Systems and methods for electronic and optical monitoring of biological samples

Abstract

A system for electronically and optically monitoring biological samples, comprising: a multiwell plate having a plurality of wells configured to receive a plurality of biological samples, each of the wells having a set of electrodes and a transparent window on the bottom of the wells that do not have electrodes; an illumination module configured to illuminate the wells; a cradle configured to receive the multiwell plate, the cradle having openings on its bottom that expose the transparent windows of the wells; and an optical imaging module movable between different wells of the same multiwell plate to capture images through the windows.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority to U.S. Patent Application No. 16 / 833,651, filed on March 29, 2020. The entire content thereof is incorporated herein by reference in its entirety.

[0002] The present invention generally relates to systems and methods for electronically and optically monitoring biological samples, and more particularly, to systems and methods for continuously and in real - time electronically and optically monitoring the same biological sample within a single well.

Background Art

[0003] Cell - based assays provide a preliminary assessment of the therapeutic effects on the human body. Many cell - based assays are endpoint assays, limited to a single point in time, but techniques known as cell - substrate impedance monitoring enable continuous monitoring of cells. Cell - substrate impedance monitoring evaluates the interaction between cells and electrodes that results in detectable changes in cell adhesion, growth, morphology, and motility on the electrodes. For this purpose, cell - substrate impedance monitoring is a powerful tool for evaluating cell proliferation and cytolysis.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Cell - substrate impedance monitoring techniques can reveal the dynamics of cell responses to potential therapies, but they have limitations. In particular, they are limited to detecting changes that occur when cells are cultured on the electrode surface. Therefore, there is a need to develop further improvements to expand the capabilities of cell - substrate impedance monitoring for evaluating the effects of therapies on cell biology. [Means for solving the problem]

[0005] In one aspect of the present invention, a system is provided for electronically and optically monitoring a biological sample, comprising: a multiwell plate having a plurality of wells configured to receive a plurality of biological samples, each of the wells including a set of a plurality of electrodes and a transparent window on the bottom surface of the electrodeless well, and an illumination module configured to illuminate the well; a cradle configured to receive the multiwell plate, the cradle having an opening in the bottom configured to expose the transparent windows of the wells; and an optical imaging module movable between different wells of the same multiwell plate to capture images through the windows via the open bottom. In some embodiments, a set of electrodes is configured to monitor cell matrix impedance.

[0006] The lighting module may be a single light source, but it is preferable that the lighting module includes multiple lights configured to independently illuminate one or more of the wells. More preferably, the lighting module includes an array of light-emitting diodes (LEDs). Most preferably, each LED is arranged to illuminate a single well. In a preferred embodiment, the lighting module is a bright-field lighting module.

[0007] The cradle is configured to receive and preferably cover the multiwell plate, and thus can provide a hinged cover. Preferably, the lighting module is bonded to the inner surface of the cover. The cradle may include a contact sensor for sensing the receipt of the plate. In some embodiments, the cradle electronically engages with both the multiwell plate for electronically communicating with the set of electrodes and the lighting module for communicating lighting commands.

[0008] Since the bottom of the cradle is open, exposing the transparent windows of the wells, the optical imaging module may be positioned beneath the cradle and configured to move from well to well, acquiring images through the corresponding transparent windows. In some embodiments, the optical imaging module is configured to acquire one or more images from a single well at a time. In some embodiments, images are acquired under bright-field illumination of the well, for example, to determine the number of cells or the cell confluence parameter (e.g., confluence percentage). In other embodiments, images, such as fluorescence images, are acquired after molecular excitation. The optical imaging system itself may include an excitation light source configured to excite one or more molecules, such as fluorescent molecules bound to antibodies or antibody fragments, for binding to biomolecules such as proteins, polypeptides, or nucleic acids, either inside or on the cell surface. Examples of excitation light sources include one or more lights selected from the group consisting of ultraviolet, violet, blue, green, yellow, orange, and red light.

[0009] In some embodiments, the optical imaging module includes a camera capable of continuously acquiring a series of cell images at sufficiently short time intervals to show cell movement. In some embodiments, the camera acquires at least 30 images per second. In other embodiments, the camera acquires at least 60 images per second. In either or both embodiments, the optical imaging module may include a camera, a bandpass filter, a tube lens, and an objective lens. High-speed imaging not only allows for a visual representation of cell movement but also enables the superposition of images from different light sources (e.g., bright-field illumination and multiple light sources of different wavelengths, each to excite fluorescent molecules and produce monochromatic fluorescence images) to reveal cellular changes occurring during motion. In some embodiments, images acquired from bright-field illumination and fluorescence of three or more colors are superimposed to form a single image for each point in time. From this imaging, the number of cells or cellular parameters, as well as the presence or absence of fluorescence, total fluorescence intensity, and average fluorescence intensity can be determined, thereby further revealing detailed cellular dynamics.

[0010] The system also includes, or may be, a computer processor communicatively coupled to a cradle for selectively manipulating each of the electrode sets, optionally for electronically monitoring the cellular substrate impedance in one or more wells, an illumination module for selectively illuminating one or more wells, and an optical imaging module for selective motion and acquisition and reception of images from one or more wells. In some embodiments, the computer processor is programmed to acquire images from one or more wells via the optical imaging module in response to one or more wells reaching or following an impedance-based value or parameter set from the electronic monitoring. In some embodiments, the computer processor is configured to electronically monitor the cellular substrate impedance and optically monitor the same well, and to pair the impedance and optical data for display or analysis. In some embodiments, the computer processor is configured to electronically monitor the cellular substrate impedance over a period including a specific time interval between two consecutive electronic impedance measurements / monitoring, and optically monitor the same well over a period within the electronic monitoring period, or over a period different from the electronic monitoring. Exemplary impedance monitoring periods can be programmed to range from short periods of a few minutes to longer periods of several hours, days, or even weeks. The impedance monitoring time interval between two consecutive impedance measurements can be specified or programmed to range from a few seconds or less than one second to a maximum of one minute, several minutes, one hour, or even several hours. The optical monitoring period can be programmed to be inside, outside, or over the same period as one or more impedance monitoring periods.

[0011] The system may be used with a single plate, but the system may also comprise two additional multiwell plates, each having multiple wells configured to receive multiple samples, with each well having a pair of electrodes and a transparent window on the bottom surface of the electrodeless wells; two additional illumination modules configured to illuminate the wells of the two additional multiwell plates; and two additional cradles configured to receive the two additional multiwell plates, each having an open bottom that exposes the transparent windows of the wells of the two additional multiwell plates, where an optical imaging module is movable across all wells to capture images from all windows through the exposed bottom.

[0012] The system may also include a cell or tissue culture vessel, not configured for electronic monitoring, in which an optical imaging module is configured to capture images from within the cell or tissue culture vessel. In an exemplary embodiment, the system may include a multiwell plate having a plurality of wells configured to receive a plurality of biological samples, each well having a transparent bottom surface; an illumination module configured to illuminate the wells; a cradle configured to receive the multiwell plate, having an opening in the bottom configured to expose all of the transparent bottom surfaces of the wells; and an optical imaging module that is movable between different wells of the same multiwell plate to acquire images through a window via the exposed bottom.

[0013] In a related embodiment of the present invention, a method for monitoring cells is provided, the method comprising the steps of electronically monitoring cells in the wells of a multiwell plate, wherein each well has a pair of cell substrate impedance monitoring electrodes and a transparent window on the bottom surface of the electrodeless wells, and acquiring an image from at least one of the electronically monitored wells through the transparent window. In some embodiments, the images are acquired periodically at a fixed time interval between two consecutive image acquisitions, or irregularly over a period within the electronic monitoring period, the method optionally includes the step of acquiring an image between two consecutive image acquisitions at the same time interval as the time interval between two consecutive electronic measurements of cells.

[0014] In some embodiments, prior to the step of acquiring an image from at least one well, an electronic monitoring step outputs results from at least one well that satisfy a setpoint and instructs the optical imaging module to acquire an image from at least one well. Examples of setpoints may include predetermined impedance-based values.

[0015] In some embodiments, the images taken are bright-field images of cells. In such embodiments, the method may also include the steps of counting cells from the bright-field images and optionally deriving cell confluence numbers or parameters (e.g., a percentage of confluence) from the bright-field images.

[0016] In other embodiments, the acquired image includes a fluorescence image of a cell. In such embodiments, the method may include the step of determining fluorescence parameters from an image, which is arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. The fluorescence parameters are calculated or determined individually for fluorescence images of each color, such as blue, green, and red.

[0017] In yet another embodiment, the images taken include bright-field images and fluorescence images of cells. In such embodiments, the method also includes the steps of: deriving a cell confluence number or parameters from the bright-field images and optionally counting cells from the bright-field images; determining fluorescence parameters from the fluorescence images, optionally selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity; and optionally superimposing bright-field images of one or more colors of one or more wells with fluorescence images.

[0018] In a related embodiment, a method for monitoring cells is provided, comprising the steps of electronically monitoring cells in the wells of a multiwell plate over a period of time, wherein each well has a set of cell-substrate impedance monitoring electrodes and the bottom of the well has a transparent window without electrodes, and acquiring images through the transparent window over a period of time during or outside of the electronic monitoring period. The period used for electronic monitoring may be the same as or different from the period used for image acquisition. These electronic and optical monitoring periods may be specified or programmed from as little as less than one minute to as long as several hours, several days, or even several weeks. During the electronic monitoring period, the electronic monitoring may be continued at specified fixed time intervals between two consecutive electronic measurements. Optical monitoring may be performed continuously over a period of time at specified fixed time intervals between two consecutive image acquisitions. These time intervals may be specified or programmed from as little as several seconds or less than one second to as long as one minute, several minutes, one hour, or even several hours.

[0019] In some embodiments, the images taken are bright-field images of cells. In such embodiments, the method may also include the steps of counting cells from the bright-field images and optionally deriving cell confluence numbers or parameters (e.g., confluence percentages) from the bright-field images.

[0020] In other embodiments, the captured image includes a fluorescence image of the cells. In such embodiments, the method may include determining fluorescence parameters from an image optionally selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. The fluorescence parameters are calculated or determined separately for the fluorescence images of each color such as blue, green, red, etc.

[0021] In yet other embodiments, the captured image includes a brightfield image of the cells and a fluorescence image of the cells. In such embodiments, the method also includes deriving a cell confluence number or parameter from the brightfield image and optionally counting the cells from the brightfield image, determining fluorescence parameters from the fluorescence image, optionally selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity, and optionally overlaying the brightfield image and the fluorescence image of one or more colors for one or more wells.

