surface of the tapered portion 64D may also be flush with the top surface 11D of the lens of the objective lens 1330D.

[0497] According to an embodiment, the inner surface of the first portion 60D may include threads.

[0498] Depending on the embodiment, the top surface 10D of the sleeve 1332D may be above or below the top surface 11D of the lens of the objective lens 1330D. For example, the top surface of the top wall 53D may be above or below the top surface 11D of the lens of the objective lens 1330D, and the top surface of the tapered portion 64D may be flush with the top surface of the top wall 53D.

[0499] According to embodiments of the present disclosure, various alternative or additional embodiments of confocal microscopes may be provided. For example, a laser point scanning confocal system may be provided. A laser point scanning confocal microscope may involve focusing a single laser point through a small aperture (pinhole) and sequentially scanning the sample point by point in a zigzag pattern. The sample emits fluorescence, and the light is transmitted back through an optical system. The light is then read point by point by a detector, which may be a photomultiplier tube (PMT), but other light-measuring sensors may also be used. The signal from the sensor is recorded point by point, with each point constituting a single pixel in the image. Compared to spinning disk confocals, laser point scanning systems have advantages and disadvantages. Laser point scanning systems are generally slower than spinning disk confocals and, therefore, in many cases, are not suitable for high-throughput applications or live cell imaging. On the other hand, laser point scanning confocal systems penetrate deeper into the sample, providing better axial and lateral resolution. Recently, improvements have been made to laser point scanning systems to increase their speed, thereby beginning to compete with spinning disk speeds while still providing greater depth of penetration. The speed of laser point scanning confocal systems is limited by the scanning speed of the motor driving the system's scanning mirror.

[0500] Depending on the embodiment, the confocal subsystem of the present disclosure may include a laser point scanning confocal microscope and a spinning disk confocal microscope. The spinning disk confocal microscope is used for live sample imaging and high-throughput applications, while the laser point scanning confocal microscope can be used to penetrate deeper into the sample with higher resolution. Just as widefield imaging or other measurement methods are used to identify a "hit," embodiments of the present disclosure enable the spinning disk confocal microscope to rapidly scan a 3D sample and locate a few points of interest. The laser point scanning confocal microscope can then be used to capture more detailed images of the region of interest. Both the laser point scanning confocal microscope and the spinning disk system are commercially available as separate devices. However, using two separate devices in this manner presents several issues. First, the cost of both a spinning disk microscope and a laser confocal microscope would make the aforementioned workflow impractical. Furthermore, there is the technical challenge of repositioning the microscope to the region of interest on a spare microscope. By implementing the laser point scanning confocal microscope and the spinning disk system in the same device, the "hit" can be identified, and then the optical system can switch and scan the region of interest without moving the stage. Finally, there is the issue of studying living cells, as samples can change over time. Relative to the rate of biological change, moving samples to different devices takes too long. By the time a sample is moved to another device, the "hit" region of interest may have changed and may no longer be relevant.

[0501] Another advantage of having both a laser point scanning confocal and a spinning disk confocal in the same setup is that the laser point scanning confocal system can be used not for imaging, but to target specific areas of the sample for photobleaching. The laser point scanning confocal system and the specific control of the XY scanning mirrors provided therein allow the laser to be targeted to a very small and specific area of ​​the sample. This could be a point or a block defined in a zigzag scan. Then, once photobleaching occurs, the setup can be quickly switched to the spinning disk confocal to monitor fluorescence recovery after photobleaching (FRAP). Some specific applications include: (a) analysis of intracellular molecular diffusion (e.g., studying F-actin diffusion in primary dendritic cells after photobleaching of the region of interest); (b) quantifying the fluidity of biological membranes (e.g., membrane fluidity in Caenorhabditis elegans); and (c) protein binding analysis (e.g., monitoring the dynamic binding of chromatin proteins in vivo).

[0502] According to the embodiments of the present disclosure, the precise positioning accuracy of a laser point scanning confocal system is combined with the imaging speed of a spinning disk system to address unmet market needs in FRAP detection.

[0503] Figure 23 is a functional block diagram illustrating modal control of a device according to an embodiment.

[0504] The operation of the modality may be controlled by a central control unit (eg, a processor, a CPU, a microprocessor, etc.) Depending on the embodiment, the central control unit may also be referred to as a controller (eg, controller 1000).

[0505] The central control unit 900 can be connected to communicate with and control the components of the embodiments of the present disclosure. For example, the central control unit 900 can be connected to communicate with and control the components of the sample environment 90A, the components of the sample selection and positioning 90B, the components of the monochromator module 90C, the components of the imager module 90D, the external light source module 932, and the injection module 934.

[0506] As described above, elements of the controlled sample environment 90A may provide temperature control (902) and gas control (904).

[0507] Sample selection and positioning 90B can be controlled by using motors for positioning the sample in any X and Y directions (906 and 908).

[0508] Elements of the controlled monochromator module 90C may include monochromator excitation (910), monochromator emission (912), monochromator PMT (916), fiber selection (918), and a light source such as a flash lamp 914.

