A full-scale "electric-calcium-machine" coupling drug efficacy evaluation system

Abstract

This application discloses a full-scale electro-calcium-mechanical coupling pharmacodynamic evaluation system. The system includes: an optical mapping microscopy module configured to acquire calcium fluorescence, voltage-sensitive dye fluorescence, and infrared bright-field image sequences; a multi-channel electrophysiological detection module, located at the bottom of a sample recording bath, comprising a semi-transparent microelectrode array with a transparent conductive glass substrate and containing fine-diameter wires and nanoring electrodes, and a synchronous data acquisition front-end; and a main control and analysis module, including synchronous control logic components configured to issue a global trigger signal to lock the light source pulse, exposure window, and sampling clock, and to extract action potential curves, calcium transient curves, and mechanical contraction curves on the same time axis, and calculate the electro-calcium-mechanical coupling delay parameters of cells. This application enables unobstructed and interference-free high-synchronous extraction of multimodal physiological signals in space and time, quantifying the intervention targets and toxic effects of drugs on the cell excitation-contraction coupling mechanism.

Description

Technical Field

[0001] This application relates to the fields of preclinical cardiovascular drug development, in vitro cellular multimodal physiological monitoring, and cardiotoxicity safety evaluation. Specifically, it relates to a full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system. Background Technology

[0002] In the field of preclinical cardiovascular drug development and cardiotoxicity safety evaluation, obtaining comprehensive and accurate information about the physiological state of cardiomyocytes is crucial. Cardiomyocyte activity is a complex excitation-contraction coupled process, encompassing action potentials manifested by electrical signal transduction, calcium transients caused by intracellular ion changes, and ultimately cell contraction leading to mechanical deformation.

[0003] Currently, it is difficult to effectively extract the aforementioned multidimensional features simultaneously on the same timeline. Existing physiological monitoring equipment is often single-function, typically only able to acquire cellular electrophysiological signals individually using traditional patch-clamp or opaque microelectrode arrays, or perform only fluorescence calcium imaging using optical microscopy. This makes it impossible to perform interference-free, simultaneous in-situ recording of electrical activity, calcium signals, and mechanical contraction of the same specimen on the same timeline. Furthermore, due to the opacity of traditional electrode materials, they severely obstruct the optical mapping field of view in physical space. Therefore, current technologies suffer from dimensional gaps, spatial misalignment, and data fragmentation in evaluating drug-induced arrhythmias, abnormal myocardial contractility, and other toxic reactions, failing to accurately reflect the specific intervention targets and toxic manifestations of drugs on the cellular excitation-contraction coupling mechanism. Summary of the Invention

[0004] The purpose of this application is to provide a full-scale "electro-calcium-mechanical" coupled drug efficacy evaluation system to solve the problems of existing equipment having single function and spatiotemporally fragmented and mutually interfering multimodal signals.

[0005] To achieve the above objectives, this application provides a full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system, comprising:

[0006] An optical mapping microscopy module includes a multi-channel beam splitter, a dual-channel excitation source, and a multi-back-illuminated complementary metal-oxide-semiconductor camera array. The optical mapping microscopy module is configured to acquire calcium fluorescence, voltage-sensitive dye fluorescence, and infrared bright-field image sequences.

[0007] A multi-channel electrophysiological detection module is located at the bottom of a sample recording bath. The multi-channel electrophysiological detection module includes a semi-transparent microelectrode array and a synchronous data acquisition front end. The substrate of the semi-transparent microelectrode array is made of transparent conductive glass. The semi-transparent microelectrode array includes fine-diameter wires and nanoring electrodes.