Brief Description of the Drawings

[0022] [Figure 1A] A photograph of an exemplary system 10 for electronically and optically monitoring biological samples, where an optional user interface 200 is displaying cell image data. [Figure 1B] A diagram showing a main housing 12 with five accessible cradles 14. [Figure 2A] A diagram showing a cradle 14 in a closed configuration. [Figure 2B] A diagram showing a cradle 14 in an open configuration. [Figure 2C] A diagram showing a dual cradle 14 in open and closed positions. [Figure 3] A diagram showing a brightfield illumination module 26 coupled to a cradle cover 14. [Figure 4] A diagram depicting an exemplary multi-well plate 20. [Figure 5]A schematic diagram of a set of electrodes 42 adapted for use with a transparent window 44 disposed centrally (Panel A), a photograph of a well 32 from a multi-well plate 20 (see FIG. 4) having a set of electrodes 42 on the bottom surface and a transparent window 44 without electrodes on the bottom surface (Panel B), and an image of cells taken through the transparent window 44 (Panel C). [Figure 6] FIG. showing an exemplary optical imaging module 26. [Figure 7] A cross-sectional view of an exemplary optical imaging module 26. [Figure 8] FIG. showing an exemplary optical imaging support 60 for the imaging module 26 of FIG. 6 that enables two-dimensional linear motion (including motion along an axis parallel to the linear track 66 and motion along another axis parallel to the linear track 70). [Figure 9A] A graph based on impedance over time after treating MCF7 breast cancer cells transfected with a lentivirus expressing a red fluorescent protein, where NK92 cells are at different effector:target (E:T) ratios over time. [Figure 9B] FIG. showing the number of red fluorescent cells (indicating live target cells) over time from the same assay. [Figure 9C] FIG. showing an image captured under brightfield illumination within the same well with an E:T ratio of 2.5:1 before the addition of NK92 cells. [Figure 9D] FIG. showing an image captured under brightfield illumination within the same well with an E:T ratio of 2.5:1 12 hours after the addition of NK92 cells. [Figure 9E] FIG. showing an image captured under brightfield illumination within the same well with an E:T ratio of 2.5:1 30 hours after the addition of NK92 cells. [Figure 10]The images show illustrative images of captured and superimposed light. The left panel superimposes three captured fluorescence images (red, green, blue) onto a captured bright-field illuminated image, while the right panel superimposes three captured fluorescence images (red, green, blue) without bright-field illumination. The cells shown are exposed to fluorescent markers for annexin V (red), caspase 3 (green), and the cell nucleus (blue). [Figure 11A] This graph shows the monitoring of cell substrate impedance over a period of time to demonstrate drug-induced apoptosis in real time. A549-Blue cells were titrated with MG132 (Figure 11A). DMSO was used as the negative control. Error bars represent the standard deviation of three samples. Impedance was measured continuously at 15-minute intervals, but to prevent overlapping error bars at adjacent time points, data points are displayed only once per hour here. [Figure 11B] This graph shows the monitoring of cell substrate impedance over a period of time to demonstrate drug-induced apoptosis in real time. A549-Blue cells were titrated with staurosporine (Figure 11B). DMSO was used as the negative control. Error bars represent the standard deviation of three samples. Impedance was measured continuously at 15-minute intervals, but data points are displayed only once per hour here to prevent overlapping error bars at adjacent time points. [Figure 12A] This figure provides a graph plotting impedance monitoring over a period of time, combined with live cell imaging at different time points within the impedance monitoring period. After treating A549-Blue cells with DMSO (Figure 12A), the impedance signal and the number of blue nuclei were tracked. Representative images are shown before treatment and at 1 or 20 hours post-treatment. Error bars represent the standard deviation of the sample run three times. [Figure 12B]This figure provides a graph plotting impedance monitoring over a period of time, combined with live cell imaging at different time points within the impedance monitoring period. After treating A549-Blue cells with 50 μm MG132 (Figure 12B), the impedance signal and the number of blue nuclei were tracked. Representative images are shown before treatment and 1 or 20 hours post-treatment. Error bars represent the standard deviation of the sample run three times. [Figure 12C] This figure provides a graph plotting impedance monitoring over a period of time, combined with live cell imaging at different time points within the impedance monitoring period. After treating A549-Blue cells with 1 μm staurosporine (Figure 12C), the impedance signal and the number of blue nuclei were tracked. Representative images are shown before treatment and 1 or 20 hours post-treatment. Error bars represent the standard deviation of samples run three times. [Figure 13] This figure provides plots based on impedance before and after treatment with 5.5 μm MG1322. Images at 0 hours, 20 hours, and 40 hours post-treatment are also shown. The top images were taken under bright-field illumination, while the bottom images were taken under fluorescence, showing annexin V staining (red), activated caspase 3 (green), and nuclear localized BFP (blue fluorescent protein). Arrows indicate large membrane blebs containing phosphatidylserine in the outer leaflet. [Figure 14A] A graph showing image-based sequential tracking of MG132-induced apoptosis in A549-Blue cells using the number of blue nuclei. Error bars represent the standard deviation of samples run three times. [Figure 14B] A graph showing image-based sequential tracking of MG132-induced apoptosis in A549-Blue cells using % confluence. Error bars represent the standard deviation of samples run three times. [Figure 14C] A graph showing image-based sequential tracking of MG132-induced apoptosis in A549-Blue cells using the number of green (caspase-3 activated) cells. Error bars represent the standard deviation of samples run three times. [Figure 14D] A graph showing the image-based, sequential tracking of MG132-induced apoptosis in A549-Blue cells using the number of red (annexin V-bound) cells. Error bars represent the standard deviation of samples run three times. [Figure 15] This graph shows a comparison of the relative rates and relative abundances of different apoptotic phenomena in cells treated with 50 μM MG132. The error bars represent the standard deviation of the samples from three trials. [Figure 16] This figure provides tables and graphs illustrating the use of dose-response curves to calculate EC50. Using real-time impedance (represented by normalized cell indices) data (Figure 11A) and live-cell imaging data (green: cell count, red: cell count, blue: nucleus count) (Figures 14A-C), the area under the curve was calculated and plotted here as a function of MG132 concentration. To determine EC50, the data were fitted to a logistic equation with four parameters. [Modes for carrying out the invention]

[0023] The systems and methods described herein enable continuous monitoring of cell health and behavior within the same well from very different perspectives, namely real-time electronic monitoring and live cell imaging. The streamlined workflow, high reproducibility, and quantitative dynamics of the systems make them ideal for a variety of cell-based assays, including, but not limited to, monitoring cell health, proliferation, cytotoxicity, apoptosis, immune cell death, cell receptor activation, and cell differentiation, including stem cell differentiation.

[0024] The continuous nature of the system and method offers two major advantages. Firstly, real-time tracking ensures that no important phenomena are missed, in contrast to endpoint assays which only provide a snapshot of the process. Secondly, the continuous nature of the technical approach significantly reduces the practical time required to run the assay. Once cells are seeded and processing is added, no further involvement is needed.

[0025] The systems and methods are preferably used to evaluate the effects on biological samples or cells. The cells may be primary cells isolated from any species or cells from a cell line. The cells may be genetically engineered cells. For example, these include cells from genetically modified organisms, such as from a “gene knockout” organism, or cells engineered to overexpress endogenous genes or transgenes, or cells in which normal gene expression is manipulated (e.g., by the use of antisense molecules or RNA silencing), cells modified by CRISPR and / or other gene editing technologies, or cells engineered to express therapeutic proteins such as CHO cells.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. All patents, applications, published applications, and other publications referenced herein constitute part of this specification by reference. If any definition in this section contradicts or is inconsistent with any definition in any patent, application, published application, or other publication that constitutes part of this specification by reference, the definition in this section shall prevail over the definition in any publication that constitutes part of this specification by reference.

[0027] As used herein, "a" or "an" means "at least one" or "one or more."

[0028] An "electrode" is a structure that has high electrical conductivity, that is, electrical conductivity that is much higher than that of the surrounding material.

[0029] As used herein, “electrode structure” refers to a single electrode, an electrode having a complex structure (e.g., a helical electrode structure), or a collection of at least two electrode elements electrically connected to one another. All electrode elements within an “electrode structure” are electrically connected.

[0030] As used herein, “electrode element” refers to a single structural feature of an electrode structure, such as a finger-like projection of an interdigitated electrode structure.

[0031] As used herein, “electrode array” or “electrode structure unit” refers to two or more electrode structures constructed to have dimensions and spacing such that they can act as a unit that generates an electric field in the spatial region surrounding the electrodes when connected to a signal source. Preferred electrode structure units of the present invention can measure impedance changes due to cell adhesion to the electrode surface. Non-limiting examples of electrode structure units are comb-type electrode structure units and concentric electrode structure units. Additional examples of electrode structures include a pair of small measuring / recording electrodes (e.g., circular microelectrodes with diameters between a small size of less than 10 microns and a large size of 100 or several hundred microns), and a much larger reference electrode. Multiple small recording electrodes forming a microelectrode array may share a common reference electrode. Such recording electrodes can be used to perform extracellular recording by amplifying and recording the voltage signal between the small recording electrodes and the much larger reference electrode. In extracellular recording embodiments, an extracellular recording system (including a voltage signal amplifier and other electronic hardware circuits for measuring voltage, and signal processing algorithms implemented in software and / or firmware) is used, rather than an impedance measurement system (e.g., an impedance analyzer system including electronic hardware circuits for measuring current and voltage, as well as software and / or firmware signal and data processing algorithms).

[0032] As used herein, "electrode bus" refers to a portion of an electrode that connects individual electrode elements or substructures. The electrode bus provides a common conduction path from individual electrode elements or individual electrode substructures to another electrical connection. In the device of the present invention, the electrode bus can contact each electrode element of the electrode structure and provide an electrical connection path to an electrical trace leading to a connection pad.

[0033] As used herein, “electrode trace,” “conductive trace,” or “electrical trace” is a conductive path extending from an electrode, electrode element, or electrode structure toward one end or boundary of a device or apparatus for connecting the electrode, electrode element, or electrode structure to an impedance analyzer. The end or boundary of the device may correspond to a connection pad of the device or apparatus.

[0034] As used herein, “connection pad” is an area on an apparatus or device (e.g., a multiwell plate) that is electrically connected to at least one electrode or all electrode elements within at least one electrode structure on the apparatus or device and can be operably connected to an external electrical circuit (e.g., an impedance measurement circuit or signal source). The electrical connection between the connection pad and the impedance measurement circuit or signal source can be made directly or indirectly via suitable electrical conduction means such as leads or wires. Such electrical conduction means may also pass through electrodes or electrical conduction paths located in other areas of the apparatus or device.

[0035] As used herein, "interdigitated" means having projections coming from one direction that intersect with projections coming from different directions, like the fingers of a folded hand (however, it should be noted that interdigitated electrode elements preferably do not come into contact with each other).

[0036] As used herein, “electrodes (or electrode structures) having substantially the same surface area” means that the surface areas of the referenced electrodes are substantially the same as those of the other electrodes (or electrode structures) mentioned, and therefore, an impedance change due to cell attachment or growth on any one of the electrodes (or electrode structures) mentioned contributes to an overall detectable change in impedance to the same or similar extent as an impedance change due to cell attachment or growth on the other electrodes (or electrode structures) mentioned. In other words, if electrodes (or electrode structures) have substantially the same surface area, any one of the electrodes may contribute to an overall change in impedance when cells attach or grow on the electrode. In most cases, the ratio of the surface areas of the largest and smallest electrodes having “substantially the same surface area” is less than 10.

