Method and System for Drug Evaluation

Abstract

The present application discloses a method and a system for drug evaluation. Among them, the method includes: obtaining original imaging data corresponding to a target area under electric field stimulation, wherein the target area includes a plurality of cells to be tested cultured with a candidate drug, and the plurality of cells to be tested have fluorescence labels; determining a target fluorescence change commonly corresponding to the plurality of cells to be tested from the original imaging data, wherein the target fluorescence change is used to quantitatively represent the average change degree of the membrane potential of the plurality of cells to be tested under electric field stimulation; evaluating the candidate drug according to the target fluorescence change to obtain an evaluation result. The present application solves the technical problem that the drug screening method in the related art has low evaluation efficiency due to the limitation of throughput in the evaluation of cell electrophysiological characteristics.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering technology. Specifically, it relates to a method and system for drug evaluation. Background Art

[0002] The patch clamp technique is a traditional method for evaluating the electrophysiological characteristics of cells. It can apply current stimulation to cells or clamp the cell membrane potential, and can detect the voltage and current values of the entire cell membrane or even a single channel, with high accuracy and time resolution. With the development of biomedical engineering, the patch clamp has gradually evolved from initially clamping and recording a single cell to dual-channel, four-channel, or even multi-channel patch clamp recording. However, when performing electrophysiological detection on a large number of cells, the patch clamp technique is still limited by throughput, resulting in too low efficiency of drug screening and evaluation.

[0003] In response to the above problems, no effective solution has been proposed yet. Summary of the Invention

[0004] The embodiments of this application provide a method and system for drug evaluation to at least solve the technical problem that the drug screening method in the related art has low evaluation efficiency due to being limited by throughput in the evaluation of cell electrophysiological characteristics.

[0005] According to one aspect of the embodiments of this application, a method for drug evaluation is provided, including: obtaining original imaging data corresponding to a target area under electric field stimulation, where the target area includes a plurality of cells to be measured cultured with a candidate drug, and the plurality of cells to be measured have fluorescence labels; determining a target fluorescence change commonly corresponding to the plurality of cells to be measured from the original imaging data, where the target fluorescence change is used to quantitatively represent the average change degree of the membrane potential of the plurality of cells to be measured under electric field stimulation; evaluating the candidate drug based on the target fluorescence change to obtain an evaluation result.

[0006] In some embodiments of this application, determining a target fluorescence change commonly corresponding to the plurality of cells to be measured from the original imaging data includes: segmenting the plurality of cells to be measured in the original imaging data to obtain cell areas respectively corresponding to the plurality of cells to be measured; determining a first fluorescence change corresponding to each cell area from the original imaging data, where the first fluorescence change is used to quantitatively represent the response degree of the cell area to electric field stimulation within a preset statistical period under electric field stimulation; determining a target fluorescence change commonly corresponding to the plurality of cells to be measured based on the first fluorescence changes respectively corresponding to all cell areas.

[0007] In some embodiments of the present application, determining the first fluorescence change corresponding to each cell region from the original imaging data includes: determining the second fluorescence change corresponding to each cell region, where the second fluorescence change is used to quantitatively represent the trend of the fluorescence intensity in the cell region changing with time; determining the background region corresponding to each cell to be measured based on each cell region, and determining the third fluorescence change corresponding to the background region, where the third fluorescence change is used to quantitatively represent the trend of the fluorescence intensity in the surrounding region of each cell region changing with time; and determining the first fluorescence change according to the second fluorescence change and the third fluorescence change.

[0008] In some embodiments of the present application, determining the background region corresponding to each cell to be measured based on each cell region includes: expanding each cell region outward by a first number of pixels to obtain the first region corresponding to each cell region; determining the region in the original imaging data except for all the first regions as the second region; based on the second region, expanding each cell region outward by a second number of pixels to obtain the third region corresponding to each cell region, where the first number is less than the second number; and determining the intersection of the second region and the third region as the background region.

[0009] In some embodiments of the present application, the original imaging data includes multiple frames of imaging data. Segmenting multiple cells to be measured in the original imaging data includes: statistically averaging the fluorescence intensities at each time point within a preset statistical period in the multiple frames of imaging data to obtain the average value projection corresponding to the original imaging data, where the average value projection is an image formed after statistical averaging; and segmenting multiple cells to be measured in the average value projection.

[0010] In some embodiments of the present application, after obtaining the cell region corresponding to each cell to be measured, the method further includes: comparing the attribute information of the cell region with a first preset condition to obtain a first comparison result, where the first preset condition is used to screen the size and / or brightness of the cell region; and determining the cell region as an invalid region when the first comparison result indicates that the attribute information does not meet the first preset condition.

[0011] In some embodiments of the present application, determining the target fluorescence change commonly corresponding to multiple cells to be measured according to the first fluorescence changes respectively corresponding to all cell regions includes: comparing the first fluorescence change with a second preset condition to obtain a second comparison result, where the second preset condition is used to screen the response degree of the cells to be measured corresponding to the cell region to the electric field stimulation; adding the cells to be measured corresponding to the cell region to the target cell set when the second comparison result indicates that the first fluorescence change meets the second preset condition; and determining the target fluorescence change according to the first fluorescence changes of the cell regions corresponding to the cells to be measured in the target cell set.

[0012] In some embodiments of the present application, acquiring the original imaging data corresponding to the target area under electric field stimulation includes: moving the stage for carrying a plurality of cells to be tested to the first position corresponding to the target area, where the first position is above the microscope objective; lowering the electrode to the second position, where the electrode contacts the upper surfaces of the plurality of cells to be tested at the second position; under the illumination of the light source, performing electric field stimulation on the plurality of cells to be tested using the electrode; acquiring the original imaging data corresponding to the plurality of cells to be tested acquired by the image acquisition device under electric field stimulation.

[0013] In some embodiments of the present application, a plurality of cells to be tested are located in any one of the wells of the target well plate. Moving the stage for carrying the plurality of cells to be tested to the first position corresponding to the target area includes: receiving a selection instruction of the target object, where the selection instruction includes the well number of any one well corresponding to the target well plate; acquiring the size parameters of the target well plate, where the size parameters include the center distance between adjacent wells; determining the third position corresponding to the stage as the coordinate origin, and determining the coordinate information corresponding to the well number according to the center distance between wells; based on the third position, moving the area corresponding to the well number to the first position according to the coordinate information.

[0014] In some embodiments of the present application, before moving the stage for carrying a plurality of cells to be tested to the first position corresponding to the target area, the method further includes: comparing the fourth position corresponding to the electrode with a third preset condition to obtain a third comparison result, where the third preset condition is used to detect whether the electrode meets the movement requirement; in the case where the third comparison result indicates that the fourth position does not meet the third preset condition, raising the electrode from the fourth position to the fifth position so that the electrode is disengaged from the plurality of cells to be tested, where the vertical distance between the fifth position and the second position meets a preset threshold.

[0015] In some embodiments of the present application, at least one well of the target well plate has a plurality of cells to be tested cultured under a candidate drug, and each well corresponds to at least one candidate drug, where the target well plate includes a 24-well plate and / or a 96-well plate and / or a 384-well plate.

[0016] According to another aspect of the embodiments of the present application, there is also provided a system for drug evaluation, including: an imaging subsystem, electrodes, and a processor. Among them, the imaging subsystem includes a light source and an image acquisition device. The light source is connected to the processor and is used to provide illumination conditions for electrode stimulation before the start of electrode stimulation; the electrodes are connected to the processor and are used to perform electric field stimulation on multiple cells to be measured cultured in a candidate drug in a target area, where the multiple cells to be measured have fluorescent labels; the image acquisition device in the imaging subsystem is connected to the processor and is used to collect the original imaging data corresponding to the target area under electric field stimulation and send the original imaging data to the processor; the processor is connected to the imaging subsystem and is used to receive the original imaging data and determine the target fluorescence change commonly corresponding to the multiple cells to be measured from the original imaging data, where the target fluorescence change is used to quantitatively represent the average change degree of the membrane potential of the multiple cells to be measured under electric field stimulation; evaluate the candidate drug according to the target fluorescence change to obtain an evaluation result.

[0017] In some embodiments of the present application, the system further includes a mobile device. Among them, the mobile device includes an electric motor. The electric motor is respectively connected to the controller and the electrodes and is used to receive the instructions sent by the controller and control the electrodes to move in the Z-axis direction of the coordinate axis according to the instructions; the mobile device further includes a stage. The stage is connected to the processor and is used to carry the target well plate and move the designated area in the target well plate to the target area.

[0018] In some embodiments of the present application, the system further includes a stimulation isolator. Among them, the stimulation isolator is connected to the electrodes and is used to isolate and amplify the voltage signal sent by the electrodes and output a pulsed voltage to the multiple cells to be measured.

[0019] In some embodiments of the present application, the processor is further used for: segmenting the multiple cells to be measured in the original imaging data to obtain the cell regions respectively corresponding to the multiple cells to be measured; determining the first fluorescence change corresponding to each cell region from the original imaging data, where the first fluorescence change is used to quantitatively represent the response degree of the cell region to electric field stimulation within a preset statistical period under electric field stimulation; determining the target fluorescence change commonly corresponding to the multiple cells to be measured according to the first fluorescence changes respectively corresponding to all cell regions.

