System and method for detection

Abstract

To provide a device, a system, and a method for detecting a sensor such as an optical detectable sensor.SOLUTION: A device or a system described in the present description may include an illumination assembly configured to direct light to at least one well of a plurality of wells in an array of wells, and a detection assembly configured to detect a signal from the well.SELECTED DRAWING: Figure 3a

Description

[Technical field]

[0001]

[0001] This application is a joint application of U.S. Provisional Patent Application No. 62 / 444, filed on January 10, 2017. No. 564, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Systems for analyzing cell arrays are useful in medical research, life sciences, and other fields. Existing systems for measuring cellular output typically involve the measurement of The system includes an array of wells, such as a microplate, into which cells are added. Individual wells are measured in sequence using a detector configuration including a microscope and a CCD camera. These systems typically provide single-cell resolution but do not provide the ability to measure from multiple wells. Other systems lack the ability to acquire cell output in parallel from multiple wells of an array. However, these systems are typically not time-resolved. Lacking ability. Summary of the Invention [Problem to be solved by the invention]

[0003] [Means for solving the problem]

[0004]

[0003] One aspect of the present disclosure provides a system. In some embodiments, the system The illumination device is configured to direct light from an excitation source to a plurality of wells in the array of wells. In some embodiments, the light source may include a light source assembly and a detection assembly. Each well of the cell may be configured to receive a cell. At least a portion of the light illuminates at least a portion of each well of the plurality of wells to at least In some embodiments, the detection assembly may form a well that is partially illuminated. The detector (i) collects a signal from each at least partially illuminated well of the plurality of wells. and (ii) transmitting each signal to a corresponding detector. In an embodiment, the acquisition of each signal in the plurality of signals may occur substantially in parallel. In some embodiments, the lens focuses at least a portion of the signal onto a corresponding detector. In some embodiments, the transfer of each signal may be substantially in parallel. In some embodiments, the signal from each well of the plurality of wells may At least a portion may be forwarded to a separate detector. The sampling rate of the signal across the wells is greater than about 100 Hertz (Hz). Good too.

[0005] In some embodiments, the collection of signals from each well of the plurality of wells is synchronized. In some embodiments, the assay may be performed twice from each well of the plurality of wells. The collection of one or more signals may occur substantially in parallel. The number of wells may be greater than five. In some embodiments, the number of wells may be greater than five. The collection of two or more signals from each of the wells may occur within a time frame of less than about 20 seconds. In some embodiments, the system provides transmitted light to each well of the plurality of wells. In some embodiments, the detection assembly may be configured to The optical path may be substantially parallel to the incident path of the transmitted light to provide a system having The device may be configured to collect a signal from each well of a plurality of wells along the long axis.

[0006] In some embodiments, the detector may be an optical detector. In some embodiments, the signal may be a light signal. In some embodiments, the sampling rate is about 8 In some embodiments, the sampling frequency may be from about 1,000 Hz to about 12,000 Hz. The ring rate may be about 10,000 Hz. The assembly may include signal collection optics. In some embodiments, the assembly may include a signal collection optics. The aperture may be from about 0.2 to about 0.8. In some embodiments, the signal collection optics The numerical aperture of the system may be about 0.5.

[0007] In some embodiments, at least one well of the plurality of wells is completely In some embodiments, the surface of the signal that may be transferred to the detector may be illuminated. The ratio of the area of ​​the well from which a signal may be collected to the product is about 1:0.5 to about 1:1.5 In some embodiments, the ratio may be about 1:1. In embodiments, the area that may be illuminated may include cells. The area that may be illuminated may include the bottom surface of the well.

[0008]

[0007] In some embodiments, the illumination assembly may include excitation optics. In some embodiments, the focal length of the excitation optics for each of the plurality of wells is may be longer than the focal length of the signal collection optics that collects signals from the corresponding wells of In some embodiments, changes in the light intensity of the excitation source have a response delay of less than about 1 millisecond. It may have.

[0009] In some embodiments, the illumination assembly may include two or more excitation sources. In some embodiments, the two or more excitation sources may be different. In some embodiments, the two or more excitation sources may be the same. , about 5 milliwatts per square millimeter (mW / mm 2 ) and multiple In some embodiments, the ion exchange membrane may be provided in one or more of the wells. The excitation source provides approximately 5 mW / mm2 to each of the wells. 2 Provides light intensity exceeding In some embodiments, the light may be applied for about 10 -7 About 10 -5 Meters In some embodiments, the light may include wavelengths of about 400 to about 800 nanometers. In some embodiments, the detection assembly may include wavelengths in meters (nm). Collect signals from each of the multiple wells at wavelengths from about 400 nm to about 1000 nm It is possible.

[0010] In some embodiments, the signal from each well of the plurality of wells corresponds to a corresponding In some embodiments, the corresponding detector may be a photodiode. In some embodiments, the photodiode may include a pin PIN) photodiodes, pn (PN) photodiodes, avalanche photodiodes The photodiode may include a Schottky photodiode, a Schottky photodiode, or any combination thereof. In some embodiments, the photodiode is a fluorescent, phosphorescent, luminescent, or any of the above. Any combination can be detected. In some embodiments, corresponding detection The vessel is about 10 -7 About 10 -5 Even if it is configured to detect light containing wavelengths of 1 meter In some embodiments, the detection assembly may include multiple corresponding detectors. In some embodiments, each corresponding detector of the plurality of corresponding detectors includes a plurality of U In some embodiments, multiple corresponding wells may be used. Each corresponding detector of the detectors may correspond to a unique well of the plurality of wells. .

[0011] In some embodiments, the plurality of wells may be an array of wells. In some embodiments, the plurality of wells is at least about 16 wells, 32 wells, or more. The plate may contain 1, 96 wells, or 384 wells.

[0012] In some embodiments, the system may include a filter. In some embodiments, the filter may include an emission filter. The system may include multiple lenses and multiple detectors. In some embodiments, Each detector of the plurality of detectors is operatively connected to one lens of the plurality of lenses. In some embodiments, the lens may include a focusing lens. In this embodiment, the lens includes a single collimation lens. In some embodiments, the detection assembly may include an amplifier. In some embodiments, the amplifier may include an on-board transimpedance amplifier. stomach.

[0013] In some embodiments, each well of the plurality of wells may contain a plurality of cells. In some embodiments, at least some of the plurality of wells include a light-detectable sensor. In some embodiments, the system may detect a time period ranging from about 1 millisecond to about 1 minute. The presence or absence of activation of a light detectable sensor within each well of the plurality of wells can be detected. In some embodiments, the system detects about a 5 / 10 mV cell membrane potential change in less than about 1 millisecond. The signal from the optically detectable sensors in each well of a multi-well array is detected with a signal-to-noise ratio exceeding that of The change in intensity can be measured.

[0014] In some embodiments, the light may include a timed pulse of light. In some embodiments, the timed light pulse is less than about 100 milliseconds in duration. In some embodiments, the system may include a timed light pulse. In an embodiment, the timed light pulse may include one or more wavelengths.

[0015] In some embodiments, the change in intensity of the signal from the light detectable sensor is At least one of the cells is subjected to a Chop1 protein, a Chop2 protein, or a Chop3 protein. at least about 52% sequence homology to protein, Chop1 or Chop2 protein sequence homology, approximately 52% sequence length, or a combination thereof. contacting the expressed protein, LiGluR, or any combination thereof; In some embodiments, the signal from the light detectable sensor may result from Changes in the intensity of the signal indicate (i) light-mediated ligand activation of ion channels or G proteins, (ii) Photoinduction of caged ligands or enzymatic substrates or (iii) any combination thereof. In some embodiments, the enzyme substrate or salt thereof is glutamine. phosphate, calcium, nucleotide-phosphate, any salt thereof, or any salt thereof It may be a combination.

[0016] In some embodiments, the optically detectable sensor detects (i) a cell membrane potential. (ii) intracellular ion concentration; (iii) protein conformation; (iv) any combination thereof. In some embodiments, the sensor detects a change in an ion concentration within a cell. In some embodiments, the sensor may be fura-2, Indian -1 (indo-1), fluo-3 (fluo-3), fluo-4, fluo-5F, fluo-5N, load -2 (Rhod-2), Calcium Green, Calcium Red, FuraRed, Quinn -2 (Quin-2), any salt thereof, or any combination thereof. In some embodiments, the sensor may detect changes in the cell membrane potential. In one embodiment, the sensor comprises JC-1 iodide (CAS number 47729-63-5) , JC-1 (CAS number 3520-43-2), di-3-ANEPPDHQ, di-4 -ANEPPS (CAS number 90134-00-2), di-8-ANEPPS (CAS No. 157134-53-7), DiBAC4(3) (CAS No. 70363-83-6 ), BeRST, Di-4-ANBDQBS, VF2.1.C1, RH237 (CAS number No. 83668-91-1), RH414 (CAS No. 161433-30-3), RH4 21 (CAS number 107610-19-5), RH795 (CAS number 172807-1 3-5), any salt thereof, or any combination thereof. In some embodiments, the sensor can detect changes in ion concentrations within a cell. In some embodiments, the ions are calcium ions, sodium ions, potassium ions, ions, hydrogen ions, chloride ions, or any combination thereof.

[0017] In some embodiments, the plurality of wells contains a plurality of cells and media. In some embodiments, at least a portion of the medium comprises a light detectable sensor. In some embodiments, the system may include a time period ranging from about 1 millisecond to about 1 minute. The presence or absence of activation of the light detectable sensor in each of the multiple wells can be detected within a period of time. In some embodiments, the optically detectable sensor can detect (i) a cell membrane potential; (iii) protein structure, or (iv) any combination thereof. A sensor for detecting the change may be included.

[0018] In some embodiments, the plurality of cells includes spontaneously electrically active cells, optically active cells, In some embodiments, the cells may include excitable cells, paced excitable cells, or a combination thereof. In an embodiment, the plurality of cells may include spontaneously electrically active cells. In embodiments, the spontaneously electrically active cells are cardiomyocytes, cortical neurons, dorsal root neurons, Some embodiments may include transganglionic neurons, or any combination thereof. In some embodiments, the plurality of cells may include optically paced excitable cells. In an embodiment, the optically paced excitable cells are ventricular muscle cells, skeletal muscle cells, or other It may also include combinations of these.

[0019] In some embodiments, the plurality of cells in each well of the plurality of wells comprises about 2 cells. In some embodiments, each well of the plurality of wells may contain from about 10 to about 50 cells. The plurality of cells may be less than about 500 cells, less than 400 cells, less than 300 cells, or less than about 2 It may be less than 00 cells, less than 100 cells, less than 50 cells or less. In some embodiments, the plurality of cells in each well of the plurality of wells comprises at least about 1 The number of cells may be up to 1,000.

[0020] Another aspect of the present disclosure provides kits. In some embodiments, the kits include In some embodiments, the kit includes a light detection system and instructions for use. In some embodiments, the kit may include a container. In some embodiments, the container may include a light-detectable sensor. In some embodiments, the kit may include a database. The instructions are for the human Ether-a-go (hERG) gene, which is a gene encoding the human delayed rectifier potassium ion channel. Some embodiments may include instructions for (go-Related Gene) screening. In the method, the instructions include screening the molecule or a salt thereof against intact cardiomyocytes. In some embodiments, the instructions for use include instructions for cardiomyocyte safety. In some embodiments, the kit may include instructions for a sexual pharmacology screen. The device includes one or more light-detectable sensors and a system for using the light-detectable sensors in conjunction with the system. In some embodiments, the kit includes intact cardiomyocytes. may include:

[0021]

[0020] Another aspect of the present disclosure provides a method for producing a kit. In some embodiments, the method comprises: The method may include combining the system with instructions for use. In some embodiments, the method includes manufacturing an illumination system. Another aspect of the present disclosure may include forming a detection assembly. A method for ERG screening is provided. In some embodiments, the method comprises: Another aspect of the present disclosure may include performing hERG screening using the The present invention provides a method for screening a molecule or a salt thereof against intact cardiomyocytes. In some embodiments, the method includes administering a molecule or molecules thereof to intact cardiomyocytes using the system. Another aspect of the present disclosure is to screen the salts of the photoactivatable markers in a plurality of cells. A method for detecting a detectable sensor is provided. In some embodiments, the method comprises: The present disclosure may also include detecting a plurality of intracellular optically detectable sensors using a method for detecting an optically detectable sensor in a plurality of intracellular optically detectable sensors. The present embodiment provides a method for detecting a light-detectable sensor in a plurality of cells. In some embodiments, the method may include collecting signals from a plurality of wells. In some embodiments, the harvesting is performed substantially in parallel. The sampling rate across the wells is greater than about 100 Hz.

[0022] Another aspect of the present disclosure provides a method for detecting an optically detectable sensor in a plurality of cells. In some embodiments, the method includes a scanning array providing an array of wells comprising a plurality of wells. and directing light from an excitation source to each of the wells of the plurality of wells. illuminating at least a portion to form an at least partially illuminated well; collecting a signal from each well of the plurality of wells; and transmitting the signal to a corresponding detector. and each well of the plurality of wells may include at least one cell of the plurality of cells. In some embodiments, the harvesting may occur substantially in parallel. In an embodiment, a lens focuses at least a portion of the signal from each well onto a corresponding detector. In some embodiments, the at least one of the plurality of cells may be configured to Some may include a light detectable sensor. In some embodiments, the signal may be a light detectable sensor. In some embodiments, the transfer may be substantially In some embodiments, sampling across multiple wells may occur in parallel. The ring rate may be greater than about 100 Hz.