[0037] As used herein, “detectable change in impedance between electrodes or between electrodes” (or “detectable change in impedance between electrode structures or between electrode structures”) means that when a molecular bonding reaction occurs at the electrode surface, the impedance between or between the electrodes (or electrode structures) will have a significant change that can be detected by an impedance analyzer or impedance measuring circuit. The change in impedance refers to the difference in impedance values ​​when cells are attached to the electrode surface, when cells are not attached to the electrode surface, or when there is a change in the number, type, activity, adhesion, or morphology of cells attached to the surface including the electrodes of the apparatus. In most cases, the detectable change in impedance is greater than 0.1%. Preferably, the detectable change in impedance is greater than 1%, 2%, 5%, or 8%. More preferably, the detectable change in impedance is greater than 10%. The impedance between electrodes or between electrodes is usually a function of the frequency of the electric field applied for measurement. “Detectable change in impedance between electrodes or between electrodes” does not require a change in impedance at all detectable frequencies. “Detectable change in impedance between electrodes or between electrodes” requires only a detectable change in impedance at any single frequency (or multiple frequencies). Furthermore, impedance has two components: resistance and reactance (reactance can be divided into two categories: capacitive reactance and inductive reactance). A "detectable change in impedance between or between electrodes" requires only that either the resistance or the reactance has a detectable change at any single or multiple frequencies. In this disclosure, impedance refers to electrical or electronic impedance.Such impedance measurement methods are achieved by (1) applying a voltage between or between electrodes at a predetermined frequency (or having multiple frequencies or a specific voltage waveform), monitoring the current passing through the electrodes at that frequency (or having multiple frequencies or a specific waveform), dividing the voltage amplitude by the current amplitude, and deriving an impedance value; (2) applying a current of a single frequency component (or having multiple frequencies or a specific current waveform) to the electrodes, monitoring the voltage between or between the electrodes at that frequency (or having multiple frequencies or a specific waveform), dividing the voltage amplitude by the current amplitude, and deriving an impedance value; and (3) other methods capable of measuring or determining electrical impedance. Note that in the explanation above, "dividing the voltage amplitude by the current amplitude to obtain the impedance value," the "division" is performed on the current amplitude and voltage amplitude values ​​at the same frequency. Such electrical impedance measurement is an electronic or electrical process that does not involve the use of reagents.

[0038] As used herein, “at least two electrodes have substantially different surface areas” means that the surface areas of any two electrodes are not similar to one another, and therefore, the change in impedance due to cell attachment or growth on the larger electrode does not contribute to the overall detectable impedance to the same or similar extent as the change in impedance due to cell attachment or growth on the smaller electrode. Preferably, the change in impedance due to cell attachment or growth on the larger electrode (typically called the “counter electrode”) is significantly smaller than the change in impedance due to cell attachment or growth on the smaller electrode (typically called the “working electrode” or “measuring electrode”). Typically, the ratio of the surface areas between the largest and smallest electrodes is 10 or greater. Preferably, the ratio of the surface areas between the largest and smallest electrodes is greater than 20, 30, 40, 50, or 100. An example of “at least two electrodes have substantially different surface areas” is the “Electronic Cell-Substrate Impedance Sensing (ECIS)” approach developed by Giaver and Keese.

[0039] As used herein, “arranged in a row-column configuration” means that, with respect to electrical connections, the location of an electrode, electrode array, or switching circuit is identified by both a row position number and a column position number.

[0040] The "Cell Index" or "CI" is a parameter that can be derived from measured impedance values ​​and can be used to reflect changes in impedance values. There are numerous methods for deriving or calculating the Cell Index. The CI has been previously described in detail in U.S. Patents 8,344,742, 7,470,533, 7,192,752, PCT / US03 / 22557, and others. Each of these, in whole, constitutes part of this Specified Publication. The "Normalized Cell Index" at a particular point in time is calculated by dividing the Cell Index at that point in time by the Cell Index at a reference point. Thus, the Normalized Cell Index is 1 at the reference point. The "Normalized Cell Index" has been previously described in detail in U.S. Patents 8,344,742, 7,470,533, 7,192,752, PCT / US03 / 22557, and others. Each of these, in whole, constitutes part of this Specified Publication. The "Delta Cell Index" at a given time is calculated by subtracting the cell index at a standard time from the cell index at a given time. Therefore, the Delta Cell Index is the absolute change in the cell index from the initial time (standard time) to the measurement time. The "Delta Cell Index" has been previously described in detail in U.S. Patents 8,344,742, 7,470,533, 7,192,752, PCT / US03 / 22557, etc. Each of these is incorporated herein by reference in whole. The "Cell Change Index" or "CCI" is a parameter derived from the Cell Index, where the "CCI" at a given time is equal to the value obtained by dividing the first derivation of the Cell Index with respect to time by the Cell Index at that time. "CCI" has been previously described in detail in U.S. Patent Nos. 8,344,742, 7,470,533, 7,192,752, PCT / US03 / 22557, and others, each of which, in whole, constitutes part of this Specification by reference.

[0041] As used herein, “dose-response curve” refers to the dependency of a cell’s response to a dose concentration of a test compound. The cell’s response can be measured using various parameters. For example, a test compound is suspected to be cytotoxic and cause cell death. After treating cells with the test compound, the cell’s response can be measured as a percentage of non-viable (or viable) cells. Plotting this percentage of non-viable (or viable) cells as a function of the dose concentration of the test compound constructs a dose-response curve. In this application, the percentage of non-viable (or viable) cells can be expressed with respect to a measured impedance value, or with respect to a cell index derived from impedance measurements, or with respect to a cell change index. For example, for a particular cell type, under specific cell physiological conditions (e.g., a particular cell culture medium), the cell index may be shown to have a linear or positive correlation with the number of viable cells in the well from which the cell index was derived from impedance measurements. Therefore, in this application, a “dose-response curve” can be constructed by plotting the cell index as a function of the dose concentration of the test compound. It should be noted that, generally, the cell index correlates not only with the number of viable cells in a well, but also with cell morphology and cell adhesion. Therefore, plotting the cell index against administered concentration provides information not only about the number of cells, but also about the physiological state of the cells (such as cell morphology and cell adhesion). Furthermore, a key advantage provided by the system and device of this application is that the system allows for continuous monitoring of cells and provides impedance measurements at multiple time points over a time range from short periods of a few minutes to periods of several days or weeks, thus enabling the acquisition of "dose-response curves" at multiple time points in a single experiment.

[0042] As used herein, “each well contains substantially the same number of cells” means that the minimum number of cells in a well is at least 50% of the maximum number of cells in a well. Preferably, the minimum number of cells in a well is at least 60%, 70%, 80%, 90%, 95%, or 99% of the maximum number of cells in a well. More preferably, each well contains the same number of cells.

[0043] Where used herein, “each well contains the same type of cells” means that, for the intended purpose, each well contains the same type of cells; it is not necessary for each well to contain exactly the same type of cells. For example, if the intended purpose is to contain mammalian cells in each well, it is acceptable for each well to contain human cells, or different mammalian cells, such as human cells and other non-human mammalian cells such as mouse, goat, or monkey cells, of the same type.

[0044] A “known compound” is a compound for which at least one activity is known. In the present invention, a known compound is preferably a compound for which one or more direct or indirect effects on cells are known. It is preferable, but not required, that the structure of the known compound be known. Preferably, the mechanism of action of the known compound on cells is known, and for example, the effects of the known compound on cells may, in non-limiting examples, be on cell viability, cell adhesion, apoptosis, cell differentiation, cell proliferation, cell morphology, cell cycle, IgE-mediated cell activation or stimulation, receptor-ligand binding, cell number, cell quality, cell cycle, etc.

[0045] The “impedance value” is the impedance measured for an electrode in a well in which cells are present or absent. Impedance is generally a function of frequency; that is, the impedance value depends on the frequency at which the measurement is taken. In this application, the impedance value refers to the impedance measured at either a single frequency or multiple frequencies. Furthermore, impedance has two components: a resistive component and a reactant component. In this application, the impedance value refers to the resistive component, or the reactant component, or both the resistive and reactant components. Therefore, when the “impedance value” is measured or monitored, it means that resistance, or reactance, or both resistance and reactance were measured or monitored. In many embodiments of the method of this application, the impedance value also refers to parameter values ​​derived from raw measured impedance data. For example, the impedance value can be represented using a cell index, or a normalized cell index, or a delta cell index.

[0046] As used herein, “liquid (fluid) sample” refers to a sample that exists naturally as a liquid or fluid, such as a biological fluid. “Liquid sample” also refers to a sample that exists naturally in a non-liquid state, such as a solid or gas, but is prepared as a liquid, fluid, solution, or suspension containing solid or gaseous sample material. For example, a liquid sample may include a liquid, fluid, solution, or suspension containing biological tissue.

[0047] As used herein, “sample” means any part that may be isolated, manipulated, measured, quantified, detected, or analyzed using the apparatus, microplate, or method of this application. A sample is preferably a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include cell culture media, urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and other cell suspensions in culture media. Biological tissues are aggregates of cells, usually together with certain types of intercellular material, that form one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, including connective tissue, epithelial tissue, muscle tissue, and nerve tissue. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cells. A biological sample may further include cell suspensions and solutions containing biological molecules (e.g., proteins, enzymes, nucleic acids, carbohydrates, and chemical molecules that bind to biological molecules).

[0048] A "test compound" is any compound whose activity or direct or indirect effect on cells is investigated in any assay. A test compound may be any compound containing, but is not limited to, small molecules, large molecules, molecular complexes, organic molecules, inorganic molecules, lipids, steroids, carbohydrates, fatty acids, amino acids, peptides, proteins, or any combination thereof, biomolecules, nucleic acids, or any combination thereof. A test compound may be a synthetic compound, a natural compound, or a derivative of a natural compound. The structure of the test compound may be known or unknown.

[0049] To begin, starting with Figure 1A, an exemplary system 10 configured to continuously monitor a sample electronically and optically in real time is shown communicating with a computer processor 100 loaded with software and a user interface 200. System 10 provides multiple perspectives for studying cellular activity by collecting and analyzing information from different distinct flows from a single assay. The richness of information in this multiplex assay lies not only in the number of parameters it reports, but also in the clarity / uniqueness of the perspectives it provides. System 10 described herein enables simultaneous monitoring of cellular health and behavior within the same well from very different perspectives of real-time cellular impedance and live cell imaging.

[0050] Moving to Figure 1B, it is preferable that System 10 be sized to fit into a commercially available cell culture incubator in order to regulate the temperature, CO2, and moisture conditions of the cells. In other words, System 10 is sized to fit into a cell culture incubator so that the incubator, rather than System 10 itself, regulates the cell culture conditions such as temperature, CO2, and humidity. The dimensions can be changed according to the user's needs, but System 10 shown in Figures 1A and 1B has an installation area of ​​approximately 430 mm × 445 mm × 410 mm.

[0051] The upper part of the primary housing 12 is embodied as a platform having five cradles 14, three cradles 14A configured for electronic (e.g., cell matrix impedance) and optical monitoring of biological samples, and two cradles 14B for optical monitoring of biological samples, but not electronic monitoring. Although a total of five cradles 14 are shown, those skilled in the art will recognize that additional cradles 14 can be added by increasing the footprint of the system 10. Furthermore, those skilled in the art will recognize that it is possible to have fewer cradles 14 than those shown in Figure 1 (e.g., four, three, two cradles 14, or a single cradle 14), including an equal or fewer number of cradles 14A (e.g., three, two, or one cradle 14A) configured for both electronic and optical monitoring of biological samples, and / or an equal or fewer number of “optical only” cradles 14B (e.g., two or one) configured for optical monitoring of biological samples only (i.e., an equal or fewer number of cradles 14A (e.g., three, two, or one cradle 14A) configured for both electronic and optical monitoring of biological samples, or an equal or fewer number of “optical only” cradles 14B (e.g., two or one) configured for optical monitoring of biological samples only, or both).