[0020] In some embodiments of the present application, the processor is further used for: determining the second fluorescence change corresponding to each cell region, where the second fluorescence change is used to quantitatively represent the change trend of the fluorescence intensity within the cell region over time; determining the background region corresponding to each cell to be measured based on each cell region and determining the third fluorescence change corresponding to the background region, where the third fluorescence change is used to quantitatively represent the change trend of the fluorescence intensity in the surrounding region of each cell region over time; determining the first fluorescence change according to the second fluorescence change and the third fluorescence change.

[0021] According to another aspect of the embodiments of the present application, a device for drug evaluation is further provided, including: an acquisition module, configured to acquire original imaging data corresponding to a target region under electric field stimulation, wherein the target region includes a plurality of cells to be measured cultured with a candidate drug, and the plurality of cells to be measured have fluorescence labels; a determination module, configured to determine a target fluorescence change commonly corresponding to the plurality of cells to be measured from the original imaging data, wherein the target fluorescence change is used to quantitatively represent the average change degree of the membrane potentials of the plurality of cells to be measured under electric field stimulation; an evaluation module, configured to evaluate the candidate drug according to the target fluorescence change to obtain an evaluation result.

[0022] According to another aspect of the embodiments of the present application, a non-volatile storage medium is further provided, and the non-volatile storage medium includes a stored computer program, wherein the device where the non-volatile storage medium is located executes the above-mentioned drug evaluation method by running the computer program.

[0023] According to another aspect of the embodiments of the present application, a computer program product is further provided, including computer instructions, and when the computer instructions are executed by a processor, the above-mentioned drug evaluation method is implemented.

[0024] In the embodiments of the present application, by adopting electric field stimulation and fluorescence labeling techniques, the differences in cell potential changes caused by different drugs under electric field stimulation are recorded through high throughput, and drugs related to ion channels are evaluated, achieving the purpose of electrophysiological detection and optical recording of a large number of cells synchronously, thereby realizing the technical effects of reducing the complexity of experimental operations and improving the efficiency of drug screening and evaluation, and further solving the technical problem that the drug screening method in the related art has low evaluation efficiency due to the limitation of throughput in the evaluation of cell electrophysiological characteristics. Description of the Drawings

[0025] The drawings described herein are used to provide a further understanding of the present application, and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation of the present application. In the drawings:

[0026] Figure 1 is a hardware structure block diagram of a computer terminal for a drug evaluation method according to an embodiment of the present application;

[0027] Figure 2 is a flowchart of a drug evaluation method according to an embodiment of the present application;

[0028] Figure 3 is an overall flowchart of a drug evaluation method according to an embodiment of the present application;

[0029] Figure 4Schematic diagram of cell segmentation in a target area of a drug evaluation method according to an embodiment of the present application;

[0030] Figure 5 Schematic diagram of the background area in a target area of a drug evaluation method according to an embodiment of the present application;

[0031] Figure 6 Schematic diagram of the average fluorescence change and sensitivity distribution of cells in a target area of a drug evaluation method according to an embodiment of the present application;

[0032] Figure 7 Schematic diagram of the distribution of representative areas of fluorescence change of highly responsive cells in a target area of a drug evaluation method according to an embodiment of the present application;

[0033] Figure 8 Flowchart of the operation of a drug evaluation method system according to an embodiment of the present application;

[0034] Figure 9 Schematic diagram of the analysis of the system operation speed of a drug evaluation method according to an embodiment of the present application;

[0035] Figure 10 Schematic diagram of the structure of a drug evaluation device according to an embodiment of the present application. Detailed implementation manners

[0036] In order to enable those skilled in the art to better understand the solution of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0037] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and do not necessarily need to be used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present application described here can be implemented in an order other than those illustrated or described here. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products, or devices.

[0038] To better understand the embodiments of the present application, the technical terms involved in the embodiments of the present application are explained as follows:

[0039] Electric Field Stimulation (EFS): In biomedical research, an external electric field is used to stimulate cells or tissues to study their electrophysiological properties or evaluate the effects of drugs on excitable cells (such as nerve cells and cardiomyocytes). In the present application, it is also referred to as electric field stimulation.

[0040] Fluorophore: A molecule that can absorb light of a specific wavelength and emit fluorescence at a longer wavelength, which is the basis of fluorescent probes and fluorescent dyes.

[0041] Fluorescence Signal: The light signal emitted by biomolecules or cell markers (fluorescent probes) after absorbing light of a specific wavelength, which is used for quantitative analysis of expression levels, activities, and positions in cell biology and biomedical research.

[0042] Membrane Potential: The potential difference generated by the concentration difference of ions inside and outside the cell membrane, which is the basis of cell electrophysiological activities and is crucial for the excitability and signal conduction of nerve, muscle, and glandular cells.

[0043] HEK 293T Cell Line (Human Embryonic Kidney 293T Cell Line, abbreviated as HEK 293T): A laboratory cell line derived from human embryonic kidney cells, which is used for gene expression, protein production, and studying cell signal transduction and drug action mechanisms.

[0044] In the related art, there are many limitations in the technologies for screening and evaluating ion channel-related drugs. First, the patch clamp technique has extremely low efficiency when dealing with a large number of cell samples because it requires independent electrode clamping and current recording for each cell. Moreover, the patch clamp needs to physically contact the cell and clamp a part of the cell membrane through the electrode, which causes certain mechanical and electrical interference to the cell and easily leads to changes in the cell physiological state, affecting the accuracy of the results. Second, EEG (Electroencephalography) and EMG (Electromyography) technologies collect electrical signals by placing electrodes on the scalp or skin surface, resulting in their inability to accurately distinguish the electrical signals of specific cell populations and lacking cell-level specificity, that is, having non-cell selectivity. In addition, animal model behavioral experiments often have characteristics such as high cost and long cycle.

[0045] To solve the above technical problems, the embodiments of the present application provide corresponding solutions, which are described in detail below.

[0046] The method embodiments for drug evaluation provided by the embodiments of the present application can be executed on a mobile terminal, a computer terminal, or a similar computing device. Figure 1 The following shows a hardware structure block diagram of a computer terminal for implementing the method for drug evaluation. As Figure 1 shown, the computer terminal 10 may include one or more processors (the processors may include, but are not limited to, processing devices such as a microprocessor MCU or a programmable logic device FPGA, shown as 102a, 102b,..., 102n in the figure), a memory 104 for storing data, and a transmission module 106 for communication functions connected by wired and / or wireless networks. In addition, it may further include: a display, a keyboard, a cursor control device, an input / output interface (I / O interface), a universal serial bus (USB) port (which may be included as one of the ports of the I / O interface), a network interface, and a BUS bus. Those of ordinary skill in the art can understand that Figure 1 the structure shown is only schematic and does not limit the structure of the above-mentioned electronic device. For example, the computer terminal 10 may further include more or fewer components than Figure 1 shown, or have a different configuration from Figure 1 shown.

[0047] It should be noted that the above one or more processors and / or other data processing circuits are generally referred to as "data processing circuits" in this article. The data processing circuit may be embodied in software, hardware, firmware, or any combination thereof, in whole or in part. In addition, the data processing circuit may be a single independent processing module, or be incorporated in whole or in part into any one of the other elements in the computer terminal 10. As involved in the embodiments of the present application, the data processing circuit is a kind of processor control (such as the selection of a variable resistance terminal path connected to an interface).

[0048] The memory 104 can be used to store software programs and modules of application software, such as the program instructions / data storage devices corresponding to the method for drug evaluation in the embodiments of the present application. The processor executes various functional applications and data processing by running the software programs and modules stored in the memory 104, that is, implements the above-mentioned method for drug evaluation. The memory 104 may include a high-speed random access memory, and may also include a non-volatile memory, such as one or more magnetic storage devices, flash memories, or other non-volatile solid-state memories. In some instances, the memory 104 may further include a memory remotely set relative to the processor, and these remote memories can be connected to the computer terminal 10 through a network. Examples of the above-mentioned network include, but are not limited to, the Internet, an enterprise intranet, a local area network, a mobile communication network, and their combinations.

[0049] The transmission module 106 is used to receive or send data via a network. Specific examples of the above-mentioned network may include a wireless network provided by a communication provider of the computer terminal 10. In one example, the transmission module 106 includes a network adapter (Network Interface Controller, NIC), which can be connected to other network devices through a base station so as to communicate with the Internet. In one example, the transmission module 106 can be a Radio Frequency (RF) module, which is used to communicate with the Internet wirelessly.

[0050] The display can be, for example, a touch-screen liquid crystal display (LCD), which enables a user to interact with the user interface of the computer terminal 10.

[0051] It should be noted here that in some alternative embodiments, the above Figure 1 illustrated computer terminal may include hardware elements (including circuits), software elements (including computer code stored on a computer-readable medium), or a combination of both hardware elements and software elements. It should be pointed out that Figure 1 is only an example of a specific specific instance and is intended to illustrate the types of components that may exist in the above computer terminal.