[0023] Another aspect of the present disclosure is a method for screening the biological activity of a molecule or a salt thereof in a plurality of cells. In some embodiments, the method includes cleaning a plurality of wells. adding a molecule or a salt thereof to at least a portion of the wells; and detecting whether the sensor is activated. The sensor may include an optically detectable sensor. In some embodiments, the plurality of cells is a myocardial In some embodiments, each well of the plurality of wells may contain a plurality of cells. In some embodiments, the cell may include at least one of the cells. At least a portion of the cells may include a light-detectable sensor. In some embodiments, the detection may occur substantially in parallel. The sampling rate across the well may be greater than about 100 Hz.

[0024] Another aspect of the present disclosure provides a system. In some embodiments, the system The system may include an illumination assembly and a detection assembly. The stem directs light to multiple wells in the array of wells and emits light from each of the multiple wells. and transferring each signal substantially in parallel to a corresponding detector. In some embodiments, sampling of signals across multiple wells may be performed. The pulling rate may be greater than about 100 Hertz (Hz). The present invention provides a method for screening the biological activity of a molecule or a salt thereof in a plurality of cells. In some embodiments, the method includes providing an array of wells comprising a plurality of wells. adding a molecule or a salt thereof to at least a portion of the plurality of wells; and directing light from the source to each of the plurality of wells at least a portion of each of the plurality of wells; illuminating the plurality of wells to form at least partially illuminated wells; and detecting whether or not the sensor in each well is activated.

[0025] In some embodiments, each well of the plurality of wells contains one or more of the cells. In some embodiments, the sample may comprise at least a portion of a plurality of cells. In some embodiments, the detection occurs substantially in parallel. In some embodiments, the sampling rate across multiple wells is: In some embodiments, the sensor is a light detectable sensor. In some embodiments, the plurality of cells may comprise cardiomyocytes. In some embodiments, the plurality of wells may be an array of wells. In the form, the array of wells may be 6 wells, 12 wells, 32 wells, 96 wells, The plate may contain 384 wells, or 384 wells.

[0026] Another aspect of the present disclosure provides a system. In some embodiments, the system The system may include an illumination assembly and a detection assembly. The stem directs light to multiple wells in the array of wells and emits light from each of the multiple wells. and transferring each signal substantially in parallel to a corresponding detector. In some embodiments, the system may be configured to operate at approximately 100 Hertz (H z) may have a sampling rate of signals across multiple wells that may be greater than In some embodiments, the collection and transfer of signals from each well of a plurality of wells is performed. The transmissions may occur simultaneously.

[0027] Further aspects and advantages of the present disclosure are set forth in the following detailed description, which are presented as illustrative embodiments only. It will be readily apparent to those skilled in the art from the following detailed description set forth herein. As such, the present disclosure is capable of other and different embodiments, and some details thereof may be learned from the disclosure. Modifications may be made in various obvious respects without departing from the spirit and scope of the present invention. , should be regarded as illustrative in nature and not as limiting.

[0028] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety. Each individual publication, patent, or patent application is specifically and individually incorporated by reference. Each of the above-mentioned references is incorporated herein by reference to the same extent as if fully set forth herein. To the extent that publications and patents or patent applications conflict with the disclosure contained herein, The Specification shall take precedence and / or prevail over any such conflicting material. The intention is to

[0029] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will now be described with reference to exemplary embodiments, in which the principles of the invention are utilized. The following detailed description and the accompanying drawings (also referred to herein as "Figure" and "Figure") provide This can be obtained by referring to [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows a block diagram of an illumination assembly that directs light from an excitation source to an array of wells where a detection assembly is configured to capture and transfer data obtained from the illuminated area. [Diagram 2] FIG. 2 shows an example of a fluorescence transmission geometry. [Figure 3a]

[0031] FIG. 3a shows the system configuration, FIG. 3b shows the optics for a single well, and FIG. 3c shows the addition of a single collimation lens. [Figure 3b] FIG. 3a shows the system configuration, FIG. 3b shows the optics for a single well, and FIG. 3c shows the addition of a single collimation lens. [Figure 3c] FIG. 3a shows the system configuration, FIG. 3b shows the optics for a single well, and FIG. 3c shows the addition of a single collimation lens. [Figure 4]

[0032] FIG. 4 illustrates a computer control system that is programmed or configured to carry out the methods provided herein. [Diagram 5]

[0033] Figure 5 shows the system configuration. [Figure 6]

[0034] Figures 6a-b show safety pharmacological assays on spontaneously beating cardiomyocytes. [Figure 7]

[0035] FIG. 7 shows dual wavelength stimulation and monitoring of sodium channel activity. [Figure 8]

[0036] Figure 8a-b show simultaneous multi-layer acquisition and automated characterization of cardiomyocyte action potentials using near-infrared voltage-sensing dyes. [Figure 9]

[0037] 9a-b show dual wavelength recording and pacing of cardiomyocyte action potentials. [Figure 10]

[0038] FIG. 10a-c show simultaneous multi-layer dual-wavelength stimulation and recording of sodium channel activity using near-infrared voltage-sensing dyes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031]

[0039] Various embodiments of the present invention are shown and described herein. It will be apparent to those skilled in the art that the present invention is provided by way of example only. Numerous variations, modifications, and substitutions may be made without departing from the spirit and scope of the present invention. It will be appreciated that various alternatives to the embodiments of the invention described herein may be employed. Please understand this.

[0032]

[0040] As used herein, the singular forms "a," "an," and "the" refer to and "the" indicate plural references unless the context clearly indicates otherwise. Any reference to "or" in this specification means "and," unless expressly stated otherwise. "and / or."

[0033]

[0041] As used herein, the term "about" refers to any number that is within the bounds of a referenced numerical table. This refers to a reference reading of ±15% of the indicated value.

[0042] As used herein, the term "well" generally refers to one or more cells. A well is a well that is configured to receive a single cell. The wells can be configured to receive a plurality of cells. The system may also be configured to accept an array of wells, such as a microplate. The array of wells may include a plurality of wells. The multiple wells may contain a single cell type or multiple cell types. One or more of the wells may contain different cell types. Multiple wells may contain different stimuli or conditions (such as different drugs or different media compositions). The array of wells may be formed in a custom-made or commercially available microplate. The system may be a microplate, such as a plate. The system may include an array of wells. The stem may be configured to receive an array of wells. The well may include glass, such as silicate glass. Contains plastics such as polyethylene, polyethylene terephthalate G, and polymethylpentene. Good too.

[0034]

[0043] As used herein, the term "cell" generally refers to the system described herein. The term refers to cells that can be added to one or more wells of a multiple well system. The cells may include cell lines such as human embryonic kidney 293 (HEK293 cells). The cells may be engineered or genetically modified cells. Engineered or genetically modified cells can be modified to produce light-gated ion channels. Channels, voltage-gated channels, ligand-gated ion channels A mechanosensitive ion channel, a temperature-dependent ion channel, or any of them. The cells can be derived from a surgical biopsy, a surgical explant, or a combination of the above. isolated from a subject, such as by resection, needle aspirate, blood sample, or a combination thereof. Cells may be collected from tissues or body fluids such as sputum, saliva, blood, urine, or a combination of these. The cells may be isolated from a patient having or suspected of having a condition, such as a cardiac condition. Cells may be isolated from a subject suspected of having a pulmonary bypass. Cells may include stem cells, neurons, myocytes (cardiomyocytes), and The cells may be cardiomyocytes or neuroblasts, or a combination thereof. The cells were electrically active, such as neurons in the cortical or dorsal root ganglion regions. The cells may be cardiomyocytes (e.g., genetically modified cardiomyocytes), neurons (e.g., cortical neurons), Neurons, dorsal root ganglion neurons, ventricular myocytes, or skeletal muscle cells. In some embodiments, electrically active cells may be paced and excitable. One or more cells, such as cells that support the electrical activity of electrically active cells and cells that support the electrical activity of electrically active cells. Cell types can be co-cultured together. For example, in some cases, cardiomyocytes and HEK29 The three cells can be co-cultured together in a single well.

[0035]

[0044] As used herein, the term "light" generally refers to one or more excitation sources. The excitation source or sources are provided as part of the system. Such as an external excitation source that may be provided to the system or that may be operatively coupled to the system. The light provided by one or more excitation sources may be separated from the system. The light may be directed toward a plurality of wells to illuminate at least a portion of each well of the well. The excitation source may be provided at a particular wavelength or within a particular wavelength range. For example, the excitation source may be at about 400 Light having a wavelength of nanometers (nm) to about 450 nm is applied to one of the multiple wells. The excitation source has a wavelength of about 400 nm to about 1000 nm. Light can be provided to one of the wells. The light can be a pulsed light, etc. The light intensity of the light may be a time constant light or a time varying light, such as an adjustable light intensity. The adjustable light intensity may be time-varying or time-variant. The light provided by the excitation source can be adjusted to a wavelength at which a signal can be detected. and exciting the light detectable sensor such that the light emitted at the light detectable sensor wavelength may be collected from the light detectable sensor. If the detection assembly includes a photodiode, The signal may be an optical signal that may be converted into an electric current. The light may be visible light, infrared light, fluorescent light, The lamps may be luminescent, phosphorescent, or any combination thereof.

[0036]

[0045] As used herein, the term "at least partially illuminated well" means Typically, one well of a plurality of wells configured to receive light from an excitation source. Light is directed from an excitation source to each well of a plurality of wells to generate at least In some cases, the light can illuminate at least some of the cells. In some cases, the light illuminates the cell or cells over substantially the entire surface area of ​​the well in which it is placed. In some cases, the light illuminates multiple areas of the well, where cells are located. In some cases, the light was irradiated to a portion of the cells in the well. In some cases, the light shines at a distance of about 0.1 mm. 2 ~about 20mm 2 Well may be The light may illuminate a surface area of ​​approximately 0.1 mm 2 ~about 10mm 2 Well may be The light may illuminate a surface area of ​​approximately 0.1 mm 2 ~about 5mm 2 It may be well The light may illuminate a surface area of ​​approximately 0.5 mm 2 ~about 10mm 2 Well may be The light may illuminate a surface area of ​​approximately 0.5 mm 2 ~ approx. 8mm 2 Table of wells that may be May illuminate the area.

[0037]

[0046] As used herein, the term "signal" generally refers to a signal detected by the detection assembly of a system. The term "signal" refers to a signal that may be collected by a sensor or other device. The signal may be a spontaneous signal. The signal may be intermittent or may be mounted in response to a fixed or defined stimulus. The signal may be continuous. The signal may follow a stimulus or input. The detection assembly may include collection optics. The detection assembly may include a photodiode. The signal may be an optical signal. The signal may be a signal that may be converted into an electrical signal. The signal may be converted into an electrical signal by a detection assembly such as a photodiode. The signal may be an optical signal emitted by a light detectable sensor. The signal may be a fluorescent signal, a phosphorescent signal, a luminescent signal, or any combination thereof. The signal may be a change in the action potential, a refractory period following the action potential, a membrane depolarization, Biological responses such as partial membrane depolarization, membrane potential or intracellular ion concentration may be indicated.

[0038]

[0047] As used herein, the term "transmission geometry" generally refers to a geometry that is For example, the incident light path (such as light from an excitation source) is A surface of a well (such as a bottom surface) of a plurality of wells. It may not be absorbed by the sample in the well (e.g., cells at the bottom of the well). The incident light may be directed approximately perpendicular to the surface of one of the wells. The detection assembly may be configured to "permeate" through the sample along the path. The ribs are substantially parallel to the incident path of the transmitted light such that the configuration may include a transmission geometry. The sample may be arranged to collect signals from the sample along a common axis.

[0039]

[0048] As used herein, the term "excitation source" generally refers to a device that provides light to a system. The excitation source may be a light source. The excitation source may be a laser, a light emitting diode, Diode (LED) lamps, flash lamps, mercury lamps, xenon arc lamps, tongue lamps The system can be equipped with one excitation source or two The two excitation sources may be the same, such as two lasers. The two excitation sources may be different, such as a laser and an LED lamp. can provide light to at least a portion of each well of the plurality of wells. The source emits a signal that may be collected by a light detectable sensor using the detection assembly of the system. The excitation source may excite or activate the light detectable sensor so as to The detection assembly can provide an excitation wavelength to excite the sensor, and an emission wavelength. For example, the excitation source may be at about 40 The detection assembly provides excitation wavelengths of about 60 to about 450 nanometers to multiple wells. Collecting signals emitted from a light-detectable sensor with emission wavelengths between 0.00 and about 700 nanometers. The detection assembly can receive signals at emission wavelengths between about 400 and about 1000 nanometers. The detection assembly can be collected as a signal of about 400 to about 800 nanometers. The detection assembly can collect signals at the emission wavelengths. The detection assembly can collect signals at emission wavelengths of about 60 nanometers. The signal can be collected at emission wavelengths from 0 to about 1000 nanometers. Approximately 400, 405, 410, 415, 420, 425, 430, 435, 440, 445 , 450, 455, 460, 465, 470, 475, 480, 485, 490, 495 , 500, 505, 510, 515, 520, 525, 530, 535, 540, 545 , 550, 555, 560, 565, 570, 575, 580, 585, 590, 595 , 600, 605, 610, 615, 620, 625, 630, 635, 640, 645 , 650, 655, 660, 665, 670, 675, 680, 685, 690, 695 , 700, 705, 710, 715, 720, 725, 730, 735, 740, 745 , 750, 755, 760, 765, 770, 775, 780, 785, 790, 795 , 800, 805, 810, 815, 820, 825, 830, 835, 840, 845 , 850, 855, 860, 865, 870, 875, 880, 885, 890, 895 The excitation source may provide an excitation wavelength of about 600 nm to about 700 nm. The excitation source may provide an excitation wavelength of about 610 nm to about 680 nm. The excitation source may provide an excitation wavelength of about 620 nm to about 660 nm. The signals emitted from the light detectable sensors are approximately 400, 405, 410, 415, 42 0, 425, 430, 435, 440, 445, 450, 455, 460, 465, 47 0, 475, 480, 485, 490, 495, 500, 505, 510, 515, 52 0, 525, 530, 535, 540, 545, 550, 555, 560, 565, 57 0, 575, 580, 585, 590, 595, 600, 605, 610, 615, 62 0, 625, 630, 635, 640, 645, 650, 655, 660, 665, 67 0, 675, 680, 685, 690, 695, 700, 705, 710, 715, 72 0, 725, 730, 735, 740, 745, 750, 755, 760, 765, 77 0, 775, 780, 785, 790, 795, 800, 805, 810, 815, 82 0, 825, 830, 835, 840, 845, 850, 855, 860, 865, 87 Detector with emission wavelengths of 0, 875, 880, 885, 890, 895, or 900 nm The signal may be collected by a fluoroscopy. The signal is detected at an emission wavelength of about 600 nm to about 900 nm. The signal may be collected by an emission assembly. The signal may be collected by the detection assembly at a wavelength between about 650 nm and about 850 nm. The emission wavelength may be collected by a detection assembly. may include a photodiode that converts an optical signal into a current signal.