[0509] Elements of the controlled imager module 90D may include an objective selector 930, an image acquisition device such as a camera 920, a focus drive 924 for the objective, an LED and filter cube selector 922 for widefield imaging, a confocal cube selector 928 and a spinning disk module and controls (926) (e.g., selection and focusing), and a laser scanning confocal module controls (927).

[0510] Figure 24 is a flowchart of a control method of a multi-detection system according to an example embodiment.

[0511] Control of the devices may be coordinated through the use of controllers, as described above with respect to e.g. Figure 23 and / or Figure 32A-Figure 32B Input to the device (step S1805) may be accomplished through a local user interface of the device (such as a touchpad or graphical display) or by communicating with the device through a wired or wireless connection (such as a network).

[0512] In the case of input to the device, the input may be performed by using a user interface or a graphical user interface displayed on a computer or other terminal executing a control application.

[0513] The input may be user input, such as settings and parameters for performing device control.

[0514] In response to receiving input, control of the device may be achieved through various elements of the device, for example, as described above with respect to Figure 23 and / or Figure 32A-Figure 32B For example, in response to receiving user input, the device may be controlled to execute a gas control process of the gas module (step S1810), a sample positioning control program for controlling sample positioning (step S1820), a monochromator control program for controlling the operation of the monochromator (step S1830), and an imager control process for controlling the imager (step S1840), and output control results for each component of the device (step S1850).

[0515] Although control Figure 24 As shown, the elements can be controlled individually in any order, and it is not necessary to control all elements. Thus, multiple modalities of the device can be controlled in a single assay.

[0516] Figure 24The control methods shown and other functions described herein that can be performed by the controller can be implemented by executing one or more control programs to control the execution of a processing unit (e.g., a CPU) of the components of the device. The programs can be stored in a memory (i.e., RAM, ROM, flash memory, etc.) or other computer-readable media (i.e., CD-ROM, disk, etc.). The programs can be executed locally by the device or by a control device, such as a computer that transmits commands to be executed by the device.

[0517] Reference Figure 33 , embodiments of the present disclosure may include a display, and the controller may be further configured to cause the display to display a user interface. Figure 33 An example user interface is shown for a device with various optical mode combinations. Element 2300 is an image of the sample. Element 2301 is a drop-down menu for selecting the magnification factor. Element 2302 is a checkbox for enabling / disabling water immersion. If selected, and the objective lens is configured for water immersion, the controller can automatically pump water to the objective lens and automatically remove the water when imaging is complete or when the checkbox in element 2302 is deselected. Element 2303 is a drop-down list for selecting the EM wavelength. Figure 33 It is shown that a selection between 4 different EM wavelengths can be provided, but any number of EM wavelength selections can be provided. Element 2304 is a drop-down list for EX wavelength selection. Figure 33 It is shown that a selection between four different EX wavelengths can be provided, but any number of EX wavelength selections can be provided. Element 2305 is a drop down menu that allows the user to select between various modes of instruction. Figure 33 23. The system includes a rotating disk, laser scanning, and widefield modalities. Depending on the embodiment, the modalities listed in element 2305 may depend on the modalities present in the system. For example, the system may have any combination of the above modalities (and / or additional modalities), or only a single modality. Element 2305 may not be provided where only a single modality is provided. Depending on the embodiment, elements 2301, 2302, 2303, 2304, and 2305 are not limited to drop-down menus and selection boxes, and may indicate options for selection in any manner known to one of ordinary skill in the art.

[0518] In accordance with embodiments, the interface may include a display element that enables a user to select multiple modalities to be automatically executed in sequence. For example, based on one or more user inputs to the interface, the controller may be configured to control the sequence of automatically executed modalities. The sequence may include any sequence of modal operations, including the sequences of modal operations described in the present disclosure. For example, an operation using a spinning disk or widefield imaging system may be performed followed by an operation using a laser point scanning confocal system.

[0519] The components and features of the optical module are further described in US Patent 7,782,454, entitled "Universal Multi-Detection System for Microplates," which is incorporated herein by reference in its entirety for all purposes.

[0520] For example, according to one aspect, an optical module is provided, comprising a first optical device that transmits narrowband light, and a first optical filter and a first monochromator that provide alternative paths for the narrowband light. The optical module may also include a light source that generates light as broadband excitation light, wherein the first optical device transmits a narrow band of the broadband excitation light and blocks other bands of the broadband excitation light through the first optical filter or the first monochromator; a second optical device that directs the narrow band of broadband excitation light onto a sample and receives emission light from the sample; a third optical device that transmits the narrow band of emission light; and a detector that converts the narrow band of emission light into an electrical signal; wherein the third optical device includes a second optical filter and a second monochromator that provide alternative paths for the narrow band of emission light.