[0052] Under the above operating environment, an embodiment of a method for drug evaluation is provided in an embodiment of the present application. It should be noted that the steps shown in the flowchart of the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although the logical order is shown in the flowchart, in some cases, the steps shown or described can be executed in a different order than here.

[0053] Figure 2 is a flowchart of a method for drug evaluation according to an embodiment of the present application. As Figure 2 shown, the method includes the following steps:

[0054] Step S202, obtaining original imaging data corresponding to a target area under electric field stimulation, where the target area includes a plurality of cells to be tested cultured under a candidate drug, and the plurality of cells to be tested have fluorescence labels.

[0055] In the above step S202, in a high-throughput drug screening experiment, the target area specifically refers to the cell culture area in one or more well positions of a microplate, and these areas contain cell populations for testing candidate drugs.

[0056] Multiple cells to be measured with fluorescence labeling means using fluorescent probes or fluorescent proteins to label cells, enabling the visualization of specific physiological states of cells (such as membrane potential changes) under an optical microscope, and reflecting the response of cells to electrical field stimulation through the intensity change of fluorescence signals.

[0057] In some embodiments of the present application, the original imaging data corresponding to the target area under electrical field stimulation can be obtained through the following steps. Specifically: move the stage for carrying multiple cells to be measured to the first position corresponding to the target area, where the first position is above the microscope objective; lower the electrode to the second position, where the electrode is in contact with the upper surface of the multiple cells at the second position; under the illumination of the light source, use the electrode to apply electrical field stimulation to the multiple cells; obtain the original imaging data corresponding to the multiple cells collected by the image acquisition device under electrical field stimulation.

[0058] The stage is used to carry samples (such as cell culture well plates) and can move in the x-axis and y-axis directions of the coordinate axis according to experimental requirements to accurately locate to the target area. The first position refers to the position where the stage moves directly above the microscope objective. For example, it is the starting position of the stage when preparing to collect the fluorescence signal of cells in a specific well, ensuring that the objective can be directly facing the target area and providing stable and accurate focusing for high-resolution imaging. In some embodiments of the present application, the stage can be controlled to move to the preset first position through the interface between the MATLAB environment and the stage driver, and this first position ensures that the microscope objective is directly facing the target area.

[0059] The electrode is a device used to generate an electrical field and apply it to cells. For example, it can be made of conductive materials such as platinum electrodes. By contacting the electrode with the cells, an electrical field can be formed around the cells, thereby simulating the scenario of cells being stimulated and evaluating the effects of candidate drugs on the electrophysiological characteristics of cells. In some embodiments of the present application, MATLAB drives through the controller to lower the electrode to the second position in contact with the upper surface of the cells, ensuring that the electrical stimulation can act precisely on the target cells and avoiding uneven electrical field distribution caused by improper electrode position.

[0060] The light source refers to the light source that provides sufficient brightness during microscope imaging to observe fluorescently labeled cells, and it can be an LED light source or a laser light source. In some embodiments of the present application, the MATLAB environment can control both electrode stimulation and light source illumination simultaneously, so that while the electrode applies electrical field stimulation, the light source provides stable illumination, ensuring that the image acquisition device can record clear fluorescence signals during the stimulation process. For example, MATLAB controls the light source output through a data acquisition card, and at the same time, outputs an electrode stimulation command and outputs a pulsed voltage through a stimulation isolator. The two are precisely synchronized through TTL (Transistor-Transistor Logic) signals.

[0061] When the electrode starts to stimulate, the image acquisition device is triggered by the data acquisition card and starts to synchronously acquire data according to parameters such as the preset sampling rate, exposure time, and bin value (binning, pixel merging method), obtaining the original imaging data.

[0062] To avoid experimental errors caused by improper electrode position, before moving the stage for carrying multiple cells to be measured to the first position corresponding to the target area, the position of the electrode can also be detected to see if it meets the movement requirements. Specifically: compare the fourth position corresponding to the electrode with the third preset condition to obtain the third comparison result, where the third preset condition is used to detect whether the electrode meets the movement requirements; in the case where the third comparison result indicates that the fourth position does not meet the third preset condition, raise the electrode from the fourth position to the fifth position so that the electrode is disengaged from contact with the multiple cells to be measured, where the vertical distance between the fifth position and the second position meets the preset threshold.

[0063] Since the electrode needs to dive at least to the position where it touches the upper surface of the cell during stimulation, after the electric field stimulation, the electrode may still be in or close to the position in contact with the cell. The fourth position refers to the actual position of the electrode after the stimulation is completed.

[0064] The preset threshold refers to the vertical distance required for the electrode to move from the second position to the fifth position after stimulation, which is set to avoid damage to the cells or contact with the well plate during the movement of the electrode. In some embodiments of the present application, during the experiment, there are situations such as switching fields within a well (switching fields within the same target well) and switching fields between wells (switching the field from the first target well to the second target well). The preset threshold (the first preset threshold) for switching fields within a well is less than the preset threshold (the second preset threshold) for switching fields between wells. The determination of the preset threshold can be based on the time consumed by the electrode displacement distance. A shorter displacement distance can reduce the time consumed during the field switching process.

[0065] In the case where multiple cells to be measured are located in any one of the wells of the target well plate, precise positioning of any well in the target well plate can be achieved through the following steps: receive a selection instruction of the target object, where the selection instruction includes the number of any well corresponding to the target well plate; obtain the size parameters of the target well plate, where the size parameters include the center distance between adjacent wells; determine the third position corresponding to the stage as the coordinate origin, and determine the coordinate information corresponding to the well number according to the center distance between wells; based on the third position, move the area corresponding to the well number to the first position according to the coordinate information.

[0066] The selection instruction of the target object is an instruction provided by the target object through a software interface or a script, which is used to specify the specific well number in the well plate for imaging and stimulation. For example, in the MATLAB environment, the target object can specify the target well by using the GUI (Graphical User Interface) or directly inputting the well number at the command line.

[0067] In some embodiments of the present application, the control program in the MATLAB environment can set the default stage position (the third position) as the coordinate origin (0, 0). When it is necessary to locate to other wells, the position coordinates of the target well relative to the coordinate origin can be calculated according to the target well number and the well center distance. Specifically, after the stage is initialized, in the preset state, the stage will move to the position where the starting well of the target well plate (such as a 96-well plate), for example, the A1 port of the 96-well plate, is located above the objective lens, and set this position coordinate as (0, 0). At this time, the relative coordinates of the center positions of each well can be obtained according to the size parameters of the target well plate and loaded into the coordinate table in the sub-function. Taking a 96-well plate (the bottom diameter is 6.4 mm, and the well center distance is 9 mm) as an example, starting from A1, every other row (the letter increases by 1), the ordinate decreases by 9 mm; starting from A1, every other column (the number increases by 1), the abscissa decreases by 9 mm. When it is desired to switch wells, the number of the well to be imaged (such as "A8", "G12") can be directly input. For example, when moving from the A1 well to the A8 well, the MATLAB program will move the stage 7×9 mm = 63 mm in the y-axis direction according to the well center distance of 9 mm, so as to move the A8 well to the position directly below the microscope objective lens (the first position).

[0068] In some embodiments of the present application, there are multiple cells to be tested cultured in at least one well of the target well plate, and each well corresponds to one treatment condition (such as concentration and time) of at least one candidate drug. Among them, the target well plate includes a 24-well plate and / or a 96-well plate and / or a 384-well plate.

[0069] The target well plate refers to a multi-well plate used for culturing cells to be tested in a high-throughput drug screening experiment. According to the flux requirements of the experiment, the target well plate can be a 24-well plate, a 96-well plate, a 384-well plate, etc. Each well can independently culture cell samples and perform different drug treatments.

[0070] Step S204, determining the target fluorescence change commonly corresponding to multiple cells to be tested from the original imaging data, where the target fluorescence change is used to quantitatively represent the average change degree of the membrane potential of multiple cells to be tested under electric field stimulation.

[0071] In the above step S204, the target fluorescence change is obtained by statistically analyzing the fluorescence changes of the identified cells in the original imaging data, which reflects the overall response of the cell population to the stimulus. The average change degree refers to the overall change amplitude and trend of the fluorescence signal when the cell population undergoes a change in membrane potential caused by an electric field stimulus. For example, by comparing the calculated target fluorescence change with the baseline fluorescence without stimulation, the amplitude of the fluorescence change, i.e., the ΔF / F value, can be obtained, which is used to reflect the degree of change in the membrane potential of the cell under an electric field stimulus or a drug. By averaging the fluorescence change amplitudes ΔF / F of all cells, the target fluorescence change can be obtained. For each stimulation pulse, the peak value of the target fluorescence change can be used to represent the intensity of the response, while the time for the fluorescence signal to return to the baseline can reflect the duration of the response.

[0072] In some embodiments of the present application, the target fluorescence change can be determined through the following steps: segmenting multiple cells to be measured in the original imaging data to obtain cell regions corresponding to the multiple cells to be measured respectively; determining a first fluorescence change corresponding to each cell region from the original imaging data, where the first fluorescence change is used to quantitatively represent the response degree of the cell region to the electric field stimulus within a preset statistical period under the electric field stimulus; and determining the target fluorescence change commonly corresponding to the multiple cells to be measured according to the first fluorescence changes corresponding to all cell regions respectively.