[0052] As clearly shown in Figures 2A to 2C, each cradle 14A configured for electronic monitoring preferably has a hinged cover 16 that can be closed and locked using a movable engagement handle 18. When the cover 16 is closed and locked, a tight electrical engagement is ensured between the electronic plate 20 (see Figure 4) and the pogo pins 22 (see Figure 2B), which are connected to an impedance analyzer or impedance measurement circuit for monitoring the impedance within the system 10, and finally communicate with an external computer processor 100 (Figure 1A) for electronic monitoring. However, as shown in Figure 2C, each cradle 14B configured solely for optical imaging does not require an electronic engagement between the plate and the cradle components, and therefore does not require a mechanism to close and lock the cover 16. In both cases, the outside of the hinged cover 16 can be marked with an indecia 24 that matches the intended format of the plate or container to be added to the cradle 14.

[0053] Inside the cradle, preferably along the cover 16, is an illumination module 26 configured to illuminate the interior of the cradle 14. As better shown in Figure 3, in a preferred embodiment, the illumination module 26 consists of an array of light-emitting diodes (LEDs) formed from a plurality of separate LEDs 28 that receive commands from a computer processor 100 (Figure 1A) via electrical contacts 30. In some embodiments, the LED array can be swapped with different multi-well plates (e.g., 6-well, 12-well, 20-well, 48-well, 96-well, 384-well) or other culture vessels by screws. Preferably, each diode 28 is assigned to a single well 32 of a 96-well plate (see Figures 4-5) and is therefore configured to independently illuminate the corresponding well 32 for imaging. By positioning the diode 28 directly above the well 32, the system 10 ensures that the entire well 32 is sufficiently illuminated to guarantee high-quality imaging. In a preferred embodiment, the illumination module 26 is configured for bright-field illumination, which means that the illumination module 26 emits white light. Similar to conventional optical microscopy, differences in the amount of white light passing through the biological sample, depending on the camera magnification, provide sufficient contrast to identify cellular components such as cell membranes, nuclei, and other details, and can therefore be used for cell counting and / or cellular confluence analysis, or to confirm that cells are properly settled at the bottom of the well 32 before electronic monitoring. Imaging can be further enhanced by staining the biological sample with an image-enhancing dye that improves the contrast or identification of living cells under bright-field illumination to assess cytotoxicity, cell viability, and / or cell proliferation (for example, dead cells absorb trypan blue).

[0054] Illumination control is performed by a computer processor 100. For this purpose, the computer processor 100 can selectively illuminate one or more diodes 28 in response to results from electronic monitoring of a biological sample. For example, living cells can be monitored using cell-matrix impedance monitoring to assess cell proliferation, and when a set value or parameter indicating an established cell monolayer or established cell population is reached, the computer processor 100 can use one or more LEDs 28 to illuminate one or more electronically monitored wells 32 to acquire a cell image. Furthermore, during experimental treatment, such as administration of a test compound to assess its cytotoxicity, the computer processor 100 can selectively illuminate one or more LEDs 28 at a predetermined time, or in response to changes in cell-matrix impedance monitoring for cell imaging, to confirm a decrease in the cell population. In addition, the computer processor 100 can use one or more diodes 28 from the illumination module 26 to provide bright-field illumination and simultaneously acquire an image while performing electronic monitoring (e.g., cell-matrix impedance monitoring).

[0055] Returning to Figures 2B and 2C, the white light passing through the biological sample eventually exits the cradle 14 through the open bottom 34 of the cradle 14. "Open" means that the bottom 34 is not completely opaque. As will be described in more detail in the following paragraphs, this open bottom 34 exposes a transparent window (see Figure 5) that provides a path for image acquisition. In embodiments configured for electronic monitoring of cells, a slotted protective shield 36 may span the open bottom 34 to protect the pogo pins 22, particularly when loading and unloading multiwell plates 22 into or from cradles 14, 14A.

[0056] For perfection, the development of a cradle 14 adapted for electronic monitoring required overcoming challenges inherent in the electronic measurement of cells using multiwell plates. For example, conventional electronic monitoring of biological samples requires a docket station with a complex electrode selection circuit of its own to communicate with different sets of electrodes in different wells of the plate and switch between measurements, in a container such as a multiwell plate with electrodes. Many of these circuits for switching and selecting measurement electrodes had to move to provide the necessary openings and therefore had to be imaged through the bottom 34 of the cradle 14. For example, Figure 2B shows the use of pogo pins 22 (120 shown for communication with a 96-well electronic plate 20) lining the outer periphery of the cradle 14. However, moving or rewiring this circuit to form the necessary openings affects the electrical properties of the system 10. In particular, lengthening electrical wires to move the necessary electrode switching circuits away from the pogo pins increases the system's resistance, which can lead to interference from electronic noise from other electrical components (such as the circuits for driving the linear motion stages 66 and 70 in system 10 in Figure 8), ultimately affecting the resolution and accuracy of electronic monitoring. This is especially important in preferred electrode configurations, where all wire resistances for switching and selecting each set of electrodes across all wells must be equal or nearly equal. Furthermore, another challenge with respect to electronic monitoring of cells is that the electrode switching circuits increase localized heat generation within system 10, which was previously dissipated within each cradle under the docking station. Therefore, moving or reconfiguring the electrode switching / selection circuits and wiring away from their conventional arrangement also affects the heating of system 10, presenting further challenges in the development of such system 10. Thus, special technical development of system 10 is required to achieve a system suitable for both electronic monitoring and optical imaging of samples in the same well, so that the electrode switching and selection circuits can be relocated within system 10 without causing excessive localized heat generation and without affecting the performance of electronic monitoring.

[0057] Through numerous innovative engineering design steps, the electrode switching and selection circuits were positioned close to the pogo pins 22. The position and orientation of the electrode switching chips were designed to maximize the distance between chips despite the limited available space, in order to minimize localized heat generation. Furthermore, the printed circuit board (PCB board) was designed using an appropriate electrical trace layout across different layers of the electrical conduction surface to minimize variations in electrode resistance between the pogo pins 22 and the circuit switching chips for each set of electrodes across all wells on the multiwell plate. In addition, electrical signal wires / lines were designed in appropriate relationships to the electrical ground line and electrical ground plane on the PCB board to minimize electrical interfaces from other circuits in the system.

[0058] Referring here to Figures 2B and 2C, each cradle 14 may include a mechanical sensor 38 that, when pressed, signals both the receipt of the multiwell plate 20 and the closing of the cover 16. Once a signal is sent, the cradle 14 is ready for electronic and / or optical monitoring.

[0059] Moving to Figures 4 and 5, preferably, each well 32 of the electronic plate 20 has a set of electrodes 42 and a transparent window 44 at the bottom of the well 32 where the electrodes 42 are not present. By providing a region with electrodes 42 and a transparent region without electrodes 42, at least two distinctly different assays can be performed in a single well 32. In particular, electronic monitoring (e.g., cell-substrate impedance monitoring) can be performed using the set of electrodes 42, and optical imaging can be performed by focusing an optical imaging module 46 (Figure 6) through the open bottom 34 of the cradle (see Figures 2B and 2C) and through the transparent window 44. As shown in Figure 4, the electronic plate 20 is preferably a multi-well plate 20 and can be described as having “multiple wells” configured for the electrical connection or electronic monitoring of the sample. Those skilled in the art will understand that in addition to these “multiple wells,” there may be one or more wells 32 that do not have electrical connections or are not for electronic monitoring. In such cases, the wells 32 that do not have electronic monitoring capabilities function as control wells or as “optical imaging only” wells 32. For this purpose, the electronically monitored well 32 can be described as having a non-conductive substrate (e.g., the bottom of the well), an electrode array defined by a set of electrodes 42 fabricated on the substrate, and a transparent display window 44 on the substrate where the electrodes 42 are not present.

[0060] The configuration of the electrode 42 can be modified according to the user's needs or requests, as long as the transparent window 44 for sample imaging is retained. In some embodiments, the electrode array is a microelectrode array (MEA) corresponding to the electronic cell-substrate impedance sensing (ECIS) system described by Gaiver and Keese, where a single large reference electrode is paired with multiple small working or measuring electrodes. In ECIS, a small alternating current is applied to the entire electrode array. This allows the potential between the electrodes to be measured by the ECIS instrument. When cells are added to the ECIS array and adhere to the measuring electrodes, they act as insulators and increase the impedance. As cells grow and cover the measuring electrodes, the current is obstructed in ways related to the number of cells covering the electrodes, the morphology of the cells, and the nature of cell adhesion. As cells proliferate or die, the impedance changes.

[0061] An alternative, more preferred approach has been previously described (e.g., U.S. Patent No. 8,344,742, U.S. Patent No. 7,470,533, U.S. Patent No. 7,192,752, and elsewhere, each of the listed patents is incorporated herein by reference in its entirety) in which each electrode array comprises two electrode structures 42A, 42B, each electrode structure 42A, 42B comprising an electrode element 42C, but the innermost electrode element 42C is removed to form a transparent window 44 (see schematic diagram shown in panel A of Figure 5). The electrode structures 42A, 42B are electrically coupled to a connection pad or interface and are located at the edges of the substrate and thus configured to be electrically connected to pogo pins 22 on the cradle 14. Each electrode array has substantially uniform electrode resistance throughout the array. In contrast to the ECIS approach, where significant changes in cellular matrix impedance are monitored only on the working or measuring electrode (not a large-area reference electrode), cell adhesion or growth on any comb-shaped electrode interferes with the current in a detectable manner, depending on the number of cells covering the electrode, the morphology of the cells, and the nature of cell adhesion. That is, in this configuration, all electrodes 42 function as either measuring or working electrodes. Thus, the electrode array can consist of two or more electrode structures 42A, 42B constructed to have dimensions and spacing such that they can act as units that generate an electric field in the area of ​​space surrounding the electrode structures 42A, 42B when connected to a signal source via a computer processor 100 and a cradle 14, etc.

[0062] If the electronic monitoring is cell substrate impedance monitoring, the electronic circuit connects the set of electrodes 42 to a computer processor 100 via the cradle 14. Preferably, in such embodiments, the computer processor 100 communicates with an impedance measurement circuit or impedance analyzer that can be fully integrated into the system 10. The impedance analyzer may include electronic hardware circuitry for measuring current and voltage, and firmware and / or software signal and data processing algorithms. When connected to the impedance analyzer, the system 100 can measure the difference in impedance values ​​related to the behavior of cells. For example, the system 10 can measure the difference in impedance values ​​when cells are attached to the electrode array and when cells are not attached to the electrode array, or it can detect differences in impedance values ​​when the number, type, activity, adhesion, or morphology of cells attached to the electrodes 42 changes. In particular, cell-substrate impedance monitoring can reveal information about the cell adhesion or bonding state on the substrate (e.g., degree of cell diffusion, cell adhesion area, degree of cell adhesion, cell morphology), including the growth or proliferation state of cells on the electrode 42, the number of living and / or dead cells in the wells (i.e., the number of living cells or dead cells in the wells or both), changes and rearrangements of the cytoskeleton, and the number of cells undergoing apoptosis and / or necrosis (i.e., apoptosis or necrosis or both).

[0063] In some embodiments, the impedance analyzer measures impedances from 0.1 ohms to 10 ohms in the frequency range of 1 Hz to 1 MHz. 5 It is possible to measure impedance between ohms. More preferably, the impedance analyzer can measure impedance from 0.1 ohms to 10 ohms in the frequency range of 100 Hz to 100 kHz. 3 The impedance between ohms can be measured. Preferably, the impedance analyzer can also measure both the resistive and reactant components (capacitive reactance and inductive reactance) of the impedance.