[0040]

[0049] As used herein, the term "optically detectable sensor" generally refers to a well or refers to a light-detectable sensor that can be added to a cell. The cell contains a light-detectable sensor. The medium in the well may include an optically detectable sensor. The optically detectable sensor may include: The detection assembly of the system detects light emitted by the light-detectable sensor at the emission wavelength. The light detectable sensor may be a fluorescent sensor, a luminescent sensor, The light detectable sensor may be a near-field sensor, a phosphorescent sensor, or any combination thereof. Infrared sensor, panchromatic sensor, blue-green sensor, ultraviolet sensor, or any of these The light-detectable sensor can detect light within a wavelength range of about 80 nm, a combination of wavelengths of about 100 nm, and a wavelength of about 100 nm. 0nm ~ approx. 1000nm, approx. 700nm ~ approx. 900nm, approx. 600nm ~ approx. 800nm, About 500 nm to about 700 nm, about 400 nm to about 600 nm, or any combination thereof The light-detectable sensor can detect light within a wavelength range such as 100 nm, ... Or it may include a sensor that may detect multiple characteristics. For example, a light-detectable sensor may , cell membrane potential, intracellular ion concentration, protein structure, or any combination thereof. The device may include a sensor for detecting changes in the

[0041]

[0050] As used herein, the term "ion" generally refers to any positively or negatively charged An ion refers to an atom or molecule that bears an electric charge. In some cases, an ion is an intracellular or extracellular ion. The ions may be calcium ions, sodium ions, potassium ions, hydrogen ions, etc. ion, chloride ion, magnesium ion, iron ion, manganese ion, biocarbonate The ions may include, for example, ions thiophene, ... Any ions that may flow into and / or out of cells through the channels may be included. good.

[0042]

[0051] As used herein, the term "molecule" generally refers to a molecule that is attached to one another by chemical bonds. Any peptide, amino acid, small molecule or any The molecule may be a drug or an investigational drug. The molecule may be administered to a subject with a condition. The molecule may be a therapeutic compound used to treat a subject having a condition. The compound may be a therapeutic compound under evaluation for treating

[0043]

[0052] As used herein, the term "sequence identity" generally refers to a sequence that is optimal for comparison purposes. To do this, the sequences are aligned (e.g., gaps can be introduced into the sequence of the first sequence). ) between two or more nucleotide or amino acid sequences. This refers to the calculation of "homology" or "percent homology." The percent identity between the two sequences is calculated based on the number of identical positions shared by the sequences. may be a function of the number of identical positions (i.e., % homology = number of identical positions / total number of positions × 100). For example, a position in a first sequence is filled with the same nucleotide as the corresponding position in a second sequence. The percent alignment between the two sequences may be occupied by a position other than the one at which the molecules are identical. Sequence homology is the number of gaps that need to be introduced for optimal alignment of two sequences. A function of the number of identical positions shared by the sequences, taking into account the number and length of each gap In some embodiments, the sequence lengths of the sequences aligned for comparison may be At least about 30%, at least about 40%, at least about 50%, At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least At least about 80%, at least about 85%, at least about 90%, at least about 91%, at least At least about 92%, at least about 93%. At least about 94%, at least about 95%, at least at least about 96%, at least about 97%, at least about 98%, or at least about 95% A BLAST® search may determine the homology between two sequences. The two sequences can be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, etc. The actual comparison of the two sequences may be performed using, for example, a mathematical This can be accomplished by well-known methods, such as by using a mathematical algorithm. A non-limiting example of a genetic algorithm is given in Karlin, S. and Altschul, S., roc.Natl.Acad.Sci.USA, 90-5873-5877(1993) Such an algorithm is described in Altschul, S., et al., Nucleic Ac As described in Ids Res, 25:3389-3402 (1997), N.B. It can be incorporated into the LAST and XBLAST programs (version 2.0). When using the BLAST and Gapped BLAST programs, Any relevant parameters of the BLAST search engine (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set as score=100, word length=12. or can be varied (e.g., W=5 or W=20). Yers and Miller, CABIOS (1989), ADVANCE, ADA In another embodiment, two algorithms are included: M, BLAT, and FASTA. The percent identity between amino acid sequences can be determined using, for example, the GCG software package (Access This can be achieved using the GAP programme within the LRYS Centre, Cambridge, UK. .

[0044]

[0053] The advantages of the systems, devices, and methods described herein include: This may include substantially parallel or simultaneous multi-layer acquisition of signals such as signals. A system that provides transmitted light or transillumination fluorescence to multiple wells or samples in a This structural feature of the transmission geometry may be achieved in part by the Systems lacking the above may lack sufficient illumination intensity required for multi-layer acquisition. Other advantages provided by the systems, apparatus, and methods include: (a) substantially concurrent or simultaneously for a sustained period of time, repeated stimulation and response signal monitoring across multiple wells (b) performing short reactions across multiple wells in substantially parallel or simultaneous fashion; Accurate recording of signals with response times (e.g., sub-millisecond scale perturbations) (c) achieving high resolution on multiple wells with low signal-to-noise ratios in substantially parallel or simultaneous fashion; This may include combining a fast response probe with a fast sampling rate. The system is (a) capable of sampling one image at a time with a high sampling rate and acceptable signal-to-noise ratio; or (b) a slow response probe or slow These conventional systems are configured to measure multiple samples at the same sampling rate. In contrast, the systems described herein may not be able to perform both functions. The apparatus and method can achieve both.

[0045]

[0054] As shown in FIG. 1, the system described herein includes a lighting assembly 102, a welded The illumination assembly may include an array of illumination tubes 101, and a detection assembly 103. As shown in FIG. 5, one or more illumination sources 501, one or more illumination optical systems 502, Light from the illumination assembly is directed to one or more samples 503. In some cases, the sample may include one or more cells. The system also includes emission optics 504 and detector 506 for collecting signals that may be emitted from the sample. The computer 506 may include an illumination source 501, one or more 503, detection electronics 505, or any combination thereof. Or it may be operatively connected to multiple system elements.

[0046]

[0055] The illumination assembly may include one or more excitation sources. An external excitation source can be operably coupled to the system. The illumination assembly is A well in an array of wells or a plurality of wells in an array of wells or a microplate Each well of the plurality of wells may be configured to direct light to a portion of one or more The light may be emitted from an excitation source to a small number of wells in the array of wells. The light may be directed at least partially into the well. The light can be fully illuminated or partially illuminated. The light may be directed to illuminate at least a portion of each well of the plurality of wells. The light may be emitted from at least a portion of the cells received in the well. The light may be directed to illuminate each cell received in the well. , or at least about 50%, 60%, 70%, 80% of the cells received in the well, The light may be directed to illuminate 90% or less of the cells received in the well. The light may be directed to illuminate at least about 60% of the cells received in the well. The light may be directed to illuminate at least about 70% of the wells. The light may be directed to illuminate at least about 80% of the cells. The light may be directed to illuminate at least about 90% of the cells received in the well. The light may be directed to illuminate at least about 95% of the cells in the different wells. Each well of multiple wells is then filled with irradiated cells so that the irradiated cells in the well are irradiated in parallel. The cells may be irradiated in parallel at least some of the received cells.

[0047]

[0056] The detection assembly may include one or more detectors. The detectors may be optical detectors. The detector may be a photodiode. The detector may be a photomultiplier tube. The detection assembly may include one or more photodiodes, phototransducers, or photomultipliers. The detection assembly may include an electron multiplier, a CCD camera, or a combination thereof. The detection assembly may include a detector corresponding to each well of the plurality of wells. and may be configured to collect and transmit to one or more detectors. The detection assembly may collect a signal from a portion of the well that may be illuminated. A signal may be collected from a portion of the cell that is received by the filter. The signal may be collected from a portion of the cell that is illuminated by an excitation source. The signal may be an optical signal, such as a fluorescent signal, collected from a portion of the cell that may be detected. The signal can be transformed, for example, by converting an optical signal to a current signal. This may be accomplished by a detector such as a photodiode. Signal collection may be performed in parallel, such as parallel collection of signals from 384 wells. The time required for detection assembly, including collection and transfer across multiple wells of an array of wells. Resolution is approximately 100 Hertz (Hz), 1,000Hz, 10,000Hz, 20,000Hz z, or even more.

[0048]

[0057] The characteristics of the light provided by the excitation source may be adjustable. For example, the intensity of the light The intensity may be adjustable. The wavelength of the light may be adjustable. The temporal pattern of the light may be adjustable, such as a constant light or a timed light pulse. The user can adjust the intensity, etc. The characteristics of the light can be adjusted. The system controller can command the adjustment of the light characteristics. The properties of the light can be determined by adjusting the wavelength to properly excite the light-detectable sensors in the wells. The properties of the light can be adjusted in response to a feedback signal, such as by adjusting the The characteristics of the light, such as its intensity, may be adjusted in response to the particular assay that may be performed using the light. Adjusting the sensitivity may result in a response delay of less than about 1 millisecond. The response delay may be less than about 0 milliseconds. The response delay may be less than about 5 milliseconds. The response delay may be less than about 2 milliseconds. The response delay may be less than about 1 millisecond. This may be less than about 0.75 milliseconds. The response delay may be less than about 0.5 milliseconds. The response delay may be less than about 0.25 milliseconds. The response delay may be less than about 0.1 milliseconds. It may be less than a second.

[0049]

[0058] The excitation source may provide a light intensity to each well of the plurality of wells. For example, Each well of the plurality of wells has a power output of approximately 5 milliwatts per square millimeter (mW / mm 2 ) Each well of the plurality of wells can receive a light intensity of at least about 5 mW / mm 2 Each well of the plurality of wells can receive a light intensity of The size of the pores varies by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 1%. Light intensities that do not vary by more than 0%, 15%, or 20% can be accepted.

[0050]

[0059] The detection assembly collects a signal, such as an optical signal, and directs the signal to a corresponding detector. In each well of the plurality of wells, a detection assembly may be provided. is configured to collect a signal and direct at least a portion of the signal to a corresponding detector. The detection assembly may collect a signal and detect at least about 90%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, It may be configured to direct 7%, 98%, or 99% of the signal to a corresponding detector. The detector may be a photodiode. In some cases, the photodiode detector is , pin (PIN) photodiode, pn (PN) photodiode, avalan Photodiodes, Schottky photodiodes, or any combination of these Detectors such as photodiodes may detect fluorescent, phosphorescent, luminescent, and other signals. or any combination thereof. It can be achieved.

[0051]

[0060] The systems described herein accept an array of wells, such as a microplate. The array of wells may include a plurality of wells. The wells may be at least about 1 well, 6 wells, 16 wells, 32 wells, 96 wells, The wells may contain 384 wells or more. Each well of the plurality of wells may be configured to receive one or more cells. In some cases, at least a portion of the plurality of wells may be configured to receive A light-detectable sensor, such as an extracellular light-detectable sensor or a light-detectable sensor in a liquid medium. In some cases, at least a portion of the plurality of cells may include a light detectable cell. The sensor may include a sensor.

[0052]

[0061] The systems described herein are adapted to detect the presence or absence of activation of a light detectable sensor. The optically detectable sensor can be configured to detect (i) cell membrane potential, (ii) intracellular ion potential, (iii) protein structure, or (iv) any combination thereof. The sensor may include a sensor for detecting a change in an intracellular ion concentration. The sensors are Fluo-2, Indo-1, Fluo-3, Fluo-4, Fluo- 5F, Fluo-5N, Rhod-2, Calcium Green, Calcium Red, Fura Red , quinone-2, any salt thereof, or combinations thereof. The change in cell membrane potential may be detected. The sensor is a soluble form of JC-1 iodide (CAS number 4772 9-63-5), JC-1 (CAS number 3520-43-2), di-3-ANEPPD HQ, di-4-ANEPPS (CAS number 90134-00), di-8-ANEPP S (CAS number 157134-53-7), DiBAC4(3) (CAS number 70363 -83-6), BeRST, Di-4-ANBDQBS, VF2.1.C1, RH237 (CAS number 83668-91-1), RH414 (CAS number 161433-30-3 ), RH421 (CAS number 107610-19-5), RH795 (CAS number 172 807-13-5), any salts thereof, or any combination thereof. The sensor may detect a change in intracellular ion concentration. The sensor may detect a change in intracellular ion concentration. , sodium ion, potassium ion, hydrogen ion, chloride ion, or any of them The sensor may detect a combination of the two. The sensor may be a genetically encoded sensor. Genetically encoded sensors detect changes in membrane potential, intracellular ion concentrations, and protein structure. A biological response such as a change in the level of a protein, ... or any combination thereof can be detected.