[0073] Cell segmentation is to automatically identify and separate the boundaries of each cell from the original imaging data, decomposing the complex cell population in the image into multiple independent cell regions. In some embodiments of the present application, the cpCyto2 model of Cellpose2 can be used to analyze the original imaging data, automatically identify the boundaries of the cells through computer vision algorithms, and segment them from the background to form independent cell regions. The parameters of the model include the average cell diameter (averageCellDiameter), the cell probability threshold (cellThreshold), and the flow field threshold (flowThreshold), etc. In order to efficiently divide the single cell contour under the cell density and membrane localization conditions and ensure the accuracy of segmentation, the cell threshold parameter and the flow threshold parameter can be equal.

[0074] In some embodiments of the present application, the original imaging data includes multiple frames of imaging data. Before segmenting the multiple cells to be measured in the original imaging data, the following steps can also be performed: statistically averaging the fluorescence intensities at each time point within a preset statistical period in the multiple frames of imaging data to obtain an average value projection corresponding to the original imaging data, where the average value projection is an image formed after statistical averaging; and segmenting the multiple cells to be measured in the average value projection.

[0075] For example, in the MATLAB environment, statistical averaging can be performed on multiple frames of image data within a preset statistical period through data processing functions. Specifically, all the images within the statistical period can be superimposed, and then the fluorescence intensity of each pixel point can be averaged to obtain an average projection image. For example, for a specific statistical period (such as the time window after a single electrical stimulation), all the image frames within this period are superimposed, and then the fluorescence values of each pixel point are averaged to reduce signal noise and fluctuations, resulting in a clearer image of cell fluorescence expression.

[0076] Through average projection, the fluorescence expression of cells within the statistical period can be seen more clearly. Using the cpCyto2 model of Cellpose2 to perform cell segmentation on this image can more accurately identify and separate the boundaries of each cell to be measured. For example, based on the average projection image, the cpCyto2 model can identify cell contours and separate different cell regions from the background, facilitating subsequent calculation of the first fluorescence change.

[0077] After obtaining the cell regions corresponding to each cell to be measured, the following steps can also be performed: comparing the attribute information of the cell regions with a first preset condition to obtain a first comparison result, where the first preset condition is used to screen the size and / or brightness of the cell regions; in the case where the first comparison result indicates that the attribute information does not meet the first preset condition, determining the cell region as an invalid region.

[0078] The attribute information of the cell regions refers to the characteristic data of each cell region obtained through cell segmentation, including but not limited to the size (area) and brightness information of the cell regions. In some embodiments of the present application, in the MATLAB environment, image processing and data analysis functions can be used to measure the attribute information (such as size and brightness) of each cell region and compare it with the preset screening conditions. For example, by calculating the area and average brightness of each cell region, it is determined whether they fall within the preset size and brightness ranges.

[0079] In high-throughput drug screening experiments, it is crucial to accurately and effectively analyze the fluorescence signal changes of each cell to be measured. However, not all cell regions obtained through cell segmentation are valid or suitable for further analysis. Cell regions with too small a size or too low a brightness may be due to the failure of cells to successfully express fluorescent probes or some technical limitations during the imaging process (such as insufficient resolution or background noise).

[0080] Some cell regions are marked as invalid regions because they are too small in size or too low in brightness and do not meet the preset screening conditions. These excluded invalid regions can still play a role in the determination of the subsequent background region. By performing a dilation operation on all the initially segmented cell regions and then taking the inverse, a global background region containing the pixels around all the cell regions can be obtained (for the specific implementation, refer to the description of the second region below). When determining the global background region, the cell regions corresponding to the invalid regions are regarded as normal cell regions, and the dilation and inverse operations are also performed on the invalid regions, so that even when the cell regions are marked as invalid, these regions can still be used to determine the background signal, reducing data misreading caused by inaccurate subtraction of the background signal and ensuring that the calculation of the first fluorescence change and the target fluorescence change is based on pure cell signals.

[0081] The first fluorescence change refers to the change in fluorescence intensity within a preset statistical period under the action of an electric field stimulation for a single cell region, including the true fluorescence intensity change of each cell region after removing the background influence, and is used to quantitatively represent the response degree of this region to the electric field stimulation. In some embodiments of the present application, for each segmented cell region, the average fluorescence intensity change during the electric stimulation process can be calculated, where the preset statistical period is the duration of each electric stimulation plus the time interval between stimulations. For example, under the condition that the stimulation frequency is 3.28 Hz and each stimulation lasts for 10.18 milliseconds, the statistical period will include a complete stimulation and recovery process. By analyzing the change curve of the fluorescence intensity over time within the cell region, the first fluorescence change can be calculated, including parameters such as the peak fluorescence intensity, the amplitude of the fluorescence intensity change, and the recovery time, to evaluate the sensitivity and response degree of the cell to the stimulation.

[0082] In some embodiments of the present application, the first fluorescence change can be determined in the following manner: determine the second fluorescence change corresponding to each cell region, where the second fluorescence change is used to quantitatively represent the change trend of the fluorescence intensity over time within the cell region; determine the background region corresponding to each cell to be measured based on each cell region, and determine the third fluorescence change corresponding to the background region, where the third fluorescence change is used to quantitatively represent the change trend of the fluorescence intensity over time in the surrounding region of each cell region; determine the first fluorescence change according to the second fluorescence change and the third fluorescence change.

[0083] The second fluorescence change refers to the change trend of the internal fluorescence intensity over time for each cell region under the action of an electric field stimulation. The second fluorescence change can be determined by analyzing the change of the average fluorescence intensity over time within each cell region in a preset statistical period. For example, by calculating the change of the average fluorescence intensity of all pixel points within the cell region over time, the second fluorescence change can be obtained.

[0084] When determining the background region corresponding to each cell to be measured, based on the cell regions obtained after cell segmentation, a certain dilation operation (such as using a structuring element) can be used to define the surrounding background region of each cell, and the change trend of the fluorescence intensity of the background region over time is extracted from the original imaging data, that is, the third fluorescence change.

[0085] In some embodiments of the present application, the second fluorescence change can be corrected by subtracting the third fluorescence change (i.e., the fluorescence change of the background region) of each cell region to obtain the first fluorescence change.

[0086] Through two dilation operations, the background region corresponding to each cell to be measured can be determined. Specifically: each cell region is dilated outward by a first number of pixels to obtain a first region corresponding to each cell region; the region in the original imaging data other than all the first regions is determined as the second region; on the basis of the second region, each cell region is dilated outward by a second number of pixels to obtain a third region corresponding to each cell region, where the first number is less than the second number; the intersection of the second region and the third region of each cell is determined as the background region of the cell.

[0087] The first number of pixels refers to the number of pixels for the preliminary dilation operation (the first dilation) of each cell region when determining the background region, which is usually small and is used to preliminarily define the boundary range of the cell to avoid directly including the cell region in the background region. The first region is the region formed after each cell region is dilated outward by the first number of pixels, and this part of the region includes the cell region and the non-cell part near the cell boundary. In some embodiments of the present application, a specific structuring element (such as an octagonal structuring element) can be combined to perform a dilation operation on the cell region to define the preliminary background range near the cell boundary.

[0088] The second number of pixels refers to the number of pixels for the further dilation operation (the second dilation) of each cell region after determining the second region. The second number is greater than the first number and is used to more comprehensively define the background region of each cell to ensure that the background region is large enough to accurately reflect the non-specific fluorescence change. The third region is the region formed after each cell region is dilated outward by the second number of pixels.

[0089] In some embodiments of the present application, the first dilation and the second dilation can use the same structuring element or different structuring elements. Each cell region can use the same structuring element for the dilation operation, and different structuring elements can also be used between multiple cell regions, which is not limited herein. For example, based on the morphological information (shape parameters) of each cell region, such as the aspect ratio, convexity, area, etc., the shape and size of the dilation structuring element can be automatically adjusted. Specifically:

[0090] Step 1: Extract the morphological features of each cell region.

[0091] (1) Load cell region data: Load the contour information of all cell regions from the binary images output by Cellpose2 or other cell segmentation algorithms.

[0092] (2) Calculate morphological features: Calculate feature data such as aspect ratio, convexity, and area for each cell region.

[0093] Step 2: Design and generate shape-adaptive structuring elements.

[0094] (1) Determine the dilation shape: Based on the first feature data of the cell region (such as aspect ratio and orientation), determine the shape of the dilation structuring element. For example, if the cell region is elongated, an elliptical or rectangular structuring element can be used; if the shape of the cell region is more complex, a polygonal structuring element can be considered.

[0095] (2) Calculate dilation parameters: Based on the second feature data of the cell region (such as area and convexity), calculate the size of the structuring element. Larger cells may require larger structuring elements to fully cover their edges, while cells with lower convexity (indicating irregular shapes) may require more refined structuring elements.

[0096] (3) Generate structuring elements: Generate the structuring element corresponding to each cell region based on the dilation shape and dilation parameters.

[0097] Step 3: Perform shape-adaptive dilation operations.