[0064] Furthermore, the system 10 includes electronic switches that can switch the connection to each set of electrodes 42 on and off for selective monitoring. These switches are preferably controlled by a software program loaded onto a computer processor 100. The software program instructs the connection of the electrode array to an impedance analyzer to monitor the cell substrate impedance from the electrodes 42. During impedance monitoring, the impedance analyzer can monitor impedance at one or more frequencies. In most cases, impedance monitoring is performed at multiple time points for a particular assay. Therefore, the system can connect individual arrays to the impedance analyzer to monitor one, some, or all of the arrays at one or more time points. In addition, the switches allow selected individual arrays to be monitored quickly and sequentially at each required monitoring time point. Each monitoring time point is, in practice, a narrow time frame (e.g., milliseconds to minutes) of measurement in the assay in which impedance monitoring is performed. In some embodiments, the software is programmable to instruct impedance monitoring of any of the wells 32 of the plate 20 containing the arrays at selected time intervals.

[0065] To advance the above, System 10 can be used to efficiently and simultaneously perform multiple assays using the circuitry, and to digitally switch from monitoring cell-substrate impedance across an array in one well 32 to monitoring cell-substrate impedance across an array in another well 32, whether from the same electronic circuit plate 20 or a different electronic plate 20. In some embodiments, the software-controlled system can complete impedance measurements of individual wells 32 within approximately 1 second at a single frequency. In further embodiments, cell-substrate impedance is monitored with millisecond resolution. Approaches to monitoring cell-substrate impedance with millisecond resolution are described in U.S. Patent No. 10,533,985, U.S. Patent No. 10,012,636, and U.S. Patent No. 9,709,548, among others. Each of the listed patents is incorporated herein by reference. Thus, in some embodiments, two consecutive impedance measurements are monitored within 40 ms of each other. In some embodiments, two consecutive impedance measurements are monitored within 20 ms of each other. In some embodiments, two consecutive impedance measurements are monitored within 10 ms of each other. In some embodiments, two consecutive impedance measurements are monitored within 1 ms of each other. In some embodiments, two consecutive impedance measurements are monitored less than 1 ms apart from each other.

[0066] While the system is primarily described in relation to monitoring the cellular substrate impedance of cells, those skilled in the art will recognize that the system can also be adapted for performing extracellular recording. Extracellular recording can be performed by amplifying and recording the voltage signal between a small recording electrode and a much larger reference electrode (it should be noted that the use of such a small recording electrode and a large reference electrode is similar to that used in ECIS). In embodiments of extracellular recording, an extracellular recording system (including a voltage signal amplifier and other electronic hardware circuits for measuring voltage, and signal processing algorithms implemented in software and / or firmware (i.e., software or firmware or both)) is used rather than an impedance measurement system (e.g., an impedance analysis system including electronic hardware circuits for measuring current and voltage, and signal and data processing algorithms in software and / or firmware (i.e., software or firmware or both)).

[0067] Moving on to Figures 6 and 7, an exemplary optical imaging module 26 is capable of capturing images from each well 32. In a preferred embodiment, the optical imaging module 26 is located under the cradle 14 within a primary housing 12 (see Figure 1B). The exemplary optical imaging module 26 includes a long working distance objective lens 48 at one end and a sealed CMOS camera 50 at the opposite end. In some embodiments, the camera captures 30 images per second. In some embodiments, the camera captures 40 images per second. In some embodiments, the camera captures 50 images per second. In some embodiments, the camera captures 60 images per second. In some embodiments, the camera captures 70 images per second. In some embodiments, the camera captures more than 70 images per second. High-speed imaging allows for the superposition of images captured under different shooting conditions and filters. Tube lenses 52A, 52B and a bandpass filter 54 for enhancing imaging are also shown. The optical imaging system 26 receives commands from the computer processor 100 and transmits images to the computer processor 100. Therefore, the bright-field illumination of the well 32 allows the optical imaging system 26 to capture images through the open bottom 34 of the cradle 14, even when the cradle 14 is closed (see Figures 2A to 2B).

[0068] Preferably, the optical imaging system 26 also includes an excitation light source 56, which is shown as a set of LEDs that excite molecules in the well 32 by guiding light through a focusing lens 58 or by inducing fluorescence for fluorescence imaging. LEDs corresponding to yellow 56A, ultraviolet 56B, and blue 56C are shown, but any number of 1 to 7 lights can be included for excitation. For example, the excitation light source 56 may include one or more lights including ultraviolet, violet, blue, green, yellow, orange, and red light. Thus, the system 10 is configured to capture not only bright-field contrast images of cells but also fluorescently tagged markers such as fluorescently tagged antibodies, antibody fragments, or other molecules that bind to cells. For this purpose, the system can provide imaging of various stages, such as cell proliferation, cell death, cell apoptosis, effector cell killing, intercellular interactions, cell binding, DNA / RNA / protein upregulation, and DNA / RNA / protein downregulation, by adding a suitable fluorescent dye or fluorescently labeled molecule to the cell sample and capturing the fluorescence via the imaging module.

[0069] As part of the optical imaging system 56, the excitation light source 56 is also controlled by a software-loaded computer processor 100. Therefore, the computer processor 100 can instruct the on / off switching of each LED 56A, 56B, and 56C, and to instruct high-speed image acquisition via the camera 50. In the operation of optical monitoring of cells, one LED is turned on at a time, and a fluorescence image of the corresponding color is acquired by the monochromatic (black and white) CMOS camera 50. For example, the yellow LED 56A, ultraviolet LED 56B, and blue LED 56C correspond to red, blue, and red fluorescence images, respectively. The acquired monochromatic image is displayed with pseudocoloring to represent the corresponding fluorescence color. Furthermore, the computer processor 100 can determine the number of viable cells from the image and determine parameters such as the presence or absence of fluorescence (+ / -), the total fluorescence intensity of cells in each well or multiple wells, and the average fluorescence intensity of cells in one well or multiple wells. Additionally, the computer processor 100 can overlay fluorescence images (or single or multiple colors) and bright-field illumination images for comprehensive analysis.

[0070] Referring together to Figures 5, 6, and 8, the optical module 56 is mounted on a movable support 60 to move across different windows 44. Generally, only movement along the X and Y axes is required, as slight differences in focal length between the wells 32 can be accounted for by lens adjustment or computer-controlled focusing. Therefore, the movable support 60 moves in two directions using an X-axis motor and timing belt 62, an X-axis cable rack 64, an X-axis linear motion guide 66, a Y-axis cable rack 68, and a Y-axis linear motion guide 70. A magnetic linear encoder 72 helps to identify the X and Y coordinates of the optical imaging module 56.

[0071] As already mentioned, the monitoring system 10 can also store and display data. The data can be displayed on screen 200, as printed data, or both. Preferably, the software can enable input and display of experimental parameters such as descriptive information including cell type, compound concentration, and time interval to be monitored. Furthermore, the data can be displayed overlaid, such as combinations showing optical imaging, bright-field illumination, and electronic monitoring data from different fluorescence channels (i.e., different fluorescence colors).

[0072] Preferably, the software can also analyze impedance data. In a preferred embodiment, the software can calculate a cell index (CI) for one or more wells at one or more time points. In some preferred embodiments, the software can calculate a cell change index (CCI) from impedance measurements of one or more wells. Preferably, the software can generate a plot of impedance data and impedance values ​​(including, but not limited to, CI or CCI) against time. The software can also perform other analyses, such as calculating cell number from CI, generating dose-response curves based on impedance data, calculating IC values ​​based on impedance values, and calculating dynamic parameters of cell proliferation or behavior based on impedance values ​​and impedance curves. The software for the impedance monitoring system can also store and display the analysis of the data, such as the calculated impedance values ​​and the dynamic parameters derived therefrom. The data can be displayed on a screen, as printed data, or both.

[0073] Similarly, the captured images can be analyzed using software. Preferably, the software can perform cell counting functions from the images and plot the data over time for statistical analysis. Preferably, the software can also store and display analyses of the data, such as counts from fluorescence imaging, bright-field illumination, and cell-versus-cell imaging results overlaid with corresponding electronic monitoring time points in different wavelength channels.

[0074] For example, if the acquired image is a bright-field image of a cell, the method of use includes the step of determining the cell confluence number or parameters from the bright-field image or counting the cells; or if the acquired image is a fluorescence image of a cell, the method of use includes the step of determining fluorescence parameters from the image of each color, which are arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity; or if the acquired image includes both a bright-field image of a cell and a fluorescence image of a cell, the method of use includes the step of deriving the cell confluence number or parameters from the bright-field image and arbitrarily counting the cells from the bright-field image, determining fluorescence parameters from the fluorescence image, which are arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity, and arbitrarily overlaying a bright-field image of one or more colors with a fluorescence image for one or more wells.

[0075] The advantages of combining electronic monitoring of cells with live cell imaging are particularly evident in Figures 11A–12C (explained in more detail in the following examples). In particular, cell-matrix impedance was able to detect the rapid staurosporine-mediated effects on A549-Blue cells, but the cellular phenomena causing this change could not be deciphered using impedance alone. However, live cell imaging revealed large-scale cytoplasmic contraction, immediately suggesting an explanation for the mechanism of the large and rapid impedance response. Similarly, impedance provided insights that would not have been obtained by imaging alone. In one such example, MG132 treatment separated cells from the electrodes but they remained in the well (cellular confluence remained >50% (Figure 14B), confluence number obtained from bright-field images by image analysis algorithm), and cell imaging showed the presence of cells in the well, but impedance (impedance signal drops to zero (Figure 11A)) alone revealed the cell-matrix adhesion strength. Beyond the advantages of having two independent measurement techniques, it is important to note the objectivity of impedance readings that are reported directly without any processing or input from the user.

[0076] Returning collectively to Figures 1-8, the non-limiting use of bound cell-substrate impedance monitoring and live cell imaging of the same cell population is described in more detail here. Specifically, a method for monitoring cells is disclosed, comprising the steps of: electronically monitoring cells in wells 32 of a multiwell plate 20, wherein each well 32 has a set of multiple electrodes 42, preferably a set of cell-substrate impedance monitoring electrodes 42, and a transparent window 44 on the bottom surface of wells 32 without electrodes 42; and acquiring an image through the window 44 from at least one well 32 that is or has been electronically monitored. From this monitoring, the method may include the steps of generating an impedance-based curve (e.g., a CI curve) from the impedance monitored over time; and displaying the impedance-based curve and a corresponding optical image. The image can be acquired from bright-field illumination or fluorescence excitation (e.g., excitation of a fluorescent molecular label bound to an antibody, antibody fragment, or other binding molecule). Thus, the data can provide quantitative dynamics that reveal detailed information about the health, behavior, and intercellular interactions of the cells.

[0077] Methods for performing cell proliferation assays are also disclosed. In these assays, an increase in monitored impedance indicates an increase in cell number, which can be confirmed by corresponding real-time imaging. By plotting impedance measurements or impedance values ​​derived from impedance measurements against time, a proliferation curve of cells growing in well 32 of a multiwell plate 20 can be obtained and presented together with images of uptake cells from the same well 32, in particular from bright-field illumination and / or from fluorescence imaging of pathways or markers associated with cell proliferation (i.e., from bright-field illumination, or from fluorescence imaging of pathways or markers associated with cell proliferation, or both).