[0521] Multimodal measurements in cloud-based systems

[0522] In certain aspects, the devices and methods disclosed herein can be used to perform a complete analysis of a cell sample by qualitatively and quantitatively measuring different parameters of the same cell sample. These methods may include measuring metabolic function, bioenergetic balance, bioenergetic capacity, and bioenergetic work of the cell, such as measuring O2, CO2, and pH using a sensor subsystem. The methods can include using an optical module, such as an automated cell imaging reader, such as Cytation™ 5 or Cytation™ 7, to visually observe sample properties, such as cell growth, cell health, cell microenvironment, morphological changes, ultrastructural changes, and marker expression, as disclosed in U.S. Patent No. 10,072,982, the entire contents of which are incorporated herein by reference for all purposes. These methods may include detecting attachment, ultrastructural changes, growth, morphological changes, cell-cell interactions by impedance measurement using sensing systems or devices described in U.S. Patents 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 Publication 2018 / 0246019 and WO2021202264A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0523] 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 at the electrode result in detectable changes. To this end, cell-substrate impedance monitoring is a useful tool for assessing cell proliferation and cell lysis. In conjunction with impedance real-time cell analysis, the brightfield and fluorescence detection optical module of xCELLigence eSight is an exemplary optical module that provides live cell imaging during impedance measurements, as described in U.S. Patent Application Publication 2021 / 0301245, which is incorporated herein by reference in its entirety for all purposes.

[0524] It will be appreciated that serial analysis may be performed by other means of performing different measurements on the same sample, such as mass spectrometry, spectroscopy, phosphorescence lifetime imaging microscopy (PLIM) and / or fluorescence lifetime imaging microscopy, including 2-photon excitation imaging, and the like.

[0525] application

[0526] The systems, consumables, and methods described herein can have various applications. Exemplary applications are described below.

[0527] Cell migration / adhesion

[0528] In one aspect, the systems, consumables, and methods described herein are used to analyze cell migration and / or adhesion.

[0529] Metastatic invasion of cancer cells is a clinical challenge in cancer therapy. Cell migration is often a bioenergetic process, and the ability to simultaneously obtain quantitative measurements related to cell migration / invasion and bioenergetic metabolic activity can open new windows for therapeutic development. Combining impedance measurements as a surrogate measure of cell migration with OCR / PER measurements will allow testing of cell metabolism modulators that induce inhibition of cell migration without affecting cell viability. These measurements can be further combined with fluorescent biosensor imaging for sensors of signaling cascades.

[0530] A similar procedure can be applied to study cell adhesion by coating plates with different extracellular matrices (ECM) and coupling changes in impedance representing cell adhesion with changes in cell metabolism.

[0531] Stem cell differentiation

[0532] In one aspect, the systems, consumables, and methods described herein are used to analyze stem cell differentiation.

[0533] Stem cell differentiation is a lengthy process lasting weeks to months, involving a change in cellular phenotype from a proliferating / undifferentiated state to a specialized state. Stem cell differentiation also involves significant changes in cell morphology and metabolic activity, which need to be controlled to generate specialized cells with the correct phenotype for use as disease models for therapeutic development or directly as cell and gene therapies for various diseases, including tissue regeneration.

[0534] Simultaneous monitoring of culture environmental conditions, cell morphological changes via impedance measurements, and metabolic activity will optimize cell model development to identify key cellular properties for stem cell-derived therapies.

[0535] Example

[0536] The embodiments may be further understood with reference to the following examples, which are intended to be illustrative rather than limiting.

[0537] Example 1: Exemplary Scenario

[0538] Cells are seeded at 50-90% confluency in the assay wells of a multi-well microplate. Suspended cells adhere to the bottom of the wells to maximize sensitivity. A 96-well sample carrier constructed and arranged to mate with the device is used in this exemplary protocol. However, the multi-well sample carrier can have any number of wells corresponding to the device, such as 1, 6, 8, 12, 24, 36, 48, 64, 72, 96, 192, 384, or other wells. The temperature of the cell suspension is controlled.

[0539] The device places a sensor probe into an analysis well. The sensor is positioned 200 microns above the well bottom, forming a transient microchamber of approximately 3 microliters, also referred to herein as the "measurement chamber." As oxygen and pH levels change, the sensor measures these changes. Measurements are typically taken over a predetermined time period between 1 and 5 minutes, for example, 3 minutes. The rate of change is automatically calculated by a computing device. After the measurement period, the sensor probe is lifted, allowing the extracellular medium to return to baseline.

[0540] The sensor cartridge also contains ports (four per well) to allow injection of modulators (target analytes) into the cell wells during the assay. When the device protocol specifies, for example, via a graphical user interface, the controller instructs the dispensing system to inject the test compound into the assay wells and perform a gentle mixing step to ensure distribution of the compound throughout the assay medium. All wells are processed simultaneously in this manner. Subsequent measurement cycles, any additional injections specified by the protocol, and rate calculations are performed automatically.

[0541] For testing purposes, an exemplary protocol was performed using THP-1 cells (human monocytes from a patient with acute monocytic leukemia). 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 shown in Figure 2. Figures 10A-10D shown.