[0098] (1) Apply the structuring element for dilation: For each cell region, perform a dilation operation using the shape-adaptive structuring element generated in Step 2 to preliminarily define the background range.

[0099] (2) Adjust the dilation range: In some embodiments of the present application, the dilation range can be further adjusted according to the brightness and size information of the cell region. For example, cells with lower brightness or smaller sizes may require a larger dilation range to ensure sufficient coverage of the background region.

[0100] The background region is the intersection of the second region and the third region. This part of the region is used to determine the surrounding environment of each cell region for subsequent fluorescence change analysis to subtract non-specific fluorescence changes.

[0101] In a specific embodiment, each segmented cell region can be first dilated outward by 9 pixels (about 15.6 microns) with an octagonal structuring element, and then the remaining region in the full field of view after deducting the dilated cell regions is defined as the global background selection region (i.e., the second region). Based on this global background selection region, the background selection region (i.e., the background region) corresponding to each cell region is defined as the intersection of the octagon formed by dilating itself outward by 108 pixels (about 187.2 microns) with the octagonal structuring element and the global background selection region obtained by dilating all cells outward by 3 pixels respectively, which can reduce the influence of uneven illumination in the imaging field of view on the accuracy of the background subtraction operation. For the background signal (i.e., the third fluorescence change) corresponding to each region, a notch operation with a frequency of 2.5 - 30 Hz and a smoothing process at an interval of 1 stimulation period (60 frames, about 305 milliseconds) can also be used to avoid introducing new noise in the operation of subtracting the background one by one.

[0102] The target fluorescence change can be determined by summing up the first fluorescence changes of all cell regions and using statistical methods such as mean, median, or specific percentiles. For example, the MATLAB program will analyze the fluorescence changes of valid cell regions, then sort them according to the change amplitude or set a threshold, and select the representative change as the target fluorescence change to evaluate the response of the overall cell population to the electric field stimulation.

[0103] In some embodiments of the present application, to improve the data precision and confidence, before determining the target fluorescence change, the first fluorescence change can also be used to screen the cells to be tested (or cell regions) to obtain a set of cells to be tested for determining the target fluorescence change. Specifically: comparing the first fluorescence change with a second preset condition to obtain a second comparison result, where the second preset condition is used to screen the response degree of the cells to be tested corresponding to the cell regions to the electric field stimulation; adding the cells to be tested corresponding to the cell regions to the target cell set when the second comparison result indicates that the first fluorescence change meets the second preset condition; determining the target fluorescence change according to the first fluorescence changes of the cell regions corresponding to the cells to be tested in the target cell set.

[0104] The second preset condition can be set such that the change amplitude of the first fluorescence change exceeds a preset percentage (such as 5%), or the response intensity of the first fluorescence change is before a preset position among all cells to be tested (such as the first 1 / 3).

[0105] The target cell set refers to the set that contains all cells to be tested that meet the conditions after being screened by the second preset condition. The target fluorescence change obtained from the first fluorescence change data of all cells to be tested (i.e., cell regions) in the target cell set is a representative fluorescence intensity change trend, reflecting the most significant response of the entire cell population to the electric field stimulation.

[0106] The above-described embodiments ensure the accurate screening of cells that respond significantly to electrical field stimulation from a large number of cell samples, and then calculate the target fluorescence change representing the response degree of these cell populations, providing more accurate data support for subsequent drug screening and evaluation. In high-throughput drug screening experiments, this method can significantly improve the screening efficiency and the reliability of the results, avoid misjudgment caused by non-specific responses or background noise interference, and ensure the screening of cell populations that are truly responsive to the drug.

[0107] Step S206: Evaluate the candidate drugs based on the target fluorescence change to obtain an evaluation result.

[0108] In the above step S206, the influence degree of the drug on the cell electrophysiological response can be evaluated by comparing the target fluorescence change with the fluorescence change of the drug-free control group. For example, by calculating the difference or ratio between the target fluorescence change amplitude (such as ΔF / F) and the fluorescence change of the control group, the influence degree of the candidate drug on the cell electrophysiological response can be quantified. One or more quantification indexes can also be defined, such as fluorescence change amplitude, response time, frequency, or duration, etc., to evaluate the drug effect. For example, an efficacy index (such as EC50 or IC50) can be set, and by fitting the relationship between the drug concentration and the first fluorescence change, the half-maximal effective concentration or inhibitory concentration of the drug can be calculated to evaluate the potency or selectivity of the drug.

[0109] In some embodiments of the present application, the drug screening process can also be optimized based on the evaluation result, that is, based on the feedback of the evaluation result, the conditions and parameters of the drug screening are optimized to improve the screening efficiency and the reliability of the results. For example, according to the evaluation result, the conditions and parameters of the drug screening, such as electric field intensity, stimulation frequency, stimulation duration, drug concentration range, etc., are adjusted to optimize the screening process and improve the screening efficiency.

[0110] Through the above steps S202 to S206, by using the electrical field stimulation and fluorescence labeling techniques, and by high-throughput recording of the differences in cell potential changes induced by different drugs under the electrical field stimulation, the ion channel-related drugs are evaluated, achieving the purpose of electrophysiological detection and optical recording of a large number of cells synchronously, thereby realizing the technical effects of reducing the complexity of experimental operations and improving the efficiency of drug screening and evaluation, and further solving the technical problem that the drug screening method in the related art has low evaluation efficiency due to the limitation of throughput in the evaluation of cell electrophysiological characteristics.

[0111] Figure 3 is the overall flowchart of a drug evaluation method according to an embodiment of the present application, as Figure 3 shown, and specifically includes the following steps:

[0112] Step 302: Input the original imaging data (image sequence) of the HEK 293T cell line expressing the genetically-encoded voltage indicator (GEVI) Cepheid1b under 10 field stimulations (frequency 3.28 Hz, stimulation duration 10.18 milliseconds, i.e., duty cycle 3.33%). This data records the change in cell fluorescence signal (600 frames, sampling rate 196.6 Hz. Total 3.053 seconds).

[0113] Step 304: Generate an average projection of the original imaging data to identify the cell distribution across the entire field of view, providing a basis for subsequent cell segmentation and signal analysis; enhance the membrane boundary contrast by sharpening and median filtering the average projection to highlight the cell contours, providing a clear image basis for subsequent analysis; use the "cpCyto2" model of Cellpose2 for cell segmentation to identify the regions of all cells in the field of view.

[0114] Step 3061: Calculate the average fluorescence trace (i.e., the second fluorescence change) for each identified region of interest (ROI) of the cell, which is the trend of the fluorescence intensity of each cell changing over time under field stimulation. For example, for each ROI, extract the corresponding time-series fluorescence signal from the stored original imaging data; calculate the average of the fluorescence intensities of all pixel points within each ROI in chronological order to obtain a time series representing the average fluorescence intensity change of the cell region, i.e., the average fluorescence trace.

[0115] Step 3081: Expand each cell region outward to 187 microns using an octagonal structuring element to form the local background region of each cell (i.e., the third region) for subsequent calculation of the background signal.

[0116] Step 3062: Expand each segmented cell region (ROI) outward by 16 microns, and then take the inverse to obtain the global background region (i.e., the second region), which is the region not occupied by cells, for the evaluation of the overall background level.

[0117] Step 3082: Calculate the average local background trace (the third fluorescence change) for each ROI from the intersection of the corresponding local background region and the global background region (i.e., the background region).

[0118] Step 310: Filter the local background trace of each ROI using a notch filter in the frequency range of 2.5 - 3.5 Hz, and at the same time perform a smoothing operation using a self-smoothing window of size 181 frames (equivalent to 0.92 seconds, corresponding to the duration of 3 stimulation cycles); subtract the background signal from the processed average local background trace and generate a normalized trace.

[0119] Step 312: Conduct data analysis and information extraction, including: screening out cell regions with low brightness and small area, and excluding these regions that may not contain valid cells or have poor cell states; calculating the average fluorescence change (i.e., the target fluorescence change) of each valid cell region during the field stimulation, which reflects the response degree of the cells to the electric field stimulation; analyzing the average fluorescence trace (the first fluorescence change) of each cell region to extract key information, such as the fluorescence change amplitude of the Ca²⁺ / voltage indicator for each stimulation pulse, or information such as the spike rate of the cells to the stimulation pulse, for evaluating the effect of the drug or the cell function state.