[0008] The main control and analysis module is communicatively connected to the optical mapping and microscopy module and the multi-channel electrophysiological detection module. The main control and analysis module includes a synchronization control logic component, which is configured to issue a global trigger signal to lock the pulse of the dual-channel excitation light source, the exposure window of the multiple back-illuminated complementary metal-oxide-semiconductor camera array, and the sampling clock of the synchronous data acquisition front end. It also extracts the action potential curve, calcium transient curve, and mechanical contraction curve of the tested specimen on the same time axis, and calculates the electro-calcium-mechanical coupling delay parameter of the cell based on the action potential curve, the calcium transient curve, and the mechanical contraction curve.

[0009] Furthermore, the multi-channel beam splitter is configured as a three-channel beam splitter, which is configured to receive the incoming infrared bright-field illumination light, the calcium fluorescence, and the voltage-sensitive dye fluorescence, and separate the three, and guide the separated light signals to the corresponding multiple back-illuminated complementary metal-oxide-semiconductor camera arrays.

[0010] Furthermore, the dual-channel excitation light source and the multi-channel beam splitter are configured as a coaxial optical architecture, and the wavelengths of the infrared bright-field illumination light, the calcium fluorescence, and the voltage-sensitive dye fluorescence do not overlap in the frequency domain.

[0011] Furthermore, the transparent conductive glass uses an indium tin oxide substrate, the nanoring electrode is attached to the surface of the indium tin oxide substrate, and the fine-diameter wires are etched along the surface of the indium tin oxide substrate.

[0012] Furthermore, the diameter of the fine-diameter wire on the surface of the indium tin oxide substrate is configured to be less than 5 μm, and the nanoring electrode and the fine-diameter wire are arrayed in the upper region of the transparent conductive glass. The overall transmittance of the semi-transparent microelectrode array for ambient light and measurement light is configured to be greater than 85%, and the semi-transparent microelectrode array and the multi-channel beam splitter are coaxially aligned.

[0013] Furthermore, the synchronous control logic component integrates a field-programmable gate array (FPGA), which is configured to emit the global trigger signal. The level type of the global trigger signal is a transistor-to-transistor logic level trigger signal.

[0014] Furthermore, the field-programmable gate array (FPGA) locks the time period during which the exposure windows of the multiple back-illuminated complementary metal-oxide-semiconductor (CMOS) camera arrays are in the open state with the sampling clock of the synchronous data acquisition front-end by issuing the transistor-to-transistor logic level trigger signal. The time difference between the opening time of the exposure window and the rising edge of the clock is configured to be less than 100 μs. The synchronous data acquisition front-end contains an amplifier. The FPGA is configured to send a forced hold command to the amplifier during the time period during which the exposure window is in the open state, instructing the amplifier to maintain the current level state.

[0015] Furthermore, the main control and analysis module is configured to perform pixel-level optical flow analysis and adaptive edge contour tracking on the infrared bright field image sequence, output a displacement curve based on the analysis results, and map the displacement curve to the mechanical contraction curve.

[0016] Furthermore, when executing the pixel-level optical flow method, the main control and analysis module extracts the optical flow field vector between adjacent frames in the infrared bright field image sequence, and locates the adaptive edge contour by combining the gray-level gradient changes of the pixels in the infrared bright field image sequence. The main control and analysis module is configured to extract the normal direction of the adaptive edge contour, integrate the optical flow field vector along the normal direction to generate the displacement curve, and register the time axis of the displacement curve to the reference clock domain of the sampling clock.

[0017] Furthermore, the electro-calcium-mechanical coupling delay parameters include the electro-calcium delay time and the calcium-contraction delay time. The main control and analysis module is configured to extract the depolarization start point of the action potential curve as the electrical signal start point, extract the rising branch start point of the calcium transient curve as the calcium signal start point, extract the contraction start point of the mechanical contraction curve as the mechanical signal start point, subtract the timestamp corresponding to the electrical signal start point from the timestamp corresponding to the calcium signal start point to obtain the difference as the electro-calcium delay time, and subtract the timestamp corresponding to the calcium signal start point from the timestamp corresponding to the timestamp corresponding to the mechanical signal start point to obtain the difference as the calcium-contraction delay time.