[0542] Figure 10A The graph shows the change of OCR measurement values ​​measured by the system disclosed in this article over analysis time. Figure 10B The graph shows the change of OCR measurement values ​​measured by the comparative system over analysis time. Figure 10C The graph shows the change in ECAR measurement values ​​measured using the system disclosed in this article over the measurement time. Figure 10D The graph shows the ECAR measurements measured using the comparative system as a function of analysis time.

[0543] The exemplary protocol was also performed using A549 cells (human lung cancer cells) and administration of 5 mM metformin as a target agent. OCR data was measured using the system described herein and the comparative system. The results are shown in FIG. Figures 11A-11B shown.

[0544] Figure 11A The graph shows the change of OCR measurement values ​​measured by the system disclosed in this article over analysis time. Figure 11B The graph shows the change of OCR measurement values ​​measured by the comparative system over analysis time.

[0545] Thus, the system having a temperature control element, a signal processing module, and a motion actuator assembly motor as described herein achieves significant improvements in lower-limit OCR detection accuracy and readability compared to comparable systems while simultaneously detecting ECAR. While not wishing to be bound by theory, it is believed that improving temperature uniformity between samples within a controlled temperature zone can improve the system's performance in sensing target analytes and cell biology.

[0546] Example 2: Water Sample Evaporation Measurement Protocol

[0547] In the system disclosed herein, six measurements were performed over a six-hour period using a known volume of water in a porous sample carrier, with a modified protocol configured to perform four measurements per hour. The evaporation of the water sample was measured using a plate reader. A standard curve was created by measuring the absorbance of known volumes of water. After each measurement, absorbance measurements were collected from the test plate immediately. The standard curve was used to calculate the amount of water in each well of the test plate to estimate the amount of water lost by evaporation during the six-hour measurement. The results were calculated as a percentage of the total volume lost. The average evaporation for each measurement was calculated as Figure 12 As shown in the table.

[0548] like Figure 12 As shown in the table, the maximum average percentage of water lost by evaporation during the 6-hour measurement was 10.04%. Therefore, the volume of sample fluid lost by evaporation was relatively low.

[0549] While not wishing to be bound by theory, it is believed that improving temperature uniformity between samples within a controlled temperature zone can reduce evaporation of sample fluid, thereby improving the performance of the system in sensing target analytes and cell biology.

[0550] Example 3: Extended Period Run in Multimodal Analysis

[0551] When the device is configured to analyze cell cultures for extended periods of time in one or more analysis modes, various environmental and sampling control elements in the device are activated to maintain consistent growth conditions over the extended period of time, and to maintain measurement conditions in sensors used to monitor growth conditions.

[0552] In various embodiments, an operator can set the extended period to last at least one hour, two hours, three hours, four hours, five hours, six hours, etc., up to twenty-four hours, forty-eight hours, seventy-two hours, etc., to analyze cell cultures and other samples for longer periods of time than previous analytical devices without requiring human intervention to maintain growth conditions for that time period.

[0553] The device includes a sensing system and a stage included in a cavity of the device. The stage is configured to receive a sample carrier having a plurality of apertures 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 in 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 in one or more of an x-axis, a z-axis, and a y-axis, which can include rotation in a yaw, pitch, or roll direction. In a multimodal analytical device, the motion actuator assembly can also move the stage and / or sample carrier or substrate between various devices or sensors, with the sample being held on the stage for sequential analysis or access by these devices or sensors.

[0554] For example, the motion actuator assembly can move the sample to a first position for access by the flux detector, a second position for access by the imaging module, a third position for access by the power measurement module, and so on. In various embodiments, the first position is provided for access by the image acquisition element, and the second position is provided for access by the impedance element. In various embodiments, as Figure 48As shown, a first location is provided in the device 5000 for access by the image acquisition element 5080 and the impedance element 5050 (connected to the sample carrier 5040 via the electrical interface 5051), and a second location is provided for access by the flux detector 5070. In various embodiments, the fluid handler 5030 can be accessed at the third location or at one or both of the first and second locations.

[0555] like Figure 48 As shown, various accessory elements of the image acquisition element 5080 and the flux detector 5070 are aligned in first and second positions, respectively. For example, excitation sources 5090a-5090b and optical adjustment elements 5095a-5095f (e.g., mirrors, lenses, filters, optical paths, etc.) are provided between the image acquisition element 5080 and the flux detector 5070 and the sample carrier 5040 (when in the associated positions) to place the wells and corresponding measurement elements in optical communication with each other. In addition, the flux cartridge 5060 can be preloaded with various compounds, growth media, and other compounds that are injected into or exchanged with the wells in the sample carrier 5040.

[0556] In various embodiments, the flux cartridge 5060 is movable relative to the sample carrier 5040 (or vice versa) along an axis that is substantially perpendicular to the axis along which the sample carrier 5040 moves between the first position and the second position, or along a plane that intersects each of a plurality of wells in the sample carrier 5040. The flux cartridge 5060 includes a plurality of heads, each directed toward a given well in the sample carrier 5040, the well including a surface proximate a surface of the sample carrier 5040, wherein the well is defined to define a closed reaction chamber when in contact with the sample carrier 5040. The closed reaction chamber is configured to maintain a seal that limits the volume of liquid contained in each sample and / or reduces the rate of evaporation volume from the reaction chamber over a period of time.