[0120] Figures 4 to 7 is a schematic diagram of imaging and analysis of a target region of a method for drug evaluation according to an embodiment of the present application, showing the high and low fluorescence signal changes of multiple cells to be measured in the target region with the field stimulation pulses, and the process of selecting cells (or more than 5% of the cells) with the top 1 / 3 fluorescence signal change amplitudes in the field of view (i.e., the target region), where Figure 4 shows that after the original image is subjected to average projection (A), its edges are sharpened to highlight the cell edges (B), and 76 cells are identified by cell segmentation through Cellpose2 (cpCyto2 model) (C), and 67 qualified cells are obtained after excluding regions with too small size and too low brightness (D). Figure 5 shows the global background region (A) obtained by dilating and inverting the 76 cells obtained by cell segmentation in this field of view. For a specific cell (B) obtained by segmentation, the intersection of the region obtained by dilating it and the blue region is its local background selection region (C). Figure 6 (A) shows Figure 4 (D) the average fluorescence brightness changes (normalized fluorescence intensity F) of the 67 qualified cells marked in (D) under 10 field stimulations (the positions shown by the red dots), and the blue triangles represent the positions of the peaks corresponding to the 10 stimulations identified by the global signal; Figure 6 (B) shows the fluorescence intensity change amplitudes of each valid cell ( ), where the gray dashed line and the black dashed line respectively represent the change amplitudes at the top 1 / 3 position of the relative fluorescence change amplitudes among all cells and the position of the relative change amplitude of 5%. Figure 7 shows the distribution in the field of view of cell regions with a fluorescence change amplitude exceeding 5% (A, a total of 53) and cell regions with a relative change amplitude ranking in the top 1 / 3 (B, a total of 23) during the field stimulation.

[0121] Specifically, in Figure 4In this study, cells were transfected with liposomes to express the membrane potential probe Cepheid1b mutant, which stably expressed Kir2.1 and Na V 1.5-channel HEK 293T cell line, which can synchronously generate global depolarization like cardiomyocyte action potential under field stimulation of 300 V / cm, pulse width of 10.18 milliseconds, and frequency of 3.28 Hz. When the user inputs the average projection of the fluorescence signal change of Cepheid1b mutant recorded at 196.6 Hz during the above process ( Figure 4 (A) ), the system will sharpen it to highlight the cell edges ( Figure 4 (B) ) and 76 cells were segmented using cellpose2 ( Figure 4 (C) ), excluding the areas with too small size and too low brightness as described above, 67 qualified cells were obtained ( Figure 4 (D)). After dilating and negating the 76 cells obtained by the initial segmentation, the global background area can be obtained ( Figure 5 Targeting a specific cell ( Figure 5 (B) The intersection of the expanded area and the blue area is its own local background selection area ( Figure 5 (C)). Then, the fluorescence signal changes obtained from the 67 qualified cell regions obtained above were subtracted from the background using the data of their local background regions, and the average fluorescence signal change of all cells in the field of view was obtained ( Figure 6 (A)) and the average sensitivity distribution of each cell calculated in turn ( Figure 6 (B)). Since the cell regions obtained by cell segmentation may not all be cells that respond to field stimulation, if one wants to evaluate the level of fluorescence brightness change in living cells expressing the mutant, the analysis program can calculate the average fluorescence brightness change of all cells during field stimulation by more than 5% ( Figure 6 (B) The black dotted line indicates the position of 53 cells, corresponding to Figure 7 (A) Each region) or the first 1 / 3 of cells ( Figure 6 (B) The gray dotted line in the right figure shows a total of 53 cells, corresponding to Figure 7 The various areas shown in (B) are used as a representative set of cells (fluorescence change levels) in the field of view to evaluate the level of fluorescence brightness change (i.e., information such as the amplitude and width of membrane potential depolarization) of the cells in the field of view under this scenario.

[0122] The present application also provides a drug evaluation system, including: an imaging subsystem, an electrode, and a processor. Among them, the imaging subsystem includes a light source and an image acquisition device. The light source is connected to the processor and is used to provide illumination conditions for electrode stimulation before the start of electrode stimulation. The electrode is connected to the processor and is used to perform electric field stimulation on a plurality of cells to be measured cultured in a candidate drug in a target area, where the plurality of cells to be measured have fluorescent labels. The image acquisition device in the imaging subsystem is connected to the processor and is used to acquire the original imaging data corresponding to the target area under electric field stimulation and send the original imaging data to the processor. The processor is connected to the imaging subsystem and is used to receive the original imaging data and determine the target fluorescence change commonly corresponding to the plurality of cells to be measured from the original imaging data, where the target fluorescence change is used to quantitatively represent the average response degree of the plurality of cells to be measured to electric field stimulation. The candidate drug is evaluated according to the target fluorescence change to obtain an evaluation result.

[0123] Through the pre-triggering of the light source, it is possible to ensure stable illumination in the target area before the start of electrode stimulation, so that the image acquisition device can clearly record the cell state. For example, before the start of electrode stimulation, the processor controls the light source to preheat it within a preset time to stabilize the light intensity; at the same time, light intensity calibration is performed to ensure the consistency and accuracy of the illumination conditions.

[0124] The processor can set the voltage parameters of electrode stimulation according to experimental requirements, including voltage amplitude, frequency, duration, etc., for stimulating cells to generate electrophysiological responses. Moreover, the processor ensures the synchronization of electrode stimulation and image acquisition through synchronous control with the image acquisition device, so as to record the real-time response of cells to stimulation.

[0125] The image acquisition device continuously records images of the target area under electrode stimulation and sends the original imaging data to the processor for analysis and processing. The processor can set the parameters of the image acquisition device, such as exposure time, readout speed, binning value, etc., to adapt to experimental conditions and ensure image quality. The image acquisition device performs real-time image acquisition under the control of the processor, records the fluorescence change of cells before and after electrode stimulation, generates the original imaging data, and sends the acquired original imaging data to the processor through the communication interface for subsequent data analysis and storage.

[0126] In some embodiments of the present application, the system further includes a mobile device. Among them, the mobile device includes an electric motor. The electric motor is respectively connected to the controller and the electrode and is used to receive the instruction sent by the controller and control the electrode to move in the Z-axis direction of the coordinate axis according to the instruction. The mobile device further includes a stage. The stage is connected to the processor and is used to carry the target well plate and move the specified area in the target well plate to the target area.

[0127] In the MATLAB environment, the processor sends instructions to the controller. After the controller parses the instructions, it drives the electric motor to control the up and down movement of the electrode, achieving precise contact or separation between the electrode and the cell sample. For example, before electrode stimulation, the processor sends instructions to the controller to control the electrode to descend to a position close to or in contact with the surface of the cell sample; after the stimulation ends, the processor sends instructions to lift the electrode away from the cell sample, preparing to move to the next target well or area.

[0128] In addition, by sending instructions to the stage driver program, the processor can control the movement of the stage in the X-axis and Y-axis directions, accurately moving the specified area in the target well plate to the center of the field of view of the imaging device, that is, the target area. For example, according to the experimental procedure and the layout of the target well plate, the processor calls the stage driver program through MATLAB to control the movement of the stage, enabling each well of the target well plate to enter the field of view of the imaging device one by one; after imaging of each well is completed, the processor controls the stage to move to the position of the next well, realizing automated well plate traversal and drug evaluation.

[0129] In some embodiments of the present application, the system further includes a stimulation isolator, wherein the stimulation isolator is connected to the electrode and is used to isolate and amplify the voltage signal emitted by the electrode and output a pulsed voltage to a plurality of cells to be measured.

[0130] The processor outputs an electrical stimulation command signal through the port of the data acquisition card. After receiving this signal, the internal circuit of the stimulation isolator isolates and amplifies the signal and outputs a set pulsed voltage to the electrode. For example, the processor can send a control signal to the stimulation isolator through the data acquisition card driver program in the MATLAB environment to trigger the operation of the internal circuit of the stimulation isolator, realizing signal isolation and amplification.

[0131] In some embodiments of the present application, the processor is further configured to: segment a plurality of cells to be measured in the original imaging data to obtain cell regions corresponding to the plurality of cells to be measured respectively; determine a first fluorescence change corresponding to each cell region from the original imaging data, wherein the first fluorescence change is used to quantitatively represent the response degree of the cell region to the electric field stimulation within a preset statistical period under the electric field stimulation; determine a target fluorescence change commonly corresponding to the plurality of cells to be measured according to the first fluorescence changes corresponding to all the cell regions respectively.

[0132] In some embodiments of the present application, the processor is further configured to: determine a second fluorescence change corresponding to each cell region, where the second fluorescence change is used to quantitatively represent the trend of the fluorescence intensity in the cell region changing over time; determine a background region corresponding to each cell to be detected based on each cell region, and determine a third fluorescence change corresponding to the background region, where the third fluorescence change is used to quantitatively represent the trend of the fluorescence intensity in the surrounding region of each cell region changing over time; determine a first fluorescence change according to the second fluorescence change and the third fluorescence change.

[0133] It should be noted that the processor in the drug evaluation system can execute Figure 2 the drug evaluation method shown, which will not be elaborated here.

[0134] In a specific embodiment, the microscope can achieve continuous imaging in well plate screening without additional media such as water or immersion oil, and at the same time provide good photon utilization. The camera (image acquisition device) needs to sample at least at a rate of about 200 Hz. To overcome the impact of the reduction of the camera field of view on the detection throughput when imaging at 200 Hz or higher speed (when imaging at about 200 Hz, the effective maximum field of view occupies half of the middle part of the microscope target surface; when the imaging sampling rate increases, the field of view (number of horizontal rows) decreases proportionally in turn. The focal lengths of the tube lenses at the excitation end and emission end of the objective lens with a default tube lens of 200 mm are selected as 125 mm and 150 mm respectively, and the distance between the emission end tube lens and the objective lens and the distance between the camera and the emission end tube lens are adjusted to 150 mm, which can achieve an equivalent magnification of 15x to complete the simultaneous recording of hundreds of positive cells at a sampling rate of about 200 Hz. Between the tube lens at the imaging end and the camera, an electric filter wheel with fluorescence filters can also be installed for filtering, which supports the requirements of high-speed and stable switching between fluorescence filters, multi-color time-lapse imaging (such as simultaneous recording of calcium signals and membrane potential fluorescence signals), and ratiometric imaging (such as various commercial calcium dyes). In the present application, the settings of the light source, dichroic mirror, and fluorescence filter selected for imaging the representative fluorophore (fluorescent label) can be configured as shown in Table 1:

[0135] Table 1: Dichroic mirror / fluorescence filter setting table.