[0018] The full-scale electro-calcium-mechanical coupling pharmacodynamic evaluation system provided in this application eliminates obstruction of the optical mapping field of view from a physical structure perspective by combining semi-transparent electrodes with coaxial high-resolution fluorescence imaging technology. Furthermore, a high-precision global clock synchronization mechanism ensures high temporal consistency of signals across the electro-calcium and mechanical contraction dimensions. By quantifying the time delay between various curves using algorithms, the system improves the comprehensiveness and signal-to-noise ratio of cardiac pharmacological characterization evaluation, directly quantifying the specific intervention targets and toxic effects of drugs on the cellular excitation-contraction coupling mechanism. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall structure provided in an embodiment of the present invention.

[0020] Figure 2 This is a control and logic architecture block diagram provided in the embodiments of the present invention.

[0021] Explanation of reference numerals in the attached figures:

[0022] 100-Full-scale "electro-calcium-mechanical" coupled drug efficacy evaluation system; 101-Optical mapping microscopy module; 102-Multi-channel beam splitter prism; 103-Dual-channel excitation light source; 104-Back-illuminated complementary metal-oxide-semiconductor camera array; 105-Sample recording bath; 106-Multi-channel electrophysiological detection module; 107-Semi-transparent microelectrode array; 108-Transparent conductive glass; 109-Nanoring electrode; 110-Fine diameter wire; 111-Infrared bright-field light source;

[0023] 201-Main control and analysis module; 202-Synchronous control logic unit; 203-Field programmable gate array; 204-Timing array generator; 205-Synchronous data acquisition front end; 206-Amplifier; 207-Environmental and drug delivery control module; 208-Temperature control heating unit; 209-Eight-channel perfusion system; 210-Power supply module; 211-Computer data processing host; 212-High-speed data transmission bus module. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this application clearer, the application will be described in detail below with reference to the accompanying drawings and specific embodiments. Here, the illustrative embodiments and their descriptions are used to explain this application, but are not intended to limit it.

[0025] This application provides a full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system. Figure 1 This is a schematic diagram of the overall structure provided in an embodiment of the present invention. Figure 2 This is a control and logic architecture block diagram provided in an embodiment of the present invention. (Reference) Figure 1 and Figure 2 As shown, the full-scale "electro-calcium-mechanical" coupled drug efficacy evaluation system 100 includes an optical mapping microscopy module 101, a multi-channel electrophysiological detection module 106, and a main control and analysis module 201. These modules work together, synchronously integrating multi-channel electrical signals and high-resolution fluorescence imaging technology, enabling the system to achieve real-time monitoring and correlation analysis of drug effects on cardiomyocytes from multiple dimensions, including electrical activity, calcium signals, and mechanical contraction.

[0026] An optical mapping microscopy module 101 is positioned above the main support, aligned with the measurement station below. The optical mapping microscopy module 101 includes a multi-channel beam splitter prism 102, a dual-channel excitation light source 103, and multiple back-illuminated complementary metal-oxide-semiconductor (CMOS) camera arrays 104. A multi-channel electrophysiological detection module 106 is located at the bottom of a sample recording bath 105. This sample recording bath 105 is used to hold detection reagents containing cell samples and culture medium. The multi-channel electrophysiological detection module 106 includes a semi-transparent microelectrode array 107 and a synchronous data acquisition front-end 205. The semi-transparent microelectrode array 107 is laid on the inner surface of the bottom end of the sample recording bath 105, allowing cell samples to directly adhere to it for extraction of local potentials and electrocardiogram (ECG) signals. The substrate of the semi-transparent microelectrode array 107 is made of transparent conductive glass 108, and the semi-transparent microelectrode array 107 includes fine-diameter wires 110 and nanoring electrodes 109. The main control and analysis module 201 establishes communication connections with both the optical mapping and microscopy module 101 and the multi-channel electrophysiological detection module 106 in its hardware architecture. The main control and analysis module 201