[0078] In connection therewith, a method is provided for generating at least one cell growth curve, the method comprising the steps of: incubating cells over time in a multiwell plate 20, where each well 32 includes a pair of electrodes 42 and a transparent window 44 on the bottom surface of the well 32 without electrodes 42; monitoring cell substrate impedance and acquiring an optical image from the same well 32 through the window 44; generating an impedance-based curve from the monitored impedance; and displaying the impedance-based curve and the corresponding optical image.

[0079] Dynamic parameters can be determined using growth curves of one or more cell types combined with real-time cell imaging. For example, the proliferation rates of different primary cancer cells can be compared, or the proliferation rates of primary cancer cells of the same type but different grades can be compared. In another example, primary cells of individuals with different genotypes can be compared. In yet another example, the proliferation rates can be compared, and primary stem cells or stem cells of cell lines can be compared. In yet another example, the proliferation curves or parameters of control cells and genetically modified cells of a cell line can be compared. In yet another example, the proliferation curves or parameters of virus-infected cells and control cells can be compared. Furthermore, growth can be confirmed using the system's imaging capabilities by performing cell counting or cell confluence calculations via images acquired by bright-field illumination, or by counting cells acquired under fluorescence imaging after being stained with fluorescent molecules or fluorescently tagged conjugated molecules.

[0080] System 10 can also be used to investigate the effects of one or more test compounds on cells. An exemplary embodiment includes the steps of incubating cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the well 32 that does not have electrodes 42; monitoring the cell substrate impedance and acquiring an optical image from the same well 32 through the window 44; adding a test compound to at least one of the wells 32; continuing to monitor the cell substrate impedance and acquiring an optical image from the same well 32 through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and optical image from the corresponding well. Changes in effect can be determined by comparing the results after compound addition with the results before compound addition, and / or by providing a vehicle control in another well 32 containing cells and comparing impedance-based curves and / or images (i.e., curves or images or both) between the wells 32 (i.e., by comparing the results after compound addition with the results before compound addition, or by providing a vehicle control in another well 32 containing cells and comparing impedance-based curves and / or images between the wells 32, or both).

[0081] A method for comparing the effects of a compound on two or more cell types is also disclosed. An exemplary method includes the steps of incubating cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the well 32 without electrodes 44, and at least one of the wells 32 accepts one cell type and at least another well 32 accepts a different cell type; monitoring the cell-substrate impedance and acquiring an optical image from each of the wells 32 having cells; adding the same test compound to the wells 32 having each cell type; continuing to monitor the cell-substrate impedance and acquiring an optical image from each of the wells 32 having cells and the test compound through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and the corresponding optical image for each of the wells 32 for comparison with each other. Wells 32 using vehicle control, as known in the art, may also be included.

[0082] A method for comparing the effects of two or more different compounds on cells is also disclosed. An exemplary method includes the steps of incubating cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the wells 32 without electrodes 42, and at least two of the wells 32 receive cells; monitoring the cell substrate impedance and acquiring optical images from each of the wells 32 having cells; adding different test compounds to different wells 32 having cells; continuing to monitor the cell substrate impedance and acquiring optical images from each well 32 having cells and test compounds through the window 44; generating impedance-based curves from the impedances monitored over time; and displaying the impedance-based curves and corresponding optical images for each of the wells 32 for comparison with each other. Wells 32 using vehicle control, as known in the art, may also be included.

[0083] Relatedly, methods for performing assays to test the effects of one or more test compounds on cells at different concentrations are also disclosed. Using such dose-response relationships, time-dependent IC5, IC10, IC20, IC30, IC40, IC50, IC60, IC70, IC80, IC90, or IC95 can be derived, all of which can be derived from dose-response curves. Typically, IC50 is the most important. Determining the time-dependent IC50 range of a compound provides information about when the compound's effect on cells is maximal. Accordingly, an exemplary method includes the steps of incubating cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the well 32 without electrodes 42, and at least two wells 32 accept cells; monitoring cell-substrate impedance and acquiring optical images from each of the wells 32 containing cells; adding different concentrations of a test compound to different wells 32 containing cells; continuing to monitor cell-substrate impedance and acquiring optical images through the window 44 from each of the wells 32 containing cells and the test compound; generating an impedance-based curve from the impedance monitored over time; and comparing the impedance-based curves and corresponding optical images for each of the wells 32, such as comparing their dose-response curves or dose-relationships, or comparing the IC50 values ​​derived from each of the dose-response curves. Wells 32 using vehicle control, as known in the art, may also be included.

[0084] A method for performing a real-time cytotoxicity assay of a compound is also disclosed. An exemplary embodiment includes the steps of: incubating cells over time in a multiwell plate 20 in which each well 32 includes a pair of electrodes 42 and a transparent window 44 on the bottom surface of the wells 32 without electrodes 42; monitoring cell-substrate impedance and acquiring an optical image from each of the wells 32 containing cells; adding a cytotoxic compound or a compound suspected of being cytotoxic to one or more wells 32 containing cells; continuing to monitor cell-substrate impedance and acquiring an optical image from each well 32 containing cells and the added compound through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and the corresponding optical image for each of the wells 32 for comparison with one another. Wells 32 using vehicle control may also be included, as is known in the art.

[0085] Methods for analyzing and comparing the time-dependent cytotoxic effects of a first compound and a second compound on a cell type are also disclosed. An exemplary embodiment includes the steps of: incubating cells over time in a multiwell plate 20, each well 32 having a pair of electrodes 42 and a transparent window 44 on the bottom surface of wells 32 without electrodes 42; monitoring cell substrate impedance and acquiring optical images from each of the wells 32 having cells; adding a first cytotoxic compound or a first compound suspected to be cytotoxic to one or more wells 32 having cells and adding a second cytotoxic compound or a second compound suspected to be cytotoxic to another one or more wells 32 having cells; continuing to monitor cell substrate impedance and acquiring optical images from each well 32 having cells and the added compound through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curves and corresponding optical images of the wells 32 associated with the first and second compounds for comparison with each other. Wells 32 using vehicle control, as known in the art, may also be included. In some embodiments, the time-dependent cytotoxic response is determined for the first compound at multiple dose concentrations. In some embodiments, the time-dependent cytotoxic response is determined for the second compound at multiple dose concentrations. In some embodiments, the time-dependent cytotoxic response is determined for both the first and second compounds at multiple dose concentrations.

[0086] In some embodiments, the first compound is a compound having a known mechanism for its cytotoxic effect, and the second compound is a compound having an unknown mechanism for its cytotoxic effect. If the time-dependent cytotoxic response from the second compound is similar to that of the first compound, the second compound may follow a similar mechanism for its cytotoxic effect as the first compound.

[0087] Various approaches can be used when comparing the cytotoxic responses of compounds. Cellular indices (or cell number indices) can be optionally calculated using acquired impedance values. In some embodiments, time-dependent IC50 can be derived for the compounds, and a comparison between their cytotoxic responses can be made by comparing their time-dependent IC50 curves based on the cell index values. If the IC50 curves follow similar time-dependent trends, the two compounds may follow similar mechanisms for inducing cytotoxic effects.

[0088] In some embodiments, a direct comparison of the time-dependent cytotoxic responses of two compounds is performed, even if the concentrations of the two compounds are the same or different. This direct comparison of time-dependent cytotoxic responses can be achieved by analyzing the gradient of the measured response change (corresponding to the first derivative of the response with respect to time) and comparing the time-dependent gradients of the two compounds. Alternatively, the time-dependent cytotoxic responses can be analyzed to determine the higher-order derivatives with respect to time. Comparing such higher-order derivatives may provide additional information about the mechanism of cytotoxicity induced by the compounds.

[0089] In some embodiments, analyzing the real-time cytotoxic response may involve deriving the time-dependent IC50 values ​​of the compound for multiple cell types. In some embodiments, analyzing the real-time cytotoxic response may involve deriving the gradient of change in the time-dependent cytotoxic response at a given compound concentration. In some embodiments, analyzing the real-time cytotoxic response may involve deriving the higher-order derivative of the time-dependent cytotoxic response with respect to time at a given compound concentration.

[0090] A method for evaluating the efficacy of a proposed anticancer drug on cancer cells is also disclosed. An exemplary embodiment includes the steps of incubating cancer cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the electrodeless well 32; adding one or more proposed therapeutic agents to the cells; monitoring the cellular matrix impedance and acquiring an optical image from each of the wells 32 containing the cells; adding effector cells, preferably from the same subject from which the cancer cells were obtained, to the wells 32; continuing to monitor the cellular matrix impedance and acquiring an optical image from each of the wells 32 through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and the corresponding optical images from the wells 32. In some embodiments, the method includes the addition of CAR-T cells as effector cells. Wells 32 using vehicle control, as known in the art, may also be included.

[0091] A method for evaluating the cytolysis of cancer cells by manipulated effector cells is also disclosed. An exemplary embodiment includes the steps of: incubating cancer cells over time in a multiwell plate 20 in which each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the well 32 without electrodes 42; monitoring cell-substrate impedance and acquiring an optical image from each of the wells 32 containing cells; adding effector cells, preferably from the same subject from which the cancer cells were obtained, manipulated to show binding sites suspected to bind to cancer cells, to the wells 32; continuing to monitor cell-substrate impedance and acquiring an optical image from each of the wells 32 through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and the corresponding optical images from the wells 32. Wells 32 using vehicle control, as known in the art, may also be included.

[0092] A method for evaluating the cytolysis of cancer cells by a bispecific engager is also disclosed. An exemplary embodiment includes the steps of incubating cancer cells over time in a multiwell plate 20, wherein each well 32 includes a set of electrodes 42 and a transparent window 44 on the bottom surface of the electrodeless wells 32; monitoring the cell matrix impedance and acquiring an optical image from each of the wells 32 containing cells; preferably adding effector cells from the same patient as the cancer cells to the wells 32; adding a bispecific engager configured to bridge the effector cells to the cancer cells, continuing to monitor the cell matrix impedance and acquiring an optical image from each of the wells 32 through the window 44; generating an impedance-based curve from the impedance monitored over time; and displaying the impedance-based curve and the corresponding optical images from the wells 32. Wells 32 using vehicle control may also be included, as is known in the art.

[0093] As illustrated in more detail in the following examples, the systems and methods herein combine highly specific readouts of live cell imaging with the simplicity, analytical sensitivity, and objectivity of real-time impedance monitoring to continuously track cellular processes with unparalleled richness of information.

[0094] [Example 1] [Real-time monitoring of immune cell-mediated death of cancer target cells] MCF7 breast cancer cells were transfected with a lentivirus expressing red fluorescent protein (eLenti Red, catalog number 8711011), seeded on E-Plate (ACEA BIOSCIENCES, San Diego, California) for 25 hours, and treated with NK92 cells at different effector:target (E:T) ratios.

[0095] Optical imaging was performed concurrently with impedance monitoring. As shown in Figures 9A and 9B, the addition of the effector induces cancer cell death in an E:T ratio-dependent manner, as indicated by impedance monitoring (Figure 9A) and optical imaging (Figure 9B). The number of fluorescent objects (Figure 9B) indicates the number of living target cells.

[0096] Figures 9C to 9E show images taken beforehand (Figure 9C) and 30 hours later (Figures 9D and 9E) at an E:T ratio of 2.5:1, illustrating target cell death over time.