[0053]

[0062] The optically detectable sensor may be within a cell or on the surface of a cell. The sensor may be in a medium in which cells may be cultured. The system may exhibit a biological response such as a change in ion flow or membrane potential. and configured to detect in parallel the presence or absence of activation of a light-detectable sensor in each of the wells. The system may measure the concentration of the ions in each well of the plurality of wells for a period ranging from about 1 millisecond to about 1 minute. The system may be configured to detect the presence or absence of activation of the light detectable sensor. Presence / absence across multiple wells in minutes, 100ms, 10ms, 1ms, or less In some cases, the system can detect approximately 5 / 10 millivolts (mV). Change in cell membrane potential of 10 / 10mV, change in cell membrane potential of 15 / 10mV , 20 / 10mV change in cell membrane potential, 25 / 10mV change in cell membrane potential, A signal-to-noise ratio of 50 / 10 mV or more of change in cell membrane potential is obtained with a resolution of approximately 1 µs. The change in intensity of the light-detectable sensor is detected in each well of multiple wells simultaneously in less than a millisecond. It can be configured as follows.

[0054]

[0063] The light from the excitation source may include constant light, timed light pulses, or a combination thereof. The timed light pulse may be less than about 100 milliseconds in duration. The timed light pulse may be less than about 10 milliseconds in duration. The timed light pulse may be less than about 1000 milliseconds in duration. The timed light pulse may have a duration of about 10 milliseconds to about 100 milliseconds. The pulse may have a duration of about 50 milliseconds to about 100 milliseconds. The timed light pulse may include one or more wavelengths. The one or more wavelengths of the timed light pulse may be the same wavelength. or at least two different wavelengths, at least three different wavelengths, or The wavelengths may be different, such as 100 nm to 150 nm or 200 nm to 300 nm.

[0055]

[0064] Multiple cells were identified, including spontaneously electrically active cells, optically paced excitable cells, and The plurality of cells may include a cell line or primary cells, or a combination thereof. The plurality of cells may comprise cells obtained from a subject. The plurality of cells may comprise: The plurality of cells may include cells obtained from a biological sample obtained from the subject. It may include cells obtained from a cell bank, tissue bank, or other population bank. Potentially electrically active cells include cardiomyocytes, cortical neurons, dorsal root ganglion neurons, or The optically paced excitable cells may include any combination thereof. The cells may include muscle cells, skeletal muscle cells, or a combination thereof.

[0056]

[0065] The systems described herein may include one or more filters, such as an emission filter. The filter may be configured to allow the detection assembly to collect the signal and filter out light from the excitation source. The system may be configured to allow for a focusing lens, a single collimator, The optical system may include one or more lenses, such as a focal length lens, a focal length lens, a focal length lens, or a combination thereof. The lens may be configured to direct the signal to the detector. The system may be configured to direct the one or more The amplifier may include an on-board transimpedance amplifier. Good too.

[0057]

[0066] The kit may include the system described herein and instructions for use. The kit may comprise the system described herein. ii) screening of molecules for intact cardiomyocytes; iii) cardiomyocyte safety pharmacology cleaning, or iv) the systems and reagents and / or combinations thereof required The kit may include one or more optically detectable sensors and instructions for use. It may also include instructions for use.

[0058]

[0067] The method may include a method of making a kit or a method of making a system. The method includes a method for screening for hERG using a system as described herein. The method may include a method of administering a therapeutic agent to intact cardiomyocytes using the system described herein. It may include a method for screening molecules.

[0059]

[0068] The method may include detecting the optically detectable sensor in the plurality of cells. may include providing an array of wells comprising a plurality of wells. Each well may contain one or more cells. The cells may contain a light-detectable sensor. In some cases, at least a portion of the one or more cells includes a light-detectable sensor. Each well of the plurality of wells may be irradiated with light from an excitation source. At least a portion of the well can be illuminated to form an at least partially illuminated well. Signals can be collected in parallel from each well of multiple wells and the signals can be Each of the multiple signals can be transmitted in parallel to a detector that can detect the light. It is possible to check whether the sensor is activated or not.

[0060]

[0069] The method includes screening the biological activity of a molecule or salt thereof in a plurality of cells. The method may include providing an array of wells comprising a plurality of wells. Each well of the plurality of wells may contain one or more cells. The salt can be added to at least some of the wells. Light can be directed at each well to illuminate at least a portion of each well. The presence or absence of activity of the functional sensor can be detected in parallel in each well of multiple wells. .

[0061]

[0070] The systems described herein can be used to rapidly detect fast response probes or optically detectable sensors. Combined with a fast sampling rate, multiple parallel inputs can be used with a high signal-to-noise ratio (SNR). A signal can be collected from the sample. For example, a light-detectable sensor can detect the signal in less than about 1 millisecond. In some cases, the response time constant may be less than about 100 microseconds. In such a case, the system may include a light-detectable The changes in the sensors can be measured in parallel. Parallel measurements across multiple wells can be performed in approximately 1 Parallel measurements across multiple wells can occur in less than about 100 microseconds. The SNR of the system is greater than approximately 5 / 10 mV of cell membrane potential change. or greater than about 25 / 10 mV of cell membrane potential change, or greater than about 50 / 10 mV of cell membrane potential change In some cases, the system may induce a monitorable change in cell membrane potential. For example, the system may generate multiple timed light pulses from an excitation source. Multiple timed light pulses can be of the same or different wavelengths to excite a light-detectable sensor. The timed light pulse may include less than about 100 milliseconds, less than about 75 milliseconds, less than about 50 milliseconds, It may be less than a second, less than about 25 milliseconds, less than about 10 milliseconds, or less than about 5 milliseconds. Light-dependent actuator proteins may induce monitorable changes in cell membrane potential. Light-dependent actuator proteins induce monitorable changes in cell membrane potential. It can be used alone or in combination with multiple timed light pulses to achieve the desired effect. The actuator proteins are Chlamydomonas chop1, Chlamydomonas chop2, or an expressed protein having at least about 52% sequence identity, about 52% sequence length, or or a combination of them with chop 1 or chop 2. The modulator protein may include a light-dependent glutamate receptor (LiGluR). .

[0062]

[0071] The signal may be collected from a region of an array of wells that contain a plurality of cells. For example, Alternatively, the regions may be individual wells containing multiple cells. 384-well, 96-well, 384-well, etc.) simultaneously or in parallel. do.

[0063]

[0072] The system may include an illumination system that includes one or more excitation sources. The system may be operatively connected to an external excitation source. The intensity of the excitation source may be controlled by the user. may be adjustable by a controller of the system. The delay time in adjusting from the first intensity to the second intensity may be less than about 10 milliseconds. The excitation source may provide each well of the plurality of wells with the same intensity. The excitation source may provide, for example, a signal that varies by less than 10% across multiple wells. The excitation source may provide a similar intensity to each well of a plurality of wells, such as a single intensity. W / mm 2 , 5mW / mm 2 , or 10 mW / mm 2 Strength that may exceed multiple wedges The antibody may be provided in each well of the tube.

[0064]

[0073] In some cases, the system may include one or more optical systems. may consist of one or more excitation optics, one or more collection optics, or a combination of both. The excitation optics may include a filter that directs light from an excitation source to multiple wells of the system. The collection optics may be configured to collect signals from multiple wells. The signals can be configured to be collected in parallel, depending on the is the focal length of the excitation optics into the well, and the focal length of the signal collection optics that collects the signal from the well. The focal length of the excitation optics can be longer than the focal length of the signal collection optics. The focal length of the excitation optics may be approximately 1%, 5%, 10%, or 20% longer than the signal. May be at least about 1%, 5%, 10%, or 20% longer than the focal length of the collection optics The focal length of the excitation optics may be the same as the focal length of the signal collection optics. The focal length of the optical system may be longer than the focal length of the excitation optical system. The separation may be about 1%, 5%, 10%, or 20% longer than the focal length of the excitation optics. The focal length of the signal collection optics should be at least about 1%, 5%, or 10% longer than the focal length of the excitation optics. %, or 20% longer.

[0065]

[0074] The system can be configured to collect signals from multiple wells in the system. The signal may be a fluorescent signal, a phosphorescent signal, a luminescent signal, a visible signal, or any combination thereof. The optical signal may be an optical signal such as a photodiode. The system may be configured such that the signal is collected in a transmission geometry and converted to a current signal by a transducer. For example, as shown in FIG. The light from the excitation source can be directed to the array of wells 201. The multiple wells can be configured to receive multiple cells. The portion of the light that may pass through the multiple welds may be a transmitted beam 204. The portion of the light that may pass through the filter may be a scattered beam 205. The system , a detector 203 for collecting a portion of the scattered beam 205. The angle θ between the optics 105 and the transmitted beam 204 may be about θ=0. The angle θ may be about θ= At about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 The angle θ may be about θ=5. The angle θ may be about θ=10. The angle θ may be about 15°. The angle θ may be less than about 5°. may be less than about 4. The angle θ may be less than about 3. The angle θ may be less than about 2. The angle θ may be less than about 1.

[0066]

[0075] In some cases, such as when the angle θ = 0, the excitation light is directed by the detection assembly In some cases, the excitation light may not be collected by the detection assembly. In some cases, a portion of the excitation light may be collected by the detection assembly. In some cases, the excitation light may also be removed so that the signal is detected but the excitation light is not. In some cases, a portion of the excitation light can be collected by the detection assembly. In some cases, the excitation light may be collected by a detection assembly. By varying the angle θ, the fraction of the excitation light that can be collected by the detection assembly is The system also includes an excitation light source that may be collected by a detection assembly. The filter may include one or more filters that may remove a portion of the excitation light. The filter can filter out a portion of the excitation light. The detector (i.e., photodiode) converts the optical signal into a current signal and not the excitation light. Therefore, the excitation light can be removed so that the

[0067]

[0076] The system includes a detection assembly including collection optics configured to collect a signal. The system may include an optical system configured to direct light to a plurality of wells. The system may include an optical system, such as an illumination assembly that includes excitation optics. The system may include a combination of collection optics and excitation optics. The numerical aperture of the optics is adjustable. The system may be capable of exchanging one or more optical systems having different numerical apertures. A larger numerical aperture allows a greater amount of signal to be focused onto the detector. For example, an optical system with a numerical aperture of 0.8 may be focused with an aperture of 0.2. It is possible to collect a larger amount of signal than optical systems that include multiple ports. The numerical aperture of the system may be about 0.2. The numerical aperture of the optical system may be about 0.3. The numerical aperture of the optical system may be about 0.4. The numerical aperture of the optical system may be about 0.5. The numerical aperture of the optical system may be about 0.6. The numerical aperture of the optical system may be about 0.7. The numerical aperture of the optical system may be about 0.8. The numerical aperture of the optical system may be about 0.2 to The numerical aperture of the optical system may be about 0.8. The numerical aperture of the optical system may be from about 0.3 to about 0.7. The numerical aperture of the optical system may be from about 0.4 to about 0.6. The numerical aperture of the optical system may be from about 0.3 to about The numerical aperture of the optical system may be from about 0.2 to about 0.7.

[0068]

[0077] The collection optics of the system converts signals, such as optical signals (i.e., fluorescent signals), into The light may be collected by a corresponding detector, such as a photodiode. The detector can convert the optical signal into a current signal. The collection optics can The signal can be collected from the area of ​​the well. The area collected from the collection optics is , about 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500mm 2 Even if stomach.

[0069]

[0078] In some cases, it may be advantageous to minimize the collection area of ​​the detector. Minimizing the collection area of ​​the detector helps minimize noise in the system. The diameter of the detector collection area is approximately 0.5, 0.75, 1.0, 1.25, 1.5, and 1 .75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, or 4.0mm The diameter of the detector collection area may be about 0.5, 0.75, 1.0, 1.25, 1. 5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, or 4.0 m The diameter of one of the plurality of wells may be less than about 0.5, 0.7, or 5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3. 0, 3.5, 4.0 mm, or more. The well diameters are approximately 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0mm or even more The diameter of the collection area of ​​the detector may be equal to the diameter of the well, for example both diameters being about The diameter of the collection area of ​​the detector may be smaller than the diameter of the well. For example, the well diameter may be about 3.0 mm and the collection area diameter may be about 2.0 mm. The diameter of the collection area of ​​the detector may be larger than the diameter of the well. For example, the well diameter may be about 1.0 mm and the collection area diameter is about 2.0 mm. The system may include a signal processor for determining the area of ​​the signal that may be transferred to the collection area of ​​the detector. Provide a magnification for the collection optics such that the ratio of signal to area of ​​the well from which it is collected is approximately 1:1. It may be configured as follows.

[0070]

[0079] The ratio is the area where signal can be collected relative to the area where signal can be transferred to the detector. The ratio may be 1:1. The ratio may be from 1:0.5 to about 1:1.5.