[0557] The sensing system includes an array of sensor cells configured to generate an electrical signal proportional to an analyte observed in a sample well. For example, a first sensor in the sensor array may monitor a first analyte proportional to the amount of gaseous O2 in a given well over an extended period of time to generate a first signal, while a second sensor in the sensor array may monitor a second analyte proportional to the pH value in the given well over a longer duration of time to generate a second signal. The sensor cells in the sensor cell array are positioned (via a motion actuator assembly) to correspond to corresponding wells on a sample carrier to analyze the contents thereof, thereby controlling and monitoring the sample held therein.

[0558] The device includes a liquid handling system for distributing various substances to the sample in each well of the sample carrier. In various embodiments, the liquid handling system is supplied by a cartridge that can be inserted into (and removed from) the device without affecting the atmosphere of the cavity. The cartridge can include tanks for different substances supplied to the wells, including water, water-based solutions (e.g., aqueous solutions of candidate compounds / substance compounds or other reagents), dyes, cell growth media, cell culture, N2, O2, CO2, etc. For example, U.S. Patent 9,170,255, entitled "Cell Analysis Apparatus and Methods," further describes exemplary components and features that can be used in cartridges, the entire contents of which are incorporated herein by reference for all purposes.

[0559] The device includes a sampling control element for controlling one or more characteristics of a sample within each well of a sample carrier measured by a sensing system over an extended period of time. The sampling control element controls the characteristics so that the characteristics of any given sample are within a predetermined amount of any other sample within another well of the sample carrier. For example, the sampling control element may include a sample temperature environment control element configured to control the temperature of the sample (e.g., to within + / - X degrees), a gas control element for controlling the gas content of at least one of the O2, CO2, and N2 content of the sample (e.g., to within + / - X parts per million (ppm)), and a humidity control element configured to control the humidity of the sample (e.g., relative humidity to within + / - X%). In various embodiments, the sample temperature environment control element is a heater.

[0560] In addition to controlling the relative characteristics of different wells in parallel (e.g., the first well versus the second well at a first time), the sampling control element can also control the characteristics of a given well (e.g., the first well at a first time and the second well) longitudinally over an extended period of time so that the characteristics in the well monitored during the analysis period remain within a controlled value range. Various consumables provided by the fluid processor and / or cartridge allow material to be added to replace lost material (e.g., due to evaporation, sublimation, sample consumption (e.g., cellular respiration), or dispersion into the environment outside the well). The material source (e.g., cartridge) can be replaced throughout the extended period to account for consumed material and provide a fresh supply of material to the well when needed to maintain the characteristics in the well or to provide additional analytical material (e.g., cell growth compound, test drug, etc.). Thus, the device can maintain consistent growth conditions for multiple samples in corresponding multiple wells simultaneously in parallel and longitudinally (e.g., the first to Nth wells each of the first to nth times).

[0561] At different times during the extended period of analysis, the device can observe various features of the sample being observed. In various embodiments, the device includes one or both of an image acquisition element (such as an optical module described herein) and an impedance element (such as an electrical measurement module described herein) that can operate at different locations within the device cavity, and a motion actuator assembly positions the sample carrier at these locations at different times. Each of the various observation modules can be located in various cavities or subcavities in the device. For example, Figure 48 As shown, the imaging collection element 5080 and the flux detector 5070 are both disposed within the chamber 5010 of the apparatus 5000, but are divided into different sub-chambers by a divider 5020. The divider 5020 may include various vents and atmosphere or environmental controls to provide different temperature, humidity, or airflow characteristics in different portions of the chamber 5010 at different times.

[0562] The image acquisition element includes one or more cameras and various camera accessory devices (e.g., mirrors, lenses, light sources) to allow images of the sample (or features of the sample) within each well of the sample carrier to be acquired. These images are acquired from the underside of the sample carrier (e.g., opposite the surface defining the well) through a transparent or translucent window. When the wells include electrodes (e.g., for electrical excitation of the sample and / or impedance measurement of the sample), the electrodes are positioned to leave gaps of predetermined size between each other, which define the windows in which imaging occurs. In various embodiments, the image acquisition element can be configured to acquire and process images of each well from the sample carrier individually (the operator can choose to image specific wells or in a specific order or to forgo imaging), or to acquire and process images of some or all wells in batches in parallel.