[0097] [Example 2] [Tracking cell death through real-time impedance multiplexing using imaging of living cells] Cell maintenance and assays were performed at 37°C / 5% CO2 in F-12K medium (ATCC, catalog no. 30-2004) containing 10% thermally inactivated FBS (Coming, catalog no. 35016CV). Impedance was measured every 15 minutes, but images were acquired once every hour. Four fields of view were captured in each well, for each channel (brightfield, red, green, blue). Exposure times were red (300 ms), green (300 ms), and blue (80 ms). The A549-Blue cell line, which stably expresses nuclear localized blue fluorescent protein (BFP), was created by transducing A549 cells (ATCC, catalog no. CCL-185) with AgilenteLenti Blue (p / n 8711012) at infection multiplicity 1. Transduced strains were selected by adding 1 μg / mL of puromycin to the growth medium from day 2 to day 11 post-infection. For real-time visualization of activated caspase 3, Agilent eCaspase 3 NucView 488 (p / n 8711005) was added to the growth medium at a concentration of 5 μm. For real-time visualization of migrated phosphatidylserine, Agilent eAnnexin V Red (p / n 8711007) was added to the growth medium at a concentration of 0.25 μg / mL. Agilent E-Plate VIEW microplates (p / n 00300601030) were also used. Stocks of MG132 (Tocris, catalog no. 1748 / 5) and staurosporine (Calbiochem, catalog no. 569396) were dissolved in DMSO.

[0098] A549-Blue cells (shown above) were seeded in E-PATE VIEW at a density of 10,000 cells / well. As the cells proliferated on day 1, they expanded the surface area of ​​the biosensor array, steadily increasing the impedance signal (Figures 11A and 11B). Left untreated, the cells proliferated to confluence, saturating the biosensor array and giving a stable impedance signal. Addition of the proteasome inhibitor MG132 or the pan-kinase inhibitor staurosporine at 25 hours significantly reduced the impedance signal in a time- and dose-dependent manner. The kinetics of these drug-induced responses, and the overall shape of the impedance trace, differed for each compound. This is consistent with extensive literature over more than 10 years, indicating that impedance responses are typically unique to each type of mechanism of action. Both MG132 and staurosporine induce apoptosis, but they trigger different cellular behaviors during the process leading to cell death, which becomes remarkably clear when real-time impedance is multiplexed with living cell imaging.

[0099] A549-Blue cells already cover most of the bottom of the well at 26 hours, but continue to proliferate for another 50 hours in the absence of the drug, packing the cells to a high density (Figure 12A). Consistently, the impedance signal plateaus at approximately 30 hours, and the total number of blue nuclei continues to increase until approximately 80 hours (Figure 12A). The number of blue nuclei is obtained by an image processing algorithm, counting the number of blue objects in the blue fluorescence image. Each blue nucleus corresponds to an A549 cell transduced to express nuclear localized blue fluorescent protein. Therefore, the number of blue nuclei in a well corresponds to the number of viable A549-blue cells present in the well. When A549-Blue cells are treated with four highest concentrations of MG132 (1.9, 5.5, 16.7, and 50 μm), the total number of blue nuclei decreases over time in a manner that correlates well with the decrease in impedance (Figure 12B, showing only data for the 50 μm treatment). In contrast, treating cells with the four highest concentrations of staurosporine (0.125, 0.250, 0.500, and 1 μm) resulted in a sharp drop in impedance within the first few minutes, but a moderate effect on the number of blue nuclei over the following 60 hours (Figure 12C, showing only data for the 1 μm treatment). One hour after staurosporine addition, A549-Blue cells shrank so severely that the cytoplasm was almost invisible, leaving only blue nuclei, consistent with their ability to expel water from the cells. Over time, these nuclei decreased in size and began to cluster together, but they remained largely intact, explaining why the number of blue nuclei remained fairly constant in Figure 12C. It is clear that the combination of impedance and imaging described above provides a more complete and nuanced understanding of drug-mediated A549 cell death than either technique alone would have yielded. Beyond simple cell counting, we then investigated the dynamics of the biochemical phenomena specific to the apoptotic death pathway.

[0100] [Example 3] [Simultaneous tracking of MG132-mediated apoptosis from five different perspectives] Induction of apoptosis by MG132 was tracked not only by impedance and blue nuclear EI count, but also by the rate of cellular confluence, caspase 3 activation (causing cells to fluoresce green), and phosphatidylsene translocation (causing cells to fluoresce red). As seen in Figure 13, drug-induced impedance reduction correlates well with the transient accumulation of these apoptosis-specific markers. The thick white arrows in the panels at 20 and 40 hours highlight large membrane blebs containing phosphatidylserine in the outer leaflet.

[0101] Next, the sequential response of A549-Blue cells to MG132 was plotted using image-based readings for each cell. The number of nuclei in blue (Figure 14A) shows a dependence on drug concentration, which closely reflects the impedance response observed in Figure 11A. Despite the extensive apoptotic response, the % cell confluence never fell below 50% throughout the course of this assay (Figure 14B), where the cell confluence number is obtained from bright-field images by an image processing algorithm. This is consistent with the fact that, unlike in vivo situations where apoptotic cells and their fragments are removed by phagocytosis, in vitro, the majority of apoptotic cells continue to occupy the bottom of the wells (Figure 12B). The fact that the impedance signal drops to zero with high concentrations of MG132 (Figure 11A), while the confluence % never falls below 50%, indicates that residual cells do not adhere to the bottom of the plate. The number of caspase-3 positive (fluorescent green) objects (Figure 14C) and annexin V positive (fluorescent red) objects (Figure 14D) increased over time, showing a clear dependency on MG132 concentration. Considering the number of seeded cells, their growth rate, and the percentage of cells displaying apoptosis markers (Figure 13), the output values ​​in Figures 14A–14C are consistent with expectations. To compare the relative rates and relative abundances of different apoptotic phenomena, impedance responses were plotted together with three different image-based readings (Figure 15). As expected, the time at which the number of blue nuclei begins to decrease (~10 hours after MG132 addition) is the same time at which caspase-3 activation and phosphatidylserine translocation become detectable. In the first 20 hours of drug treatment, the number of cells showing signals for caspase-3 and phosphatidylserine are similar, but in the following 40 hours, the number of cells activated by caspase-3 exceeds the number of cells translocated by phosphatidylserine by approximately 20%.

[0102] [Example 4] [Quantification of drug efficacy by simultaneous monitoring of cell matrix impedance and optical imaging] The EC50 of MG132 was calculated using the impedance and image-based readouts presented in Examples II-IV. The area under the curve from drug addition to 60 hours post-addition was plotted as a function of MG132 concentration to obtain the dose-response curve shown in Figure 16. The R² value was in the range of 0.96-0.98, indicating very good fitting quality for the four different readings. The calculated EC50 values ​​were in the range of 0.86-3.0 μm, which is consistent with values ​​reported in the literature. See Han, YH et al., The Effect of MG132, a Proteasome Inhibitor on HeLa Cells in Relation to Cell Growth, Reactive Oxygen Species and GSH. Oncol. Rep. 2009, 22(1), 215-21. The claims as originally filed are as follows: Claim 1: A multiwell plate having multiple wells configured to receive multiple biological samples, wherein each of the wells includes a set of electrodes, a transparent window on the bottom surface of the well without electrodes, A lighting module configured to illuminate the aforementioned well, A cradle having an opening at the bottom, configured to expose the transparent window of the well, and configured to receive the multiwell plate, To capture images through the exposed window, an optical imaging module is used that is movable between different wells of the same multiwell plate. A system for electronically and optically monitoring biological samples, equipped with [specific features / equipment]. Claim 2: The system according to claim 1, wherein the biological sample optionally includes cells, and cancer cells. Claim 3: The system according to claim 1, wherein the lighting module comprises a plurality of lights configured to independently illuminate one or more of the wells. Claim 4: The system according to claim 1, wherein the lighting module comprises an array of light-emitting diodes (LEDs), and each LED is arranged to illuminate a single well. Claim 5: The system according to claim 1, wherein the illumination module is a bright-field illumination module. Claim 6: The system according to claim 1, wherein the cradle further comprises a hinged cover, and the lighting module is joined to the cover. Claim 7: The system according to claim 1, wherein the cradle electrically engages with both the multiwell plate for electronically communicating with the set of electrodes and the lighting module for communicating lighting commands. Claim 8: The system according to claim 1, wherein the optical imaging module is configured to acquire one or more images from a single well at a time. Claim 9: The system according to claim 1, wherein the optical imaging module is located below the cradle. Claim 10: The optical imaging system further comprises an excitation light source configured to excite one or more molecules, wherein the excitation light source optionally comprises one or more lights selected from the group consisting of ultraviolet light, violet light, blue light, green light, yellow light, orange light, and red light, according to claim 1. Claim 11: The system according to claim 1, wherein the optical imaging module includes a camera. Claim 12: The system according to claim 1, wherein the optical imaging module comprises a camera, a bandpass filter, a tube lens, and an objective lens. Claim 13: A cradle for selectively operating each of the set of electrodes for electronically monitoring the cell substrate impedance in one or more wells, The lighting module for selectively illuminating one or more of the wells, The optical imaging module for acquiring and receiving images from one or more wells and for selective motion. The system according to claim 1, further comprising a computer processor competently coupled to the system. Claim 14: The system according to claim 13, wherein the computer processor is programmed to acquire images from the one or more wells via the optical imaging module in response to the one or more wells reaching or following an impedance-based value or impedance-based parameter set by electronic monitoring. Claim 15: The system according to claim 13, wherein the processor is programmed to electronically monitor cell substrate impedance and optically monitor the same well, and is configured to pair impedance with optical data for display or analysis. Claim 16: Two additional multiwell plates, each having multiple wells configured to receive multiple samples, each of the wells including a set of electrodes and a transparent window on the bottom surface of the well without electrodes, Two additional lighting modules configured to illuminate the wells of the two additional multiwell plates, Two additional cradles, each having an open bottom that exposes the transparent window of the well, and configured to receive the two additional multiwell plates. Furthermore, The system according to claim 1, wherein the optical imaging module is movable across all wells to capture images through all windows. Claim 17: The system according to claim 1, further comprising a cell or tissue culture vessel not configured for electronic monitoring, wherein the optical imaging module is configured to capture images within the cell or tissue culture vessel. Claim 18: A step of continuously electronically monitoring cells in the wells of a multiwell plate over a period of time at specific time intervals between continuous monitoring, wherein each of the wells includes a set of cell substrate impedance monitoring electrodes and a transparent window on the bottom surface of the well without electrodes. The steps include capturing an image from at least one electronically monitored well through the transparent window, and Methods for monitoring cells, including those mentioned above. Claim 19: The method according to claim 18, wherein the images are acquired periodically or irregularly over a period of time within the electronic monitoring period, and the method optionally includes the step of acquiring the images at the same time as performing electronic measurements of the cells. Claim 20: The method according to claim 18, wherein, prior to the step of acquiring an image from the at least one well, the electronic monitoring step outputs a result from the at least one well that satisfies a set value and instructs the optical imaging module to acquire the image from the at least one well. Claim 21: The method according to claim 18, further comprising the step of determining the number of cell confluences or parameters from the bright-field images and optionally counting the cells, wherein the acquired images include bright-field images of cells. Claim 22: The method according to claim 18, further comprising the step of determining fluorescence parameters from an image in which the acquired image includes a fluorescence image of a cell, and which is arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. Claim 23: The acquired images include bright-field images of the cells and fluorescence images of the cells. The steps include: deriving the number of cell confluences or parameters from the bright-field image, and optionally counting cells from the bright-field image; A step of determining fluorescence parameters from a fluorescence image, which are arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. The steps include optionally superimposing the brightfield image and the fluorescence image, or one or more colors from the same well. The method according to claim 18, further comprising: Claim 24: A step of electronically monitoring cells in the wells of a multiwell plate over a certain period of time, wherein each of the wells comprises a set of cell substrate impedance monitoring electrodes and a transparent window on the bottom surface of the well without electrodes. The steps include: capturing an image from at least one well of the multiwell plate through the transparent window over a period of time within the electronic monitoring period; A method for monitoring cells that include [this component]. Claim 25: The acquired image is a bright-field image of the cells, and optionally includes the steps of counting cells from the bright-field image, or determining the number of cell confluences or parameters. The acquired image is a fluorescence image of the cell, and the step includes arbitrarily determining a fluorescence parameter from the image, which is arbitrarily selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. The acquired image includes a bright-field image of the cell and a fluorescence image of the cell. The method of claim 24, further comprising counting cells from the bright-field image, optionally deriving cell confluence numbers or parameters from the bright-field image, optionally determining fluorescence parameters from the fluorescence image, selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity, and optionally superimposing the bright-field image and fluorescence image of one or more colors for one or more wells.