[0136]

[0137] In this application, the field stimulation electrode used can be a platinum electrode fabricated by 3D printing. The distance between the two poles can be set as required, and it is installed and fixed to the x-axis and y-axis direction adjustment brackets for fine-tuning the position in the x-axis and y-axis directions. Then, this bracket is installed on an electric motor, and the up and down (z-axis direction) displacement of the field stimulation electrode is controlled through a controller. During actual operation, the height of the z-axis can be controlled using the official driver of the controller in the MATLAB environment. The positions in the x-axis and y-axis directions can be manually adjusted by the user or controlled through a processor. In addition, the electrodes used for stimulation also output pulsed voltages through a stimulation isolator to induce the depolarization of the membrane potential of the cell sample to be measured, and it can trigger voltage pulse commands through the digital acquisition card inside the device computer for control. For example, the maximum output voltage can be 100 V, and the maximum effective field strength that can be achieved between the electrodes is approximately 333 V / cm. It should be noted that the illumination light can be turned on approximately 1 s before triggering the camera to start recording to eliminate possible unstable light intensity, and the illumination light output can be turned off after the stimulation ends to reduce photobleaching.

[0138] Figure 8 is the operation flowchart of a drug evaluation system according to an embodiment of the present application, as Figure 8 shown

[0139] Step 802: Initialize and connect the hardware in the MATLAB environment, including: camera, stage, LED or laser light source, Z-axis electric displacement stage, and the position for placing the fluorescence filter. Move the stage to well A1 of the 96-well plate (as an example), and reset the position (x, y) of the stage to (0, 0).

[0140] Step 804: Start the diagonal traversal of the stage from well A1, or move the stage to a specific well of interest for imaging.

[0141] Step 806: After the user determines the cell state and the brightness of the fluorescence probe in the current well, decide whether to perform stimulation. If "yes", lower the electrode and execute Step 808; if "no", skip the stimulation and execute Step 812.

[0142] Step 808: Perform electric field stimulation and synchronous imaging, including: lowering the electrode to a position above the cells; starting the light source illumination; performing electric field stimulation and 196.6 Hz synchronous fluorescence imaging; turning off the light source and checking the average time trajectory of the region of interest (ROI); writing the movie data to the hard disk, and raising the electrode to the default height.

[0143] Step 810: Move the stage to the next region of interest (ROI) within the well and repeat Step 808 or directly execute Step 812.

[0144] Step 812: Move the stage to the next adjacent well.

[0145] Figure 9 It is a schematic diagram of the analysis of the system operation speed of a drug evaluation according to an embodiment of the present application. As Figure 9 shown, it shows the average speed of the system for collecting, storing, and analyzing data. Among them, Figure 9 (A) is the time required to complete a total of 4.68 seconds of field stimulation commands according to the acquisition command using the maximum imaging field of view of 200 Hz, store the obtained 720 frames of data in the memory, and generate the global average fluorescence signal change curve. The data comes from the timing results of 206 consecutive acquisition commands; Figure 9 (B) is Figure 9 the time consumption when the data obtained from 206 consecutive acquisition commands in (A) is transferred from the memory to the hard disk (a video file in bin format (737 MB) and a parameter file in mat format (about 3 MB)); Figure 9 (C) is the average time required for each field of view when continuously analyzing 50 videos of 200 Hz field stimulation screening using a laboratory workstation with 64 GB of memory (green line) and a common laptop with 16 GB of memory (blue line). The error bar represents the standard deviation of 5 analyses of the same field of view.

[0146] Figure 9 (A) and Figure 9 (B) respectively show the time required for each data acquisition and writing to the memory (i.e., MATLAB workspace variables, and at the same time generate the average signal intensity change "global average trace" of the entire field of view) and writing the original imaging data in the memory and related parameters during acquisition (including the number of rows and columns of the camera, bin value, exposure time, and stage coordinates, etc.) from the memory to the hard disk during 206 consecutive standard field stimulation synchronous imaging processes. Specifically, during the 206 acquisitions, the time required to complete the acquisition command and input the data into the memory is 7.20 ± 0.10 seconds (mean ± standard deviation, the same below), and the time required to write the data to the hard disk is 0.33 ± 0.05 seconds. These data prove that the time used for data acquisition and storage is short and relatively stable. Figure 9 (C) shows the time consumption results of the automatic data analysis program for continuously processing 50 fields of view with different cells and repeating 5 times. When simulating using a workstation with 64 GB of memory and a personal laptop with 16 GB respectively, it can be found that even when using a laptop, the slowest field of view during analysis will not exceed 1 minute, while if using a workstation, most fields of view generally take 20 - 40 seconds to complete (the more independent cells segmented and recognized within the field of view, the slower the processing speed). This shows that the automatic analysis and processing program demonstrates a high data processing speed and program operation stability.

[0147] Figure 10 is a structural diagram of a device for drug evaluation according to an embodiment of the present application, as Figure 10 shown, the device includes:

[0148] An acquisition module 1002, configured to acquire original imaging data corresponding to a target area under electric field stimulation, where the target area includes a plurality of cells to be tested cultured with a candidate drug, and the plurality of cells to be tested have fluorescent labels;

[0149] A determination module 1004, configured to determine a target fluorescence change commonly corresponding to the plurality of cells to be tested from the original imaging data, where the target fluorescence change is used to quantitatively represent the average response degree of the plurality of cells to be tested to the electric field stimulation;

[0150] An evaluation module 1006, configured to evaluate the candidate drug according to the target fluorescence change to obtain an evaluation result.

[0151] It should be noted that Figure 10 the device for drug evaluation shown is used to execute Figure 2 the drug evaluation method shown, so Figure 2 the relevant explanations in the drug evaluation method also apply to Figure 10 the device for drug evaluation shown, which will not be elaborated here.

[0152] The embodiment of the present application further provides a non-volatile storage medium, which includes a stored computer program, where the device where the non-volatile storage medium is located executes the steps of the drug evaluation method in each embodiment of the present application by running the computer program.

[0153] The embodiment of the present application further provides a computer program product, including computer instructions, and when the computer instructions are executed by a processor, the steps of the drug evaluation method in each embodiment of the present application are implemented.

[0154] The embodiment of the present application further provides a computer program, and when the computer program is executed by a processor, the steps of the drug evaluation method in each embodiment of the present application are implemented.

[0155] The serial numbers of the above embodiments of the present application are only for description and do not represent the advantages and disadvantages of the embodiments.

[0156] In the above embodiments of the present application, the descriptions of each embodiment have their own emphases. For parts not detailed in a certain embodiment, reference may be made to the relevant descriptions of other embodiments.

[0157] In several embodiments provided in the present application, it should be understood that the disclosed technical content can be implemented in other ways. Among them, the device embodiments described above are merely illustrative. For example, the division of the units can be a logical function division. In actual implementation, there can be other division methods. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the displayed or discussed couplings or direct couplings or communication connections to each other can be through some interfaces. The indirect couplings or communication connections of units or modules can be in electrical or other forms.

[0158] The units described as separate components may or may not be physically separated. The components displayed as units may or may not be physical units, that is, they can be located in one place or distributed to multiple units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0159] In addition, in each embodiment of the present application, each functional unit can be integrated in a processing unit, or each unit can exist physically alone, or two or more units can be integrated in one unit. The above-mentioned integrated units can be implemented in the form of hardware or in the form of software functional units.

[0160] If the above-mentioned integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application, in essence, or the part that contributes to the prior art, or all or part of this technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions for causing a computer device (which can be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods described in each embodiment of the present application. The aforementioned storage medium includes: various media such as USB flash drives, read-only memories (ROMs), random access memories (RAMs), mobile hard disks, magnetic disks, or optical discs that can store program codes.

[0161] The above are only the preferred embodiments of the present application. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present application, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for drug evaluation, characterized in that, Including: Obtaining original imaging data corresponding to a target area under electric field stimulation, including: moving a stage for carrying a plurality of cells to be tested to a first position corresponding to the target area; lowering an electrode to a second position; under illumination of a light source, performing electric field stimulation on the plurality of cells to be tested by using the electrode; obtaining original imaging data corresponding to the plurality of cells to be tested collected by an image acquisition device under the electric field stimulation, wherein the target area includes the plurality of cells to be tested cultured with a candidate drug, the plurality of cells to be tested have fluorescence labels, the first position is above a microscope objective lens, and the electrode is in contact with the upper surfaces of the plurality of cells to be tested at the second position; Determining a target fluorescence change commonly corresponding to the plurality of cells to be tested from the original imaging data, wherein the target fluorescence change is used to quantitatively represent an average change degree of the membrane potential of the plurality of cells to be tested under the electric field stimulation; Evaluating the candidate drug according to the target fluorescence change to obtain an evaluation result.