[0563] The impedance element includes an electrode surface configured to measure impedance changes caused by sample attachment within each hole of the sample carrier, to excite the sample with an electrical signal within each hole of the sample carrier, or both. In various embodiments, the electrode surface is located at the bottom of the sample carrier (e.g., on the side opposite to the side defining the hole), and the electrode surface includes a non-conductive carrier. When positioned to interact with the sample carrier, the multiple electrode arrays included in the impedance element are positioned to contact the sample carrier. Each of these electrode arrays includes at least two electrode structures that are located on the same plane (e.g., the fragment plane) and have substantially the same surface area as each other. The electrode structures are connected (e.g., electrically connected) to corresponding components of the multiple connection pads located on the sample carrier. Therefore, when electrically communicating with the sample carrier, the impedance meter, impedance analyzer, or impedance measurement circuit of the impedance element can detect the electrical impedance changes between the electrode structures caused by the sample changes. Similarly, when electrically communicating with the sample carrier, the voltage or current source of the impedance element can excite the sample with an electrical signal.

[0564] The operation of each component and module of the device is coordinated by a controller, which can be any type of computing device (e.g., an FPGA array, a microcontroller, a processor and coupled memory, an ASIC, etc.) that is operably connected to the various components 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 via a sampling control element over an extended period of time, acquire data corresponding to the first signal and the second signal via various sensing systems (including an image acquisition element and an impedance element) for corresponding analysis at at least two time points over the extended period of time, and adjust the first and second information. In various embodiments, adjusting 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.

[0565] Figure 49 A schematic system diagram of an embodiment of a system 5100 for analyzing living cells is shown, particularly for measuring extracellular flux, impedance, and imaging for long-term measurements of cell samples, including simultaneous measurement of one or more combinations of extracellular flux, impedance, and imaging. The system incorporates a sample carrier 300 that is configured to interface with a sensor cartridge 5102. The sample carrier 300, also referred to as a well plate, includes an array of wells 200 for individually holding cell samples and an impedance and / or exciter component or array thereof. One or more wells include an array of electrodes for collecting impedance measurements at the bottom of the wells, and a window at the bottom of the wells for transmitting light through the bottom of the wells and imaging, such as by an optical subsystem 5120, such as an inverted microscope. The optical subsystem can incorporate any number of optical configurations and / or imaging modalities, such as wide-field fluorescence microscopy, wide-field brightfield microscopy, or confocal fluorescence microscopy, as well as any of the imaging systems described above. For example, exemplary configurations of windowed sample carriers with impedance and optical measurement capabilities are described in PCT patent application WO 2021202264, “Systems and methods for electronic and optical monitoring of biological samples,” which is incorporated herein by reference in its entirety for all purposes.

[0566] The sample carrier 300 can be positioned on a bracket 310 and a heating stage 360 ​​that can be moved to interact with a fluid handling system 5130 that can include a fluid injection manifold 5130 and a heater / cooler 5140 for fluid temperature control (see FIG. Figure 8 In one embodiment, the manifold is configured to grip the cartridge and move the stage up and down. A substance / medium source 5170 is connected to the manifold 5130 for delivering fluid to the sample carrier 300.

[0567] Optical components, such as filters 5160a, 5160b, focusing optics 5162a, 5162b, excitation source 5166, and detector 5164, are positioned above the sample carrier 300 and cartridge 5102. An optical manifold 5166 is an optical element (which can be an array of optical fibers, each connected to an excitation LED, or multiplexing one excitation source to many fibers, or an array of glass or plastic blocks) over which the optical elements can be scanned.

[0568] Figure 50 An exploded view of the sample carrier 300 and cartridge 5102 is shown, comprising a plurality of spikes 5104, each having an analyte sensor 5106 at its distal tip, wherein the spikes are sized and shaped to interface with the well 200 to form individual microcavities. An electrode interface / impedance reader 5108 is electrically coupled to the sample carrier for receiving and transmitting electrical signals from and to impedance electrodes in the well 200 (see FIG. Figure 53 ).

[0569] Figure 49 A schematic system diagram of an embodiment of a system 5100 for analyzing living cells is shown. Figure 50 An exploded view of the sample carrier 300 and cartridge 5102 is shown. Figure 51 and Figure 52 A planar side view of the stage 360 ​​is shown without the sample carrier 300. The platform 360 may include a heater / cooler and may include peripheral features 5112 to hold and align the sample carrier 300. One or more environmental characteristic sensors 5114 (e.g., temperature, gas, humidity, etc.) may be included on the stage.

[0570] Figure 53 Shown are top and perspective views of the bottom surface of the well 200 of the sample carrier 300. The well bottom can include a standoff in the form of a protrusion 5118, a scaffold or other engaging feature that interfaces with the ridge 5104 of the sensor cartridge, thereby forming a stop that allows the ridge to rest against the protrusion at a specified distance to define a microchamber. Figure 54 Further shown are windows 5112 disposed between electrode elements 5124 on the surface of the well, which allow imaging of the well bottom.