Claims

1. A multiwell plate having multiple wells configured to receive multiple biological samples, wherein each of the wells includes a set of electrodes and a transparent window on the bottom surface of the well without electrodes, A cradle having an opening at its bottom that is configured to expose the transparent window of the well, and configured to receive the multiwell plate, To capture an image through the exposed window, an optical imaging module is provided that is movable between different wells of the same multiwell plate, A computer processor for selectively operating each of the set of electrodes that electronically monitor the cell substrate impedance in one or more wells, A lighting module connected to the computer processor, having multiple light sources, and selectively illuminating one or more of the multiple wells. Equipped with, The optical imaging module is movable and is for acquiring and receiving images from one or more wells. The lighting module selectively illuminates one or more wells using one or more light sources from the plurality of light sources in response to changes obtained in monitoring cell substrate impedance for cell imaging, in order to identify changes in the cell population in the plurality of wells. A system for electronically and optically monitoring a biological sample, wherein the computer processor is programmed to acquire images from one or more wells via an optical imaging module in response to one or more wells reaching an impedance-based value or parameter set from electronic monitoring.

2. The system according to claim 1, wherein the biological sample comprises cells or cancer cells.

3. The system according to claim 1, wherein the lighting module comprises a plurality of lights configured to independently illuminate one or more of the wells.

4. The system according to claim 1, wherein the lighting module comprises an array of light-emitting diodes (LEDs), and each LED is arranged to illuminate a single well.

5. The system according to claim 1, wherein the illumination module is a bright-field illumination module.

6. The system according to claim 1, wherein the cradle further comprises a hinged cover, and the lighting module is joined to the cover.

7. The system according to claim 1, wherein the cradle electrically engages with both the multiwell plate for electronically communicating with the set of electrodes and the lighting module for communicating lighting commands.

8. The system according to claim 1, wherein the optical imaging module is configured to acquire one or more images from a single well at a time.

9. The system according to claim 1, wherein the optical imaging module is located below the cradle.

10. The system according to claim 1, wherein the optical imaging module further includes an excitation light source configured to excite one or more molecules, the excitation light source comprising one or more light selected from the group consisting of ultraviolet light, violet light, blue light, green light, yellow light, orange light, and red light.

11. The system according to claim 1, wherein the optical imaging module includes a camera.

12. The system according to claim 1, wherein the optical imaging module comprises a camera, a bandpass filter, a tube lens, and an objective lens.

13. A cradle for selectively operating each of the set of electrodes for electronically monitoring the cell substrate impedance in one or more wells, The lighting module for selectively illuminating one or more wells, The optical imaging module is movable and for acquiring and receiving images from one or more wells. The system according to claim 1, further comprising a computer processor that is competently connected to the system.

14. The system according to claim 13, wherein the computer processor is programmed to acquire images from the one or more wells via the optical imaging module in response to the one or more wells reaching an impedance-based value or impedance-based parameter set by electronic monitoring.

15. The system according to claim 13, wherein the processor is programmed to electronically monitor cell substrate impedance and optically monitor the same well, and is configured to pair impedance with optical data for display or analysis.

16. Two additional multiwell plates, each having multiple wells configured to receive multiple samples, each of the wells including a set of electrodes and a transparent window on the bottom surface of the well without electrodes, Two additional lighting modules configured to illuminate the wells of the two additional multiwell plates, Two additional cradles, each having an open bottom that exposes the transparent window of the well, and configured to receive the two additional multiwell plates. Furthermore, The system according to claim 1, wherein the optical imaging module is movable across all wells to capture images through all windows.

17. The system according to claim 1, further comprising a cell or tissue culture vessel not configured for electronic monitoring, wherein the optical imaging module is configured to capture images of the cell or tissue culture vessel.

18. A step of continuously electronically monitoring cells in multiple wells of a multiwell plate over a period of time at specific time intervals between continuous monitoring, wherein each of the multiple wells includes a set of cell substrate impedance monitoring electrodes and a transparent window on the bottom surface of each of the multiple wells without electrodes. A step of acquiring an image from at least one well of a plurality of electronically monitored wells through a transparent window, wherein, prior to the step of acquiring an image from the at least one well, the electronically monitoring step outputs a result from the at least one well that satisfies a set value and instructs the optical imaging module to acquire the image from the at least one well; Includes, A method for monitoring cells, the step of acquiring the image comprising selectively illuminating at least one of the multiple light sources using one or more light sources from a plurality of light sources in response to changes acquired in monitoring cell substrate impedance for cell imaging, in order to identify changes in the cell population in the plurality of wells.

19. The method according to claim 18, wherein the image is acquired periodically or irregularly over a period of time within the period of the electronic monitoring step, and the method includes the step of acquiring the image at the same time as performing an electronic measurement of the cell.

20. The method according to claim 18, wherein the acquired image includes a bright-field image of cells, and further comprises the steps of determining the number of cell confluences or parameters from the bright-field image, or counting the cells.

21. The method according to claim 18, further comprising the step of determining fluorescence parameters from an image selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity, wherein the acquired image includes a fluorescence image of a cell.

22. The acquired images include bright-field images of the cells and fluorescence images of the cells. A step of deriving the number of cell confluences or parameters from the bright-field image, or a step of counting cells from the bright-field image, A step of determining fluorescence parameters from the fluorescence image, selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity. The steps include overlaying the brightfield image and the fluorescence image, or one or more colors from the same well. The method according to claim 18, further comprising:

23. A step of electronically monitoring cells in multiple wells of a multiwell plate over a certain period of time, wherein each of the multiple wells comprises a set of cell substrate impedance monitoring electrodes and a transparent window on the bottom surface of the well without electrodes. A step of selectively illuminating one or more wells from among multiple wells using one or more light sources from among multiple light sources, The steps include: capturing an image from at least one well of the multiwell plate through the transparent window over a period of time during or outside of the electronic monitoring period; Includes, The steps of electronically monitoring cells and acquiring images are, i) Electronically monitor the cell substrate impedance, capture an image of at least one well, and pair the impedance with optical data for display or analysis. ii) Taking an image from at least one well in response to one or more wells reaching an impedance-based value or parameter set from the electronic monitoring, iii) Electronically monitoring the cell substrate impedance over a period including a specific time interval between two consecutive electronic impedance monitorings, and capturing an image of at least one well over a period within the electronic monitoring period or over a period separate from the electronic monitoring. It is combined by one or more of the following: A method for monitoring cells, comprising selectively illuminating one or more wells using one or more light sources from the plurality of light sources in response to changes acquired in monitoring cell substrate impedance for cell imaging, in order to identify changes in the cell populations in the plurality of wells.

24. The acquired image is a bright-field image of the cells, and the steps include counting the cells from the bright-field image, or determining the number of cell confluences or parameters. The acquired image is a fluorescence image of the cell, and includes the step of selecting one or more from the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity, and determining a certain fluorescence parameter from the image. The acquired image includes a bright-field image of the cell and a fluorescence image of the cell. The method according to claim 23, further comprising counting cells from the bright-field image or deriving cell confluence numbers or parameters from the bright-field image; determining fluorescence parameters from the fluorescence image selected from one or more of the group consisting of total fluorescence count, total fluorescence intensity, and average fluorescence intensity; and superimposing the bright-field image and fluorescence image of one or more colors for one or more wells.

25. The method according to claim 23, comprising the step of item ii) above, wherein the value or parameter represents an established cell monolayer or an established cell population.

26. The method according to claim 23, comprising the steps of counting cells or determining the cell confluence number of a parameter, and confirming that the cells are properly settled on the bottom surface of the well before monitoring.

27. The method according to claim 23, wherein the step of acquiring the image comprises the step of irradiating one or more light sources in accordance with the results from the electronic monitoring of the cell.

28. The method according to claim 23, wherein the step of acquiring the image includes a step of illuminating in response to changes in the cell substrate impedance for cell imaging, thereby confirming changes in the cell population, preferably a decrease in the cell population.

29. The method according to claim 28, wherein one or more light sources are diodes.

30. A multiwell plate having a plurality of wells configured to receive a plurality of biological samples, wherein each of the plurality of wells includes a set of electrodes and a transparent window on the bottom surface of the well without electrodes, An optical imaging module for imaging the plurality of wells, Computer processors and A lighting module connected to the computer processor, having multiple light sources, and selectively illuminating one or more of the multiple wells. Equipped with, The aforementioned computer processor electronically monitors the cell substrate impedance and the same web The device is programmed to optically monitor the light. i) Electronically monitor the cell substrate impedance, capture an image of the same well, and pair the impedance with optical data for display or analysis. ii) Taking an image from at least one well in response to one or more wells reaching an impedance-based value or parameter set from the electronic monitoring, iii) Electronically monitoring the cell substrate impedance over a period including a specific time interval between two consecutive electronic impedance monitorings, and capturing images of the same well over a period within the electronic monitoring period or over a period different from the electronic monitoring. One or more of these are configured to couple impedance and optical data, The lighting module selectively illuminates one or more wells using one or more light sources from the plurality of light sources in response to changes obtained in monitoring cell substrate impedance for cell imaging, in order to identify changes in the cell populations in the plurality of wells. A system for electronically and optically monitoring biological samples.

31. The optical imaging module is movable between different wells of the same multiwell plate in order to capture images through the transparent window. The system according to claim 30, wherein the computer processor is communicably connected to the optical imaging module and the illumination module, and selectively operates each of the set of electrodes for electronically monitoring the cell substrate impedance in one or more wells, selectively illuminates one or more wells, and selectively operates the optical imaging module for acquiring and receiving images from one or more wells.

32. The system according to claim 30, wherein the computer processor is programmed to acquire images from the one or more wells via the optical imaging module in response to the one or more wells reaching an impedance-based value or impedance-based parameter set from electronic monitoring, the value or parameter representing an established cell monolayer or an established cell population.

33. The system according to claim 30, wherein the lighting module comprises a plurality of lights configured to independently illuminate one or more of the wells.

34. The system according to claim 30, further comprising a cradle that electrically engages with both the multiwell plate for electronically communicating with the set of electrodes and the lighting module for communicating lighting commands.

35. The system according to claim 30, wherein the optical imaging module further includes an excitation light source configured to excite one or more molecules, the excitation light source comprising one or more light selected from the group consisting of ultraviolet light, violet light, blue light, green light, yellow light, orange light, and red light.

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