[0071]

[0080] The ratio is the fraction of signal that can be collected from a well by illuminating the well. and the ratio may be about 1:1. The ratio may be about 1:0. The ratio may be from about 1:1.5 to about 1:1.5.

[0072]

[0081] The ratio is the ratio of the illumination to the portion of the well where a signal can be collected and transferred to the detector. The wells may be part of a plurality of wells that may be used for the same purpose. The ratio is about 1:1. The ratio may be from about 1:0.5 to about 1:1.5.

[0073]

[0082] The portion of the well that may be illuminated is at least about 1, 10, 50, 100, 50 The signal may be collected from 0, 1000, 2000 or more cells. The portion of the wells that can be formed is at least about 1, 10, 50, 100, 500, 1000, 20 The active area of ​​the corresponding detector corresponds to the illumination of the well. The active area of ​​the corresponding detector may be equal to the illuminated area of ​​the well. It may be similar to:

[0074]

[0083] The advantage of the system described herein is that it can detect signals from fluorescent or optically detectable sensors. The system may provide efficient collection and detection of signals such as one or more The detection assembly of the system may include one or more lenses, such as a focusing lens. The focusing lens of the systems described herein may include a lens of about 6 mm. The lens may include a diameter of about 1 mm and a focal length of about 6 mm. Positioned by or operably coupled to a machined plate In some cases, shortening the focal length of the lens can reduce the amount of light collected by the lens. The proportion of signals that may be detected may be increased. In some cases, the proportion may be increased by 1 / 2 (1-cos(θ)).

[0075]

[0084] In some cases, when θ is small, the ratio is: ratio=sin 2 (θ) / 4 or NA 2 may be given as / 4.

[0085] where NA is the numerical aperture. In some cases, the lens is focused into a gas medium such as the surrounding air. The lens may be placed in the body. In some cases, the lens may be placed in a liquid medium.

[0076]

[0086] In some cases, the systems described herein focus the signals onto a corresponding detector. In this case, a focusing lens having a focal length similar to that of the lens that can be focused is provided. In some cases, the focal length of the collection lens can focus the signal onto the corresponding detector. The focal length may be equal to the focal length of the lens.

[0077]

[0087] The one or more detectors of the systems described herein may include one or more photodetectors. The detector or detectors may include a 300 nm to 350 nm laser diode. High response to signals containing wavelengths similar to near-infrared light, such as 300 nanometers The one or more detectors may be arranged or configured in a detector array. The array of detectors may correspond to the array of wells received by the system. For example, the system can accept a 384 well plate, and the system The detector array may be composed of one or more corresponding detectors. It may be combined with an on-board transimpedance amplifier.

[0078]

[0088] The systems described herein operate at frequencies of approximately 100 Hertz (Hz), 250 Hz, 500 Hz, z, 750Hz, 1,000Hz, 2,000Hz, 5,000Hz, 6,000Hz, 7,000Hz, 8,000Hz, 9,000Hz, 10,000Hz, 11,000H z, from wells that may exceed 12,000 Hz, 15,000 Hz, or even more The system can provide a signal sampling rate of approximately 100 Hertz (Hz ), 1,000Hz, 5,000Hz, 10,000Hz, 15,000Hz, or 100%, or more. The sampling rate may be from about 500 Hz to about 1212,000 Hz. The sampling rate may be from about 500 Hz to about 5,000 Hz. The rate may be from about 8,000 Hz to about 12,000 Hz. The sampling rate may be at least about 100 Hz to about 15,000 Hz. The sampling rate may be at least about 200 Hz to about 15,000 Hz. The sampling rate may be at least about 300 Hz to about 15,000 Hz. The rate may be at least about 400 Hz to about 15,000 Hz. The range may be from about 500 Hz to about 15,000 Hz. The grade may be at least about 1,000 Hz to about 15,000 Hz.

[0079]

[0089] FIG. 3a shows a system as described herein. FIG. 3b shows an expanded subset of FIG. In Fig. 3a, an expanded subset is shown in dotted lines. In Fig. 3b, multiple The optics for one of the wells are highlighted. A filter (such as an emission filter) is shown in FIG. 3b as 302. The filter 303 may be placed below the lens 302. The filter can be placed over the lens. A lens that can focus the signal onto a detector is shown as 305. The lens 305 may be disposed within a plate shown as 304. A detector corresponding to one of the wells (e.g., a detector corresponding to one of the wells) is shown as 306. A lens (e.g., a focusing lens) is placed over the microplate that is received by the system. The lens may be located on the optical axis 301, and the lens may be shown as 301. also includes a lens (e.g., a collimation lens) shown as 307 in FIG. A collimation lens may be disposed above lens 301. In some systems, such as those described herein, the excitation source is coupled to a collimation lens 307. The divergent light may be provided by a lens such as

[0080]

[0090] The system described herein uses cultures of spontaneously active cortical neurons. In the technical field of environmental toxicology evaluation of molecules (e.g., chemicals) that may typically be performed using the Similarity of spontaneously active or light-modulated neuronal firing patterns may be observed. Studies may also be conducted.

[0081]

[0091] The systems described herein include ligand modulation using photoactivatable ligands. It may also be used in the field of high throughput screening of targets. As an alternative to using light-sensitive voltage modulators to control ion channel target activity, Alternatively, photoactivatable ligands can be added to cell culture media and used to modulate the activity of targets. The target may be expressed either naturally (e.g., in neurons) or in cells for heterologous expression. These may be ion channels or G proteins. The biological response can be measured at one wavelength, while the effective "dose" of the ligand is By modulating the duration or intensity of light at a second wavelength that can activate the Ligands can be used to regulate the function of neurotransmitters (e.g., glutamate, GABA , acetylcholine, purine nucleotides), ions (calcium), small drug-like molecules, The ligand may be caged (permanently caged by irradiation with light). ) or photoswitchable (reversible light-induced activation / inactivation). In some cases, glutamate photoswitchable ligands bind to LiGluR ligands. This application can be extended to biochemical assays. do.

[0082]

[0092] The system described herein is indirectly modulated by a photosensitive actuator. Such a method can be used in the field of high-throughput screening of targeted proteins. In some cases, light-activated receptors (e.g., melanopsin) or enzymes (e.g., adenylyl cyclase It can regulate second messengers (e.g., phosphoinositides or The AMP molecule acts on the target, and its function is optically may be monitored.

[0083]

[0093] The systems described herein can be used in the field of safety pharmacology. For example, in the field of safety pharmacology, the system is designed to meet the U.S. Food and Drug Administration (FDA) regulatory requirements. As part of an integrated human cell study standardized in comprehensive in vitro arrhythmia assessment guidelines The system described herein can be used for optically paced The systems described herein may also be used in safety pharmacology testing in cardiomyocytes. All-optical voltage modulation and / or high-throughput screening of ion channel targets In such cases, the system can be used to synthesize non-excitable cells and heterologous expression The systems described herein may be configured for ion channel targeting. It can be used as an alternative or complement to RG screening.

[0084]

[0094] For example, cells can be plated and plated in a standard optical bottom 96 or 384 well plate. The cells may be: a) spontaneously active; a) induced pluripotent stem cells (iPSCs) or stem cell-derived cardiomyocytes, b) a photosensitive actuator ( Typically, cardiomyocytes or thyrocytes may heterologously express channel rhodopsin. c) a mixture of non-excitable cells and cardiomyocytes, which may heterologously express channel rhodopsin; The present invention provides a method for heterologously expressing a photoactivator and one or more target ion channels. Specifically engineered non-excitable cells (which can then be combined to become excitable in characteristic ways) The cells may comprise a voltage-sensitive dye, or d) any combination thereof. (or other fluorescent sensor). The molecule-containing wells may be compared to a number of control wells, or Comparisons may be made to the baseline activity of each well, measured prior to compound addition.

[0085]

[0095] Changes in voltage (or other sensors) can be monitored, e.g., spontaneous activity can be monitored for about 30 seconds using one excitation light wavelength. Cell activity is monitored in the second wave. The optical pulse register may be triggered or modulated by one or more actuating optical pulses of different lengths. The regimen involves the selection of frequency, duration, intensity, or other parameters to affect the photoinduced voltage change. This variation can be used to target ion channels or to measure the pulse duration within the well. "Non-static" or transient conformations that may differentially interact with existing molecules Pharmacological studies on cellular responses using different irradiation / activation protocols may be performed. The profile of biological effects may have predictive therapeutic or toxicological impacts. .

[0086]

[0096] To reduce electronic noise, first separate the voltage (or other) traces into one or Subjected to multiple standard electronic "long-pass" filtering algorithms Spontaneously active cell voltage traces are analyzed using our proprietary event detection algorithms. The rhythm of the action potential (or "beat" in the case of cardiac muscle cells) can be used to analyze Such events of interest are identified by the exact time start and end of each event, The upstroke duration, the maximum intensity (fluorescence change) of each event, and the intensity at a specific percentage of the maximum intensity The duration of each event in the sequence (the "action potential duration"), or the event rate, duration, or rhythm User-specified or controller-specified parameters that can guide the identification of features such as The induced or modulated activity trace can be detected based on the above. It may be analyzed using an event detection algorithm or, more generally, different A software algorithm is used to define a region of interest for the stimulation light pulse. The types of measurements may be similar to those described above.

[0087]

[0097] The system as described herein can be used to culture non-excitable cells (i.e., HEK2 Screening for ion channel targets expressed in cells such as 93 cells or CHO cells The method may be used for screening heterologously expressed ion channels. For example, screening for target ion channels, light-dependent Actuator proteins and accessory ion channels function in non-excitable cells The ion channel or auxiliary ion channel may be expressed in a voltage-gated ion channel. The present invention may include an electrochemical channel, a ligand-gated ion channel, or a combination thereof. The potential-gated ion channels are sodium channels, calcium channels, and potassium channels. channels, transient receptor potential (TRP) channels, proton channels, or any combination thereof. Ligand-gated ion channels include acetylcholine receptors, acetylcholine receptors, and combinations thereof. On-channel glutamate-dependent receptors, acid-sensing ion channels (ASICs), adeno ATP-dependent P2X receptors, anion-permeable gamma-aminobutyric acid-dependent (GABAa) receptors, or any combination thereof.

[0088]

[0098] Light-dependent actuator proteins are stimulated with a wavelength of light, such as light from an excitation source. The wavelength of the light may be about 400 nanometers to about 1000 nanometers. The wavelength of the light may be about 400 nanometers to about 800 nanometers. The length may be about 400 nanometers to about 500 nanometers. The wavelength of the light may be about 5 The wavelength of the light may be about 600 nanometers to about 600 nanometers. The wavelength of the light may be from about 700 nanometers to about The cell or cells may be irradiated with light (such as a fluorescent dye). Detectable sensors or genetically encoded sensors or combinations thereof. Thus, the cellular response of the target ion channel can be detected by a fluorescent signal from the optically detectable sensor. The monitoring may be via a signal such as a light-detectable sensor or a genetically encoded The sensor, or a combination thereof, has a wavelength of about 400 nanometers to about 500 nanometers. b) light of a first wavelength, such as about 600 nanometers to about 700 nanometers, or c) a combination thereof. The detectable or genetically encoded sensor detects changes in the cell membrane potential or the cell It provides a signal that can indicate a biological response or change, such as a change in intracellular ion concentration. Thus, the observed effects of contacting one or more cells with a molecule can be The magnitude and temporal profiles of optically detectable or genetically encoded sensors or a combination thereof, changes in the activity of such molecules on the target ion channel. It may be shown to elicit a biological response.

[0089]

[0099] The target ion channels are (i) voltage-gated sodium channels, voltage-gated cal channels, calcium channels, or voltage-gated potassium channels, or a combination of both Voltage-gated channels, (ii) inositides (TRP family), cyclic nucleases Nucleotides (HCNx, CNGx), calcium (KCax), or a combination (iii) potassium leak channels that respond to second messengers such as potassium chloride " channels (K2Px), or (iv) any combination thereof.

[0090]

[0100] Light-dependent actuator proteins are involved in the cation channel Chlamydomonas reinhardtii. CHOP1, Chlamydomonas CHOP2, sequence identity > 52%; length, or combinations thereof for chop1 or chop2 The light-dependent actuator protein may also include a light-activated glutamate receptor. LiGluR, G protein (such as melanopsin or mGluR), adenylate cyclase (bPAC), or any combination thereof.

[0091]

[0101] Auxiliary ion channels regulate the resting cell membrane potential together with the defined extracellular potassium concentration. The voltage-dependent inward rectifier potassium channel (Kirx) is used to regulate Auxiliary ion channels may also be voltage-dependent channels that are expressed in addition to the target ion channel. It may be a calcium, sodium, or potassium channel.

[0092]

[0102] The disclosed system may be used for the pharmacological evaluation of electrically excitable cells. The system can be used with spontaneously electrically active cells. For example, fluorescent Voltage or calcium sensors may be incorporated into spontaneously electrically active cells. Light at a first wavelength excites the sensor and emits fluorescent light that may be collected over time. Submillisecond-scale perturbations in response parameters allow automated heuristic algorithms to and may be used to predict potential target tissue toxicity. The spontaneously electrically active cells are stem cell or induced pluripotent stem cell-derived cardiomyocytes. The cells may be either cortical neurons, dorsal root ganglion neurons, or stem cell-derived neurons. It is also possible.