[0571] Example 4: Direct identification of mitochondrial toxicity using a novel metric based on mitochondrial oxygen consumption rate

[0572] Mitochondrial toxicity (MitoTox) is a common problem in therapeutic development, leading to compound / substance candidate attrition and post-marketing compound / substance withdrawal (Wallace, K.B., 2008, Mitochondrial off-targets of drug therapeutics, Trends in Pharmacology, 29, 361–366). Direct measurement of mitochondrial oxygen consumption using Agilent Seahorse XF technology has been shown to be a specific and sensitive marker / indicator among methods used to assess compound / substance-induced mitochondrial toxicity in compound / substance discovery and preclinical safety (Yvonne Will & James Dykens (2014) Industrial mitochondrial toxicity assessment - a decade of technological development and insights, Expert Reviews in Drug Metabolism and Toxicology, 10:8, 1061-1067, DOI:101517 / 17425255.2014.939628) (Tilmant Ka,*, Gerets Ha, De Ron Pa, Hanon Ea, Bento-Pereira Ca,b,l, Atienzar FA, In vitro screening of cellular bioenergetics to assess mitochondrial dysfunction in drug development,a,c In Vitro Toxicology 52 (2018) 374-383).

[0573] Therefore, this article discloses a standardized XF solution that can evaluate compounds that exhibit mitochondrial toxicity. As described herein, the XF Pro analyzer incorporates several novel design features that provide enhanced sensitivity, precision, and consistency. Here, we leverage these improvements to detect compound / substance-induced mitochondrial dysfunction for OCR measurements.

[0574] The Agilent Seahorse XF Mito Tox assay workflow involves continuous infusion of oligomycin and FCCP, but includes a separate control where rotenone / antimycin A is provided prior to the assay. Compounds to be assessed for mitochondrial toxicity are provided to cells at the designated times prior to the assay.

[0575] Based on the response of test compounds in basal, oligomycin, and / or FCCP OCR compared to appropriate controls, the XFMito-Tox assay can identify 3 different types of mitochondrial toxicity: direct / indirect inhibition of the ETC or other mitochondrial processes, uncoupling of the ETC from OxPhos, and (potentially) specific inhibition of OxPhos mechanisms (CV, ANT, PiT).

[0576] Significant improvements are provided by extracting a new parameter, the Mito Tox Index (MTI), derived from the oxygen consumption rate (OCR) measured by the system disclosed herein (Agilent Seahorse XF Analyzer).

[0577] Surprisingly, the improved sensing accuracy achieved by implementing the devices and methods disclosed herein has enabled this method of deriving an easily interpretable mitochondrial toxicity index, providing a simple and robust method for in vitro screening and verification of toxicity. This workflow is able to simplify the complex respiratory response into a mitochondrial toxicity index (MTI), providing two types of MTIs that score the inhibitory effect and uncoupling effect of the electron transport chain (ETC) on a positive and negative scale. The inhibitor MTI is designed to calculate the relative inhibitory effect on the maximum OCR relative to the effect of the ETC inhibitor control - rotenone / antimycin A mixture. In contrast, the uncoupler MTI is used to calculate the relative increase in the minimum OCR measured after injection of oligomycin. In compounds that do not show significant scores in both MTIs, potential ATP synthase inhibitors can be identified by monitoring specific inhibition of the basal OCR, because ATP synthase inhibitors do not affect the maximum OCR. The ability to derive defined indicators enables additional functionality, such as conveniently generating dose-response relationships or conveniently setting thresholds for "hit" identification.

[0578] Defining the Mitochondrial Toxicity Index (MTI)

[0579] To distinguish the three modes of mitochondrial toxicity described above and to quantify the extent of toxicity, the Mito Tox Index (MTI) value was derived using the improved measurement accuracy of the device disclosed herein. Inhibition-induced mitochondrial toxicity, where inhibition is defined and detected as a decrease in the FCCP OCR of the test compound compared to the maximum FCCP OCR of the vehicle group, resulting in a negative MTI value (usually between 0 and -1), such as Figures 37A-37C Shown and described.

[0580] Figure 37A Involving a situation where a test compound causes a decrease in FCCP-induced OCR compared to a vehicle (negative) control (MTI = 0), the compound is then classified as an inhibitor and has a negative MTI value (e.g., MTI = -0.8). Note that Rot / AAOCR serves as a positive (+) control for inhibition (MTI = -1). Figure 39 B- Figure 39 C provides a summary of the measurements and groupings used for inhibition controls.

[0581] Mitochondrial toxicity caused by uncoupling, where uncoupling is defined and detected as an increase in the oligomeric OCR of the test compound compared to the minimal oligomeric OCR of the vehicle group, resulting in a positive MTI value (usually between 0 and 1), as Figures 38A-38C Shown and described.

[0582] Figure 38AIt involves a situation where, compared to a vehicle (negative) control (MTI = 0), a test compound causes an increase in oligomycin-induced OCR, and then the compound is classified as an uncoupler with a positive MTI value (e.g., MTI = 0.6). Note that the vehicle FCCP OCR is used as a positive (+) control for uncoupling (MTI = 1). Figure 40 B- Figure 40 C provides a summary of the measurements and grouping for the uncoupling control.