2. The method according to claim 1, characterized in that Determining a target fluorescence change commonly corresponding to the plurality of cells to be tested from the original imaging data, including: Segmenting the plurality of cells to be tested in the original imaging data to obtain cell areas respectively corresponding to the plurality of cells to be tested; Determining a first fluorescence change corresponding to each cell area from the original imaging data, wherein the first fluorescence change is used to quantitatively represent a response degree of the cell area to the electric field stimulation within a preset statistical period under the electric field stimulation; Determining a target fluorescence change commonly corresponding to the plurality of cells to be tested according to the first fluorescence changes respectively corresponding to all the cell areas.

3. The method according to claim 2, characterized in that, Determining a first fluorescence change corresponding to each cell area from the original imaging data, including: Determining a second fluorescence change corresponding to each cell area, wherein the second fluorescence change is used to quantitatively represent a change trend of fluorescence intensity within the cell area over time; Determining a background area corresponding to each cell to be tested based on each cell area, and determining a third fluorescence change corresponding to each background area, wherein the third fluorescence change is used to quantitatively represent a change trend of fluorescence intensity in the surrounding area of each cell area over time; Determining the first fluorescence change according to the second fluorescence change and the third fluorescence change.

4. The method according to claim 3, wherein Determining a background area corresponding to each cell to be tested based on each cell area, including: Expanding each cell area outward by a first number of pixels to obtain a first area corresponding to each cell area; Determining an area in the original imaging data other than all the first areas as a second area; On the basis of the second area, expanding each cell area outward by a second number of pixels to obtain a third area corresponding to each cell area, wherein the first number is less than the second number; Determining an intersection of the second area and the third area as the background area.

5. The method according to claim 2, wherein The original imaging data includes multiple frames of imaging data. Segmenting the plurality of cells to be tested in the original imaging data includes: Statistically average the fluorescence intensities at each time point within the preset statistical period in the multi-frame imaging data to obtain the average projection corresponding to the original imaging data, where the average projection is an image formed after the statistical averaging; Segment the multiple cells to be measured in the average projection.

6. The method according to claim 2, wherein After obtaining the cell region corresponding to each cell to be measured, the method further includes: Compare the attribute information of the cell region with a first preset condition to obtain a first comparison result, where the first preset condition is used to screen the size and / or brightness of the cell region; When the first comparison result indicates that the attribute information does not meet the first preset condition, determine the cell region as an invalid region.

7. The method according to claim 2, wherein Determine the target fluorescence change common to the multiple cells to be measured according to the first fluorescence changes corresponding to all the cell regions, including: Compare the first fluorescence change with a second preset condition to obtain a second comparison result, where the second preset condition is used to screen the response degree of the cell to be measured corresponding to the cell region to the electric field stimulation; When the second comparison result indicates that the first fluorescence change meets the second preset condition, add the cell to be measured corresponding to the cell region to the target cell set; Determine the target fluorescence change according to the first fluorescence changes of the cell regions corresponding to the cells to be measured in the target cell set.

8. The method according to claim 1, wherein The multiple cells to be measured are located in any one of the wells of the target well plate. Moving the stage for carrying the multiple cells to the first position corresponding to the target region includes: Receiving a selection instruction from a target object, where the selection instruction includes any well number corresponding to the target well plate; Obtaining the size parameters of the target well plate, where the size parameters include the center distance between adjacent wells; Determining the third position corresponding to the stage as the coordinate origin and determining the coordinate information corresponding to the well number according to the center distance between the wells; Based on the third position, move the region corresponding to the well number to the first position according to the coordinate information.

9. The method according to claim 1, wherein Before moving the stage for carrying the multiple cells to the first position corresponding to the target region, the method further includes: Compare the fourth position corresponding to the electrode with a third preset condition to obtain a third comparison result, where the third preset condition is used to detect whether the electrode meets the movement requirement; When the third comparison result indicates that the fourth position does not meet the third preset condition, raise the electrode from the fourth position to the fifth position so that the electrode is disengaged from the multiple cells to be measured, where the vertical distance between the fifth position and the second position meets a preset threshold.

10. The method according to claim 8, wherein At least one well of the target well plate has multiple cells to be measured cultured with candidate drugs, and each well corresponds to at least one candidate drug, where the target well plate includes a 24-well plate and / or a 96-well plate and / or a 384-well plate.

11. A drug evaluation system, characterized in that, Including: An imaging subsystem, an electrode, and a processor, where, The imaging subsystem includes a light source and an image acquisition device. The light source is connected to the processor and is used to provide illumination conditions for the electrode stimulation before the start of the electrode stimulation. The electrode is connected to the processor and is used to perform electric field stimulation on a plurality of cells to be measured cultured in a candidate drug in a target area. Among them, the plurality of cells to be measured have fluorescent labels. The image acquisition device in the imaging subsystem is connected to the processor and is used to acquire the original imaging data corresponding to the target area under the electric field stimulation and send the original imaging data to the processor. The processor is connected to the imaging subsystem and is used to receive the original imaging data, and determine the target fluorescence change common to the plurality of cells to be measured from the original imaging data. Among them, the target fluorescence change is used to quantitatively represent the average change degree of the membrane potential of the plurality of cells to be measured under the electric field stimulation; evaluate the candidate drug according to the target fluorescence change to obtain an evaluation result; move the stage for carrying the plurality of cells to be measured to the first position corresponding to the target area; lower the electrode to the second position; under the illumination of the light source, use the electrode to perform electric field stimulation on the plurality of cells to be measured; acquire the original imaging data corresponding to the plurality of cells to be measured acquired by the image acquisition device under the electric field stimulation. Among them, the first position is above the microscope objective lens, and the electrode contacts the upper surface of the plurality of cells to be measured at the second position.

12. The system according to claim 11, wherein The system further includes a moving device, where The moving device includes an electric motor. The electric motor is respectively connected to the controller and the electrode, and is used to receive the instruction sent by the controller and control the movement of the electrode in the Z-axis direction of the coordinate axis according to the instruction. The moving device further includes a stage. The stage is connected to the processor and is used to carry the target well plate and move the specified area in the target well plate to the target area.

13. The system according to claim 11, wherein The system further includes a stimulation isolator. The stimulation isolator is connected to the electrode and is used to isolate and amplify the voltage signal sent by the electrode and output a pulsed voltage to the plurality of cells to be measured.

14. The system according to claim 11, wherein The processor is further used for: Segment the plurality of cells to be measured in the original imaging data to obtain the cell areas corresponding to the plurality of cells to be measured respectively. Determine the first fluorescence change corresponding to each cell area from the original imaging data. Among them, the first fluorescence change is used to quantitatively represent the response degree of the cell area to the electric field stimulation within a preset statistical period under the electric field stimulation. Determine the target fluorescence change common to the plurality of cells to be measured according to the first fluorescence changes corresponding to all the cell areas respectively.

15. The system according to claim 14, wherein The processor is further used for: Determine the second fluorescence change corresponding to each cell area. Among them, the second fluorescence change is used to quantitatively represent the change trend of the fluorescence intensity in the cell area over time. Based on each of the cell regions, a background region corresponding to each of the cells to be measured is determined, and a third fluorescence change corresponding to the background region is determined, where the third fluorescence change is used to quantitatively represent the trend of the fluorescence intensity of the surrounding region of each cell region changing with time; The first fluorescence change is determined according to the second fluorescence change and the third fluorescence change.

16. A device for drug evaluation, characterized in that, Comprising: An acquisition module, configured to acquire original imaging data corresponding to a target region under electric field stimulation, including: moving a stage for carrying a plurality of cells to be measured to a first position corresponding to the target region; lowering an electrode to a second position; under the illumination of a light source, performing electric field stimulation on the plurality of cells to be measured by using the electrode; acquiring original imaging data corresponding to the plurality of cells to be measured acquired by an image acquisition device under the electric field stimulation, where the target region includes a plurality of cells to be measured cultured in a candidate drug, the plurality of cells to be measured have fluorescence labels, the first position is above a microscope objective lens, and the electrode is in contact with the upper surfaces of the plurality of cells to be measured at the second position; A determination module, configured to determine a target fluorescence change commonly corresponding to the plurality of cells to be measured from the original imaging data, where the target fluorescence change is used to quantitatively represent the average degree of change of the membrane potential of the plurality of cells to be measured under the electric field stimulation; An evaluation module, configured to evaluate the candidate drug according to the target fluorescence change to obtain an evaluation result.

17. A non-volatile storage medium, characterized in that, The non-volatile storage medium includes a stored computer program, where a device where the non-volatile storage medium is located executes the method for drug evaluation according to any one of claims 1 to 10 by running the computer program.

18. A computer program product, comprising computer instructions, characterized in that, The computer instructions, when executed by a processor, implement the method for drug evaluation according to any one of claims 1 to 10.

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