[0093]

[0103] The disclosed system may also be used with optically paced excitable cells. For example, photoresponsive actuator proteins and fluorescent voltage or calcium sensors The agent can be introduced into spontaneously active or quiescent electrically excitable cells. Timed pulses of light of certain wavelengths can slow the electrical activity of excitable cells. while the second wavelength can excite the fluorescent sensor. The variation of response parameters when exposed to chemicals or proteins under can be detected by heuristic algorithms and to predict potential target tissue toxicity can be used for.

[0094]

[0104] The light-responsive actuators are Chlamydomonas CHOP1 or CHOP2, or greater than about 52% sequence identity, about 52% sequence length, or chop 1 or chop In one embodiment, the actuator is a HEK The actuators can be expressed in non-excitable cells such as 293, and the actuator-expressing cells can be electrically excited. In another embodiment, the light-responsive actuator can be co-cultured with excitatory cells. It may also be expressed directly in excitable cells.

[0095]

[0105] Optically paced excitable cells may be spontaneously active as described above. or excitable quiescent cells, including adult ventricular myocytes or skeletal myocytes. .

[0096]

[0106] In some cases, each well of the plurality of wells includes a corresponding photodiode, etc. In some cases, each photodiode has a corresponding lens. The lenses may be configured to focus a signal, such as an optical signal, onto a corresponding detector. Without a lens to focus the signal onto the detector, part of the signal will be collected by the detector. The optical signal may be collimated and may be filtered to further reduce the In such a case, the signal is collected by a detection assembly and Light from the source may not be collected. The filters may be configured in fixed or adjustable positions. The system may include lenses, detectors, filters, or a combination of these. One or more components of the system may be remotely controlled, electronically controlled, or manually controlled by a user. Each filter in the system can be wired independently. The detection assembly is In some cases, the system collects signals from fluorescent dyes, such as intracellular calcium dyes. The controller of the system is responsible for processing one or more signals collected from the system. An algorithm can be used.

[0097]

[0107] The systems of the present disclosure include one or more detectors, such as photodiodes. In some cases, the system may include at least two detectors, e.g., multiple well detectors. Each well may include a corresponding detector. In some cases, the detection assembly may include one or more The system may include at least two signal collection optics. If the signal collection optics includes a 10-channel cascade, the numerical aperture of each signal collection optic is: (i) both signal collection optics have an aperture of about 0.5; (ii) one signal The collection optics comprises a numerical aperture of about 0.5 and the second signal collection optics comprises a numerical aperture of about 0.4. The numerical aperture of the signal collection optics may be about 0.2. The numerical aperture of the signal collection optics may be about 0.3. The numerical aperture of the signal collection optics may be about 0.4. The numerical aperture of the signal collection optics may be about 0.7. The numerical aperture of the signal collection optics may be about 0.8. The numerical aperture of the signal collection optics may be at least about 0.2. The numerical aperture of the signal collection optics may be at least about 0.4. The numerical aperture of the collection optics may be at least about 0.5. The numerical aperture of the signal collection optics is about The numerical aperture of the signal collection optics may be less than about 0.9. The numerical aperture of the signal collection optics may be less than about 0.7. The numerical aperture of the signal collection optics may be less than about 0.6. The numerical aperture of the signal collection optics may be from about 0.2 to about 0.8. The numerical aperture of the signal collection optics may be from about 0.3 to about 0.7. The numerical aperture of the optical system may be from about 0.4 to about 0.6.

[0098]

[0108] The system of the present disclosure may include an excitation source such that the illumination assembly may include the excitation source. The system may include at least two excitation sources. The system may include at least The system may include as few as three excitation sources. The system may include at least four excitation sources. The stem may include at least five excitation sources, such as a first laser and a second laser. At least two of the excitation sources may be the same, such as a laser and a light-emitting diode (LED) light. In addition, at least two of the excitation sources may be different. The excitation sources may be lasers, LED lights, xenon lights, etc. In some cases, the system may be a laser or a non-arc lamp. It includes a single laser with a beam splitter to direct portions of the light to multiple wells.

[0099]

[0109] The sampling rate may be about 100 Hz. , may be about 250 Hz. The sampling rate may be about 500 Hz. The sampling rate may be about 750 Hz. The sampling rate may be about 2,500 Hz. The sampling rate may be about 3,000 Hz. The sampling rate may be about 5,000 Hz. The sampling rate may be about 6,000 Hz. The sampling rate may be about 8,000 Hz. The sampling rate may be about 9,000 Hz. The sampling rate may be about 12,500 Hz. The sampling rate may be about 15,000 Hz. The sampling rate may be about 100 Hz or greater. The sampling rate may be about 250 Hz or greater. The sampling rate may be about 500 Hz or more. The sampling rate may be about 1,000 Hz or more. The sampling rate may be about 2,500 Hz or greater. The sampling rate may be about 3,000 Hz or more. z or greater. The sampling rate may be about 5,000 Hz or greater. The sampling rate may be about 6,000 Hz or greater. The sampling rate may be about 7,500 Hz or more. The sampling rate may be about 8,000 Hz or more. The sampling rate may be about 9,000 Hz or greater. The rate may be about 10,000 Hz or higher. The sampling rate may be about 12, The sampling rate may be greater than 500 Hz. The sampling rate may be from about 200 Hz to about 10,000 Hz. The sampling rate may be from about 500 Hz to about 10,000 Hz. The rate may be from about 1,000 Hz to about 10,000 Hz. The sampling rate may be from about 2,000 Hz to about 10,000 Hz. The sampling rate may be from about 5,000 Hz to about 10,000 Hz. It may be from about 5,000 Hz to about 15,000 Hz.

[0100]

[0110] The disclosed system detects one or more of the wells using light directed from an excitation source. At least a portion of one well can be illuminated. In some cases, multiple wells can be illuminated. At least a portion of each of the wells may be illuminated by light directed from an excitation source. In some cases, a signal (such as an electrical signal) is collected from one of the wells. In some cases, the signal can be transmitted to multiple detectors. The signal from each well can be collected and transferred to a detector. The portion of the well where a signal may be collected from and transferred to a detector. In some cases, the portion of the well that may be illuminated corresponds to the same portion of the well where the signal is This may be a portion of the well that is different from the portion of the well that may be collected. The ratio of the fraction of light that may illuminate the well to the fraction of signal that can be collected from the well is approximately The ratio may be from 1:0.5 to about 1:1.5. In some cases, the ratio is about 1:1. In some cases, illumination may be provided for a portion of the well from which a signal may be collected. The ratio of the good well fraction may be from about 1:0.5 to about 1:1.5. In some cases, the ratio may be about 1:0.75 to about 1:1.25. The ratio may be from 0.9 to about 1.1. In some cases, the ratio may be about 1:1. Depending on the ratio, it may be 1:0.5, 1:0.55, 1:0.6, 1:0.65, 1:0.7 , 1:0.75, 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1: It may be 1.2, 1:1.3, 1:1.4, or 1:1.5.

[0101]

[0111] The system of the present disclosure includes: The device may include an excitation source for illuminating the device. In some cases, the excitation source may be at about 10 -5 Meters ~about 10 5In some cases, the light may be about 10 -4 meters ~ about 10 4 In some cases, the light may have wavelengths of about 10 meters. -3 M 10~approx. 3 In some cases, the light may have wavelengths of about 10 meters. -2 Meh Torr ~ approx. 10 2 In some cases, the light may have wavelengths of about 10 meters. -1 Mate ~ approx. 10 1 In some cases, the light may include visible light (e.g., The radiation may include light visible to the human eye, ultraviolet, infrared, or a combination thereof. In some cases, the light includes wavelengths from about 400 nanometers to about 1000 nanometers. In some cases, the light may be from about 400 nanometers to about 900 nanometers. In some cases, the light may include wavelengths from about 400 nanometers to about 800 nanometers. In some cases, the light may have wavelengths from about 400 nanometers to In some cases, the light may include wavelengths from about 600 nanometers to about 500 nanometers. In some cases, the light may include wavelengths from about 800 nanometers to about 800 nanometers. , may include wavelengths from about 500 nanometers to about 700 nanometers. Thus, the light may include wavelengths from about 500 nanometers to about 900 nanometers. In some cases, the light has a wavelength between about 600 nanometers and about 900 nanometers. In some cases, the light may include a wavelength of about 700 nanometers to about 900 nanometers. In some cases, the light may include wavelengths from about 800 nanometers to about 90 This may include wavelengths up to 0 nanometers.

[0102]

[0112] The systems of the present disclosure may include one or more excitation sources. The light intensity provided by the laser is approximately 1 milliwatt per square millimeter (mW / mm 2 ) The light intensity provided by the excitation source may be approximately 1.5 milliwatts per square millimeter. (mW / mm 2 The light intensity provided by the excitation source may be in millimeters squared. Approximately 2 milliwatts per millivolt (mW / mm 2 ) may be provided by the excitation source. The light intensity is about 2.5 milliwatts per square millimeter (mW / mm 2 ) may be The light intensity provided by the excitation source is approximately 3 milliwatts per square millimeter (mW / mm 2 The light intensity provided by the excitation source may be Approximately 3.5 milliwatts (mW / mm 2 The light intensity provided by the excitation source may be A power of approximately 4 milliwatts per square millimeter (mW / mm 2 ) may be used. The light intensity provided by the 2 The light intensity provided by the excitation source may be about 5 per square millimeter. Milliwatts (mW / mm 2 The light intensity provided by the excitation source may be expressed as Approximately 5.5 milliwatts per millimeter (mW / mm 2 ) may be used. The light intensity provided by the LD is approximately 6 milliwatts per square millimeter (mW / mm 2 ) The light intensity provided by the excitation source may be approximately 6.5 milliwatts per square millimeter. (mW / mm 2 The light intensity provided by the excitation source may be in millimeters squared. Approximately 7 milliwatts per millivolt (mW / mm 2 ) may be provided by the excitation source. The light intensity is about 7.5 milliwatts per square millimeter (mW / mm 2 ) may be The light intensity provided by the excitation source is approximately 8 milliwatts per square millimeter (mW / mm 2 The light intensity provided by the excitation source may be Approximately 8.5 milliwatts (mW / mm 2 The light intensity provided by the excitation source may be A power of approximately 9 milliwatts per square millimeter (mW / mm 2 ) may be used. The light intensity provided by the 2 The light intensity provided by the excitation source may be about 1 per square millimeter. 0 Milliwatts (mW / mm 2 The light intensity provided by the excitation source may be about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8. 5, 9, 9.5, or 10mW / mm 2 It could be larger.

[0103]

[0113] The systems of the present disclosure may include a detection assembly, a signal detected by the system, In some cases, the detected signal can be In some cases, the signal may be a change in cell membrane potential or an intracellular In some cases, the SNR is approximately 5 / 1. It may be greater than 0 millivolt (mV) membrane potential change. In some cases, the SNR may be greater than the cell membrane potential change of about 10 / 10 mV. NR can be greater than the cell membrane potential change of about 15 / 10 mV. In this case, the SNR may be greater than the cell membrane potential change of about 20 / 10 mV. In this case, the SNR may be greater than the cell membrane potential change of about 25 / 10 mV. Therefore, the SNR may be greater than the cell membrane potential change of about 30 / 10 mV. In some cases, the SNR may be greater than the cell membrane potential change of approximately 35 / 10 mV. In some cases, the SNR can be greater than the cell membrane potential change of about 40 / 10 mV. In some cases, the SNR can be greater than the cell membrane potential change of approximately 45 / 10 mV. In some cases, the SNR is greater than the cell membrane potential change of about 50 / 10 mV. It's possible.

[0104]

[0114] The disclosed system can provide an excitation source that provides light to multiple wells. The light may be a time-constant light. The light may be a time-varying light such as a pulsed light. In some cases, the pulsed light may be pulsed at various time intervals. The light may be pulsed at specific time intervals, such as timed light pulses. The timed light pulse may have a duration of about 200 milliseconds. The timed light pulse may have a duration of about 100 milliseconds. The timed light pulse may be approximately 80 milliseconds in duration. The timed light pulse may be approximately 60 milliseconds in duration. The timed light pulse may be approximately 40 milliseconds in duration. The timed light pulse may be about 20 milliseconds in duration. The timed light pulse may be about 10 milliseconds in duration. The timed light pulse may be about 200 milliseconds in duration. The timed light pulse may be less than about 100 milliseconds in duration. The timed light pulse may be less than about 80 milliseconds in duration. The timed light pulse may be The timed light pulse may have a duration of less than about 60 milliseconds. The timed light pulse may be less than about 20 milliseconds in duration. The limiting light pulse may be less than about 10 milliseconds in duration.

[0105]

[0115] The disclosed system is capable of adding a single well of multiple wells of the system. The protein or fragment thereof can be provided for contacting a cell capable of The protein or fragment thereof has at least about 52%, 50%, or more similarity to the known protein. 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 For example, the protein or protein may have 7%, 98%, or 99% sequence homology. The fragment has at least about 52% sequence identity to the Chop1 protein, about 52% The protein or fragment thereof may have a sequence of any one of a number of lengths, or a combination thereof. At least about 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 10 ... 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence The protein or fragment thereof may have a similarity, a similarity in length, or a combination thereof. The pieces may be at least about 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 9 9% sequence identity, sequence length, or a combination thereof. The fragments have at least about 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 9 9% sequence identity, sequence length, or a combination thereof.

[0106]

[0116] The light intensity of the excitation source may vary over time. In some cases, the system When multiple excitation sources are included, each excitation may include a light intensity that may be the same or different from the other excitation sources. The light intensity of the excitation source may be adjusted by the system controller or by the system user. The light intensity across multiple wells may be the same, but may be adjustable. For example, about 5 mW / mm for each of the multiple wells. 2 5mW / mm 2 + / -0.25mW / mm 2 As such, the light intensity across multiple wells varies by approximately 5%. The light intensity across multiple wells may not vary by approximately 1%, 2%, 3%, 4% %, 5%, 6%, 7%, 8%, 9%, or 10%. When the system includes multiple excitation sources, the light intensity across the multiple excitation sources varies by approximately 1% between each excitation source. If it does not fluctuate by more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% There is a match.

[0107]

[0117] When the controller or user can adjust the light intensity, the light If the intensity can vary over time, the delay or lag in the change in light intensity is approximately 1 The delay in the change in the light intensity of the excitation source may be less than 5 milliseconds (ms). The delay in changing the light intensity of the excitation source may be less than about 9 milliseconds. The delay in changing the light intensity of the excitation source may be less than about 8 milliseconds. The delay in the change in the light intensity of the source may be less than about 7 milliseconds. The delay in the change may be less than about 6 milliseconds. This may be less than about 5 milliseconds. The delay in the change in the light intensity of the excitation source is less than about 4 milliseconds. The delay in the change in the light intensity of the excitation source may be less than about 3 milliseconds. The delay in changing the light intensity of the excitation source may be less than about 2 milliseconds. The delay in the change in light intensity may be less than about 1 millisecond.

[0108]

[0118] The systems of the present disclosure may include microwell plates or arrays of cuvettes, etc. The array of wells may be configured to receive an array of wells, such as a commercially available microfluidic Microwell plates such as cross-well plates or custom-designed microwell plates The array of wells may be made of glass (such as borosilicate glass), plastic, or (polypropylene, polyethylene, polyethylene terephthalate G, polymethylpentene The array of wells can be formed of a flat The array of wells may include a 6-well plate. Rate, 16-well plate, 32-well plate, 96-well plate, or 384 The array of wells may include a plurality of wells. Wells are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 4 2, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75 , 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 , 135, 140, 145, 150, 155, 160, 165, 170, 175, 180 , 185, 190, 195, 200, 210, 220, 230, 240, 250, 260 , 270, 280, 290, 300, 310, 320, 330, 340, 350, 360 , 370, 380, 390, 400, 410, 420, 430, 440, 450, 460 , 470, 480, 490, 500, 550, 600, 650, 700, 750, 800 , 850, 900, 950, 1000, or more wells. The well may comprise from about 1 to about 20 wells. The plurality of wells may comprise from about 1 to about 100 The plurality of wells may include from about 10 to about 400 wells. The plurality of wells may include from about 50 to about 1000 wells. The plurality of wells may include at least three wells. The plurality of wells may include at least five wells. The plurality of wells may include at least eight wells. The plurality of wells may include at least The plurality of wells may include at least 10 wells. The plurality of wells may include at least 11 wells. The plurality of wells may include at least 15 wells. The plurality of wells may comprise at least 20 wells. The plurality of wells may comprise at least 31 wells. The plurality of wells may include at least 95 wells. The plurality of wells may include at least 100 wells. The plurality of wells may include at least 200 wells. The plurality of wells may comprise at least 383 wells.

[0109]

[0119] The system of the present disclosure may include a plurality of wells, One of the wells may be configured to receive cells. One of the wells may be configured to receive a plurality of cells. In some cases, the plurality of cells may be the same in each well of the plurality of wells. In some cases, the plurality of cells may be different in each well of the plurality of wells. Depending on the type, one of the wells may contain about 2, 5, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 , 1500, or 2000 cells.

[0110]

[0120] In some cases, the plurality of cells in the well may range from about 1 cell to about 100 cells. In some cases, the number of cells in the well may range from about 1 cell to about 200 cells. In some cases, the plurality of cells in the well may range from about 1 cell to about 500 cells. In some cases, the number of cells in a well may range from about 1 cell to about 1,000 cells. In some cases, the plurality of cells in the well may range from about 1 cell to about 2,000 cells. In some cases, the plurality of cells in the well may be about 10 cells to about 100 cells. In some cases, the plurality of cells in the well may range from about 10 cells to about 5 00 cells. In some cases, the plurality of cells in the well may be from about 500 cells to about In some cases, the number of cells in a well may be about 1,000 cells. The number of cells in the well may be 500 to about 2,000. may be about 100 cells to about 500 cells.

[0111]

[0121] In some cases, a plurality of cells in a well of the plurality of wells. In some cases, the number of cells in a plurality of wells may be at least about 10 cells. The plurality of cells in one of the wells may be at least about 50 cells. In some cases, the plurality of cells in a well of the plurality of wells is at least about 10 In some cases, the number of cells in one well of the plurality of wells may be 0. The plurality of cells may be at least about 250 cells. The number of cells in one well of several wells may be at least about 500 cells. In some cases, the plurality of cells in one well of the plurality of wells may be In some cases, the number of wells may be at least about 750 cells. The plurality of cells in a single well may be at least about 1000 cells. In the case, the plurality of cells in a well of the plurality of wells is at least about 2 000 cells.

[0112]

[0122] In some cases, a plurality of cells in a well of the plurality of wells. In some cases, one of the multiple wells may contain less than about 10 cells. The number of cells in a well may be less than about 50 cells. The plurality of cells in a well of the plurality of wells may be less than about 100 cells. In some cases, the plurality of cells in one well of the plurality of wells is about In some cases, one of the multiple wells may contain less than 250 cells. The plurality of cells in a well may be less than about 500 cells. The plurality of cells in a well of the plurality of wells may be less than about 750 cells. In some cases, the plurality of cells in one well of the plurality of wells is about 1 In some cases, one of the multiple wells may contain less than 1,000 cells. The plurality of cells in a well may be less than about 2000 cells.

[0113]

[0123] The disclosed system includes a light-detectable sensor in one of the multiple wells. The system can detect the presence or absence of activation of the sensor in each of the multiple wells. The system can detect the presence or absence of a sensor. The system can be configured to detect the presence or absence of The present invention is capable of detecting the presence or absence of a light-detectable sensor in one of a plurality of wells within minutes. The system can detect within about 40 seconds. The system can detect within about 30 seconds. The system can detect within about 20 seconds. The system can detect within about 10 seconds. The system can detect within about 1 second. The system can detect within about 100 milliseconds (ms). The system can detect within about 50ms. The system can detect within about 10 milliseconds. The system is capable of detection within about 1 millisecond.

[0114]

[0124] The system detects the number of light-detectable sensors in each well of a plurality of wells in approximately one minute. The system can detect the presence or absence of a substance in each well of multiple wells in approximately 30 seconds. The system can detect within each well of multiple wells in approximately 10 seconds. The system can detect each well in multiple wells in approximately 1 second. The system is capable of detecting each well in multiple wells within approximately 100 milliseconds. The system is capable of detecting within each well of multiple wells within approximately 50 milliseconds. The system can detect within each well of multiple wells within approximately 10 milliseconds. The system is capable of detecting within each well of multiple wells within approximately 1 millisecond.

[0115]

[0125] The system can measure multiple wells (6 wells, 12 wells, etc.) in a time frame of less than approximately 20 seconds. Collect one or more signals from each well of a 96-well, 384-well, etc. The system can be set to approximately 20 seconds, 15 seconds, 10 seconds, 5 seconds, 1 second, 100 milliseconds, 50 milliseconds, 1 µs, 10 ms, 1 ms, or less from each well of multiple wells One or more signals may be collected.

[0116]

[0126] The system collects one or more signals from each well of the plurality of wells. The system can collect multiple signals. Approximately 2, 3, 4, 5, 6, 7, 8, 9, 10 or more signals can be collected from The system can at least detect when two signals are the same, such as two trains of action potentials. The system can collect two signals at the same time: voltage-dependent events and ion-dependent events. Collect at least two signals when the two signals are different, such as a presence event. Two or more signals in a well, such as two trains of action potentials, are consecutive in time. Voltage-dependent events and ion-dependent events may occur. Two or more signals in a channel may occur substantially in parallel or simultaneously.

[0117] Computer Control System

[0127] The present disclosure also relates to a computer programmed to carry out the methods of the present disclosure. A control system is provided. FIG. 4 illustrates a system for (i) controlling an excitation source to provide light to multiple wells. (ii) controlling the direction of light, such as directing light to an area of ​​a well in a microplate; iii) Collect and / or transfer information (e.g., electrical signals) to a database or memory, etc. Controlling the detection assembly to transmit a signal to the detector The computer system 401 is, for example, (i) controlling the excitation source; (ii) controlling the detection assembly; (iii) controlling the optical path or (iv) controlling the portion of the well that receives light; and / or (iv) collecting information (e.g., an electrical signal). or control the frequency of data collection, data transfer, or otherwise, as provided herein. Various aspects of the data analysis, and data storage can be coordinated. 401 is a computer system located remotely to the user's electronic device or electronic device. The electronic device may be a portable electronic device.

[0118]

[0128] The computer system 401 includes a central processing unit (CPU, here abbreviated as “processor”). The “Computer Processor” 405 includes a single core or The processor may be a multi-core processor, or multiple processors for parallel processing. The computer system 401 also includes a memory or memory location 410 (e.g., a random access memory, read only memory, flash memory), electronic storage device 415 (e.g. a hard disk), a communications interface for communicating with one or more other systems 420 (e.g., a network adapter), and caches, other memories, data peripherals 425, such as digital storage devices, and / or electronic display adapters. The memory 410, the storage device 415, the interface 420 and the peripheral device 425 are The memory device 415 communicates with the CPU 405 via a communication bus (solid line) such as a board. The device may be a data store (or data repository) for storing data. The computer system 401 communicates with a computer network using a communication interface 420. The network may be operatively coupled to a network ("network") 430. 430 refers to the Internet, the Internet and / or an extranet, or It can be an intranet and / or an extranet that communicates with the Internet. In some cases, the network 430 may be a telecommunications and / or data network. The network 430 is a distributed computing network such as cloud computing. The system may include one or more computer servers capable of enabling routing. In some cases, the network 430 may be assisted by the computer system 401. A peer-to-peer network can be implemented by a computer system 4 Enables devices bound to 01 to act as either clients or servers It is possible.

[0119]

[0129] The CPU 405 may be implemented as a program or software. The instructions may be stored in a memory location, such as memory 410. The instructions may be directed to the CPU 405, which then executes the steps of the present disclosure. The CPU 405 can be programmed or configured to implement the method. Examples of operations performed by a 405 are fetch, decode, execute, and writeback. may include:

[0120]

[0130] The CPU 405 may be part of a circuit such as an integrated circuit. One or more other components of the circuit may be included in the circuit. The path is an application specific integrated circuit (ASIC).

[0121]

[0131] The storage device 415 stores files such as drivers, libraries, and stored programs. The storage unit 415 can store user data, such as user preferences and In some cases, the computer system 40 may store user programs. 1 communicates with a computer system 401 via an intranet or the Internet The present invention relates to a single server that is external to the computer system 401, such as a server that is located on a remote server that It may also include one or more additional data storage units.

[0122]

[0132] The computer system 401 communicates with one or more For example, the computer system The system 401 is a remote computer system of a user (e.g., a portable PC, a tablet, It can communicate with remote computer systems (mobile PCs, smartphones, etc.). Examples include personal computers (e.g., portable PCs), slates or tablets. PC (e.g., Apple's iPad, Samsung (registered trademark), phones, smartphones (e.g., Apple (registered trademark) Galaxy Tab), ) iPhone (registered trademark), Android compatible devices, Blackberry ( A user can access the computer via a network 430. The computer system 401 is accessible.

[0123]

[0133] The methods described herein may be implemented using a variety of storage devices, such as memory 410 or electronic storage device 415. Such a computer system 401 may include a machine (e.g., a computer The present invention may be implemented by machine executable code or by a processor executable code. The machine-readable code may be provided in the form of software. During use, the code In some cases, the code may be executed by processor 405. 415 and stored in memory 410 for easy access by In some circumstances, the electronic storage device 415 may be omitted and the Executable instructions are stored in memory 410 .

[0124]

[0134] The code may be used in a machine having a processor adapted to execute the code. It may be precompiled and configured for use, or compiled during run-time. The code may be executed in either a precompiled or compile-time form. The method may be provided in a programming language that the user can choose to use.

[0125]

[0135] The systems and methods provided herein, such as computer system 401 Aspects of the present technology may be embodied in programming. Typically machine (or processor) executable code and / or some type of machine-readable medium "Products" in the form of related data carried on or embodied in the The machine executable code can be considered an "article of manufacture." memory, random access memory, flash memory) or hard disks The "storage" type of media can be stored in a computer, processor, Tangible memory such as processors, or various semiconductor memories, tape drives, disk drives It can include any or all of the associated modules, such as You may provide non-transient storage for software programming at any time. The Software may be transmitted in whole or in part via the Internet or various other telecommunications networks. Such communication may be, for example, via a computer or processor. from the processor to another computer or processor, such as a management server or host computer. Transfer of software from the computer to the application server computer platform It may be possible to load software elements. Another type of medium is over wired and optical terrestrial networks, and over various air links. The optical, electrical, and This includes waves and electromagnetic waves. The physical elements that carry such waves, e.g. wires or wireless links, A network, optical link, etc. may also be considered as a medium bearing the software. , computer or machine "readable" unless limited to non-transitory, tangible "storage" media The terms "medium" and "processor" refer to any medium that participates in providing instructions to a processor for execution. Point to the body.

[0126]

[0136] Thus, a machine-readable medium such as a computer-executable code may be considered a tangible storage medium. This may take many forms, including but not limited to a body, a carrier wave medium, or a physical transmission medium. The non-volatile storage medium may, for example, implement a database or the like as shown in the drawings. Any storage device, such as any computer, that may be used for A volatile storage medium includes a hard disk or a magnetic disk. Tangible transmission media includes dynamic memory such as the main memory of a computer system. This includes wires that contain buses within a system, as well as coaxial cables such as copper wires and optical fibers. Wave transmission media are electric or electromagnetic signals, or radio frequency (RF) and infrared (IR) signals. R) may take the form of sound waves or light waves such as those generated during data communications. Thus Common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROM, any other optical media, punch card paper, having a pattern of holes Any other physical storage medium, RAM, ROM, PROM and EPROM, FLASH- EPROM, any other memory chip or cartridge, that carries data or instructions A carrier wave, a cable or link carrying such a carrier wave, or a computer This includes any other medium from which programming code and / or data can be read. Many of these forms of computer readable media include one or more instructions for execution. The method may involve conveying one or more sequences of the above to a processor.

[0127]

[0137] The computer system 401 may include: (i) determining whether a sensor, such as a photosensitive sensor, is detected; (ii) the percentage of the area of ​​the well or the percentage of cells in the well that emit the sensor (iii) system settings, such as the intensity of the light source or the type of light source used, or A user interface (UI) 44 configured to provide or receive other information 0. The UI may include or communicate with an electronic display 435. Examples include, but are not limited to, graphical user interfaces (GUIs) and web A base user interface is included.

[0128]

[0138] The methods and systems of the present disclosure are implemented by one or more algorithms. The algorithm may be implemented in software when executed by the central processing unit 405. For example, the algorithm may be implemented by (i) a detection assembly (ii) comparing the acquired information with information stored in a database of the system; It is possible to check the presence or absence of sensors such as sensors.

[0129] kit

[0139] The kits may include instructions for use and the systems described herein, e.g., detection assemblies. The kit may include a system including a lighting assembly and a lighting assembly. ray, one or more sensors (e.g., light-detectable sensors), or a combination thereof. It may further include.

[0130] Example 1: Screening for expression ion channel targets in non-excitable cells

[0140] Intracellular calcium fluorescent indicator Fluo4 and photosensitive actuator CHOP2 and multiple HEK293 cells engineered to express voltage-gated calcium channels. Add multiple HEK293 cells containing Fluo4 indicator to a microplate. The molecules are added to some of the wells of the microplate. The HEK293 cells in the wells were simultaneously illuminated with a light source having a wavelength of 494 nanometers (nm). The stimulation is provided by the lighting assembly. The answer is that the fluorescence of Fluo-4 indicator at 516 nm is detected simultaneously. The photodiode in the detection assembly detects the fluorescent emission. The length or time profile may be determined by determining whether a molecule is transduced into one or more calcium ion channels. These results support the idea that the compound acts on a global voltage-dependent structure.

[0131] Example 2: Screening for expression ion channel targets in non-excitable cells

[0141] The cell membrane potential indicator BeRST, the photosensitive actuator CHOP2, and the A complex engineered to express the sodium channel Nav1.7, a potential-gated ion channel Add a number of HEK293 cells containing the BeRST indicator. The molecules are added to some of the wells of the microplate. The HEK293 cells in each well are then imaged with a 658 nanometer (nm) wavelength. The actuators are stimulated simultaneously with a light source having 460 nm light pulses that independently activate the actuators. This induces transient repetitive activation of the sodium channel Nav1.7. The cell response in each well was measured using BeRS at a wavelength of 683 nm. The emission of the T indicator is monitored simultaneously. The diode detects the fluorescence emission. The magnitude or time profile of the fluorescence emission is The effect of the β-catenin on one or more transient voltage-dependent structures of the voltage-gated sodium ion channel Make sure you use it.

[0132] Example 3: Pharmacological evaluation of electrically excitable cells - spontaneously active cells

[0142] The voltage-sensitive dye VF2.1.C1 was applied to multiple cardiomyocytes differentiated from iPS cells. Multiple cardiomyocytes containing the VF2.1.C1 indicator are added to the wells of a microplate. Compounds are added to some of the wells of the microplate. Each well is then The cardiac muscle cells in the stimulator are stimulated simultaneously with a light source having a wavelength of 460 nanometers (nm). Excitation is provided by the illumination assembly. Cell responses in each well are monitored at a wavelength of 516 nm. The detection assay is monitored simultaneously by detecting the luminescence of the VF2.1.C1 indicator. The photodiode in the assembly detects the fluorescence emission. Submillisecond-scale perturbations in the It is used to predict potential target tissue toxicity.

[0133] Example 4: Electrically excitable cells - Pharmacological evaluation of optically paced excitable cells

[0143] Multiple cardiomyocytes were differentiated from induced pluripotent stem (IPS) cells and showed light-responsive actuators. Eta protein, engineered to express chop2. Voltage-sensitive dye BeRST Add the BeRST indicator to multiple cardiomyocytes. Multiple cardiomyocytes expressing peptide 2 are split into wells of a microplate. The cardiomyocytes in each well are then added to a portion of the wells of the plate. The first pacing light is stimulated simultaneously with a timed pulse of the second pacing light. Both the pacing and the second pacing occur at a first wavelength of 460 nm, while the A second excitation wavelength of 100 nm is provided to excite the BeRST indicator. The cell response in each well was measured using BeRST at a wavelength of 690 nm. The fluorescence of the indicator is monitored simultaneously by detecting the emission of the indicator. The perturbations in response to two different pacing conditions are detected by an automatic heuristic algorithm. and are used to predict potential heart rate-dependent target tissue toxicity.

[0134] Example 5: Safety pharmacology assays on spontaneously beating cardiomyocytes

[0144] The induced pluripotent stem cell-derived cardiomyocytes were cultured for 384 h until spontaneous beating activity was observed. The cells were grown in well microtiter plates. The cells were stained with voltage-sensitive dyes. As shown in a, the hERG inhibitor, E4031, was administered at various concentrations (400 nM, 100 nM, 25nM, 6.3nM, 1.6nM, 0nM) in a 384-well microtiter plate The change in fluorescence emission of the voltage-sensitive dye was monitored over a period of 20 seconds at 37 °C. All 84 wells were simultaneously collected. Action potentials were extracted from the collected data using a proprietary software. The action potential duration at 90% repolarization (APD90) was measured. The dose-response curves were then measured and converted to the dose-response of the hERG inhibitor, E4031, as shown in FIG. 6b.

[0135] Example 6: Dual wavelength stimulation and monitoring of sodium channel activity

[0145] HEK293 cells express Nav1.7 sodium to control the resting membrane potential. channels, photosensitive actuator ion channels, and auxiliary potassium ion channels. The cell monolayers in microtiter plates were visualized with voltage-sensitive fluorescent dyes. As shown in Figure 7, the voltage-sensitive fluorescent dye was excited and the emitted fluorescence was measured near the The cell membrane potential is monitored at infrared wavelengths, while a pulse of blue light partially depolarizes the cell membrane potential, resulting in a fluorescent readout. Transient sodium channel activity and action potential voltage traces observed in the extrusion Voltage traces from patch-clamped cells in a monolayer are shown for comparison. can be.

[0136] Example 7: Simultaneous multi-layer acquisition and automated characterization of cardiomyocyte action potentials using near-infrared voltage-sensing dyes Signing

[0146] Spontaneously beating cardiomyocytes stained with near-infrared voltage-sensitive dyes were visualized in 384-well plates. The cells were grown in single-cell microtiter plates and continuously illuminated with 660 nm light. As shown in -b, fluorescence emission was recorded for 20 s, with the onset of each action potential (solid vertical line) and The end (dotted vertical line) was detected using a proprietary algorithm. It took about 353 ± 14 milliseconds (m The decrease in action potential duration at 90% repolarization (APD90) in sec and the decrease in ) The reduction in the pulsation period of about 79.8 beats was approximately 531 ± 3 beats in the control group (Figure 8b). 7 ms and approximately 49.8 bpm, compared with the tachycardia-inducing drug nifedipine (A dalata®, Procardia®, CAS number 21829- 25-4) (Figure 8a). Two of the traces are displayed.

[0137] Example 8: Dual wavelength recording and pacing of cardiomyocyte action potentials

[0147] Cardiomyocytes were transfected with HEK293 cells expressing the light-gated bacterial channel rhodopsin. The cells were co-cultured with β-lactams in microtiter plates and stained with near-infrared voltage-sensitive dyes. As shown in Fig. 9a-b, low-frequency spontaneous action potentials were recorded (Fig. 9a), followed by the manipulation of the cardiomyocytes. Approximately 460 nanometers (nm) 0.5 Hz pulses were detected via electrical coupling of HEK293 cells. Action potentials were recorded by a train of single pulses (FIG. 9b).

[0138] Example 9: Simultaneous multi-layer dual-wavelength stimulation using near-infrared voltage-sensing dyes and sodium channels Activity Record

[0148] Bacterial channels in combination with the human Nav1.7 voltage-gated sodium channel HEK293 cells engineered to express rhodopsin were cultured in microtiter plates. The cells were grown in PBS and stained with near-infrared voltage-sensitive dyes. To stimulate electrical potentials, about 660 nanometer (nm) light and about 460 nm light are used. Wells were sequentially irradiated with 10 ms pulses at 4 Hz (Figure 10a). Two of the 96 wells The traces of the simultaneously measured fluorescence traces are shown in Figure 10b-c.

[0139]

[0149] Although preferred embodiments of the present invention have been shown and described herein, such implementations It will be apparent to one skilled in the art that the embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. Although the present invention has been described with reference to the above, the description and examples of the embodiments herein are not intended to be construed in a limiting sense. It is understood that those skilled in the art will appreciate that numerous variations are possible without departing from the invention. In addition, all aspects of the present invention may be modified in various ways. The invention is not limited to the specific depictions, configurations or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein are possible. It is understood that alternatives may be used in practicing the present invention. It is intended to cover all such alternatives, modifications, variations, or equivalents. The claims define the scope of the invention and within the scope of these claims, and structures and their equivalents are intended to be covered thereby.

Claims

1. 1. A method for screening for biological activity of a molecule or salt thereof in a plurality of cells, comprising: (a) providing a well array comprising a plurality of wells, each well of the plurality of wells comprising a cell of the plurality of cells, at least a portion of the plurality of cells comprising a sensor; (b) adding the molecule or a salt thereof to at least some of the plurality of wells; (c) directing light from an excitation source at each well of the plurality of wells to illuminate at least a portion of each well of the plurality of wells to form an at least partially illuminated well; and (d) detecting the presence or absence of activation of the sensor in each well of the plurality of wells, the detection occurring substantially in parallel and at a sampling rate across the plurality of wells greater than about 100 Hz; A method comprising:

2. The method of claim 1 , wherein the sensor comprises a light-detectable sensor.

3. The method of claim 2 , wherein the optically detectable sensor comprises a sensor that detects changes in at least one of: (i) cell membrane potential; (ii) intracellular or extracellular ion concentration; and (iii) protein structure.

4. The method of claim 2 , wherein the optically detectable sensor is located within a cell or on a cell surface.

5. The method of claim 2 , wherein the optically detectable sensor is disposed within a medium in which cells may be cultured.

6. The method of claim 2 , wherein the plurality of cells comprises cardiomyocytes.

7. The method of claim 6, wherein the screening is a cardiomyocyte safety pharmacology screening.

8. The method of claim 6, wherein the screening is a hERG screening.

9. The method of claim 2 , wherein the detecting step comprises monitoring fluorescent emissions from the sensor.

10. 10. The method of claim 9, wherein the monitoring step comprises measuring a fluorescent trace corresponding to an action potential.

11. The method of claim 1 , wherein the plurality of wells is an array of the wells.

12. The method of claim 1 , wherein the sampling rate is greater than about 1,000 Hz.

13. A system for screening the biological activity of a molecule or a salt thereof in a plurality of cells, comprising: (a) a well array comprising a plurality of wells, each well of the plurality of wells comprising one cell of the plurality of cells, at least a portion of the plurality of cells comprising a sensor, and at least a portion of the plurality of wells having the molecule or salt thereof added thereto; (b) an illumination assembly that directs light from an excitation source to each well of the plurality of wells to illuminate at least a portion of each well of the plurality of wells; and (c) a detector that detects the presence or absence of activation of the sensor in each well of the plurality of wells, wherein detection by the detector occurs substantially in parallel and a sampling rate across the plurality of wells is greater than about 100 Hz; Including, the system.

14. The system described in claim 13, wherein the sensor includes a light-detectable sensor.

15. The system described in claim 14, wherein the optically detectable sensor includes a sensor that detects changes in at least one of (i) cell membrane potential, (ii) intracellular or extracellular ion concentration, and (iii) protein structure.

16. The system described in claim 14, wherein the optically detectable sensor is positioned inside or on the surface of a cell.

17. The system described in claim 14, wherein the optically detectable sensor is positioned within a medium in which cells may be cultured.

18. The system described in claim 14, wherein the plurality of cells includes cardiomyocytes.

19. The system described in claim 13, wherein the screening is a cardiomyocyte safety pharmacology screening.

20. The system described in claim 13, wherein the screening is hERG screening.

21. The system described in claim 14, wherein the optically detectable sensor monitors fluorescent emissions from the optically detectable sensor.

22. The system of claim 21, wherein the monitoring is performed by measuring a fluorescent trace corresponding to an action potential.

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