[0583] In summary, MTI is a fractional value of the effect of a test compound compared to the corresponding control for uncoupling and / or inhibition. The uncoupler MTI is calculated as a positive exponent and is defined as the ratio of the fraction of uncoupling induced by the test compound to the maximum uncoupling fraction (FCCP OCR of the vehicle group, positive control). Note that the oligomycin OCR of the vehicle group is used as a negative control for uncoupling. In contrast, the inhibitor MTI is calculated as a negative exponent and is defined as the fraction of inhibition caused by the test compound compared to the maximum inhibition (FCCP OCR of the Rot / AA group, positi...

Claims

1. A device capable of measuring for an extended period of time, comprising: a sensing system comprising an array of sensor cells configured to generate a first signal in response to a first analyte over an extended period of time and to generate a second signal in response to a second analyte over the extended period of time, each sensor cell of the array of sensor cells being positioned to correspond to a respective 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 each other in one or more of an x-axis, a z-axis, and a y-axis; a liquid handling system that dispenses a substance into at least one well of the sample carrier; a sample control element configured to control a characteristic of a sample within at least one well of the sample carrier within a predetermined amount of another sample within another well of the sample carrier during the extended period; and a controller operably connected to the sensing system and the sample control element, the controller being configured to: controlling one or more of temperature, humidity, and gas content of the environment surrounding the sample carrier during the extended period; and Data corresponding to the first signal and the second signal is acquired for at least two points in time spanning the extended period.

2. The device according to claim 1, wherein The extended period measurement is performed in a microchamber having a reduced volume of no more than 3 microliters, the reduced volume being generated by moving the sensor cells of the sensor cell array down along predetermined positions into corresponding wells in the sample carrier.

3. The device according to claim 1, wherein The extended period measurements are performed in a non-continuous manner between a single modality selected from the group consisting of: flux measurement, impedance measurement, and imaging.

4. The device according to claim 1, wherein The extended period measurements are performed in a discontinuous manner between at least two modalities selected from the group consisting of: flux measurement, impedance measurement, and imaging.

5. The device according to claim 1, wherein The control element controls the sample environment to maintain environmental parameters at target levels for associated wells in the sample carrier.

6. The device according to claim 5, wherein The target level for the environmental parameter is programmatically changed over time measured over the extended period of time.

7. The device according to claim 1, wherein The control element controls the sample environment via at least one of direct cellular / intracellular / pericellular / proximity measurement of sample parameters to achieve a target cellular microenvironment of a biological model in the sample.

8. The device according to claim 7, wherein The cellular microenvironment is controlled on a per sample basis.

9. The device according to claim 5, wherein The target level for the sample parameter is programmatically changed over the time measured over the extended period of time.

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

11. The device according to claim 1, wherein The sample control element includes one or both of the following: a sample temperature control element configured to control the temperature of the sample; or Sample environment control elements, including one or both of the following: a gas control element configured to control the content of one or more gases among O2, CO2 and N2 in the sample, or A humidity control element is configured to control the humidity of an environment.

12. The device according to claim 11, wherein The sample control element includes a heater.

13. The device according to claim 1, wherein The first signal measures the first analyte in proportion to the O2 content in a given well, and the second signal measures the second analyte in proportion to the pH value in the given well.

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

15. The device according to claim 1, wherein The extended period 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. The apparatus according to claim 1, further comprising: an image acquisition element configured to image a sample or a feature of the sample within each of a plurality of wells defined in the sample carrier through an opening or window; Wherein, the image acquisition element is configured to acquire and process at least one image from each hole of the sample carrier.

17. The device according to claim 1, wherein The sample carrier comprises: A plurality of wells are configured to hold a predetermined amount of sample, wherein each well of the plurality of wells includes an opening or window that allows the image capture element to capture at least one image from each well of the sample carrier.

18. The apparatus according to claim 1, further comprising: an electrode surface comprising a non-conductive carrier located at the bottom of the sample carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises at least two electrode structures positioned on a single plane and having substantially the same surface area; and A plurality of connection pads are located on the sample carrier, wherein each connection pad is in electrical communication with at least one of the electrode structures within each of the plurality of wells.

19. The device according to claim 1, wherein A plurality of wells configured to hold a predetermined amount of sample are positioned above the plurality of electrode arrays, wherein each well of the plurality of wells includes an opening or window allowing the image capture element to capture at least one image from each well of the sample carrier.

20. The apparatus according to claim 1, further comprising: An impedance measuring device configured as follows: measuring impedance changes caused by sample attachment in each hole of the sample carrier; or exciting a sample in each well of the sample carrier by an electrical signal, wherein the electrode surface is located at a bottom of the sample carrier, and wherein the electrode surface comprises a non-conductive carrier; a plurality of electrode arrays positioned on the sample carrier, wherein each electrode array comprises 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, wherein each connection pad is in electrical communication with at least one of the electrode structures; wherein the impedance element detects changes in electrical impedance between or within the electrode structures, or excites the sample via an electrical signal; and The impedance element detects changes in electrical impedance between or within the electrode structures, or excitation output of the sample from an electrical signal.

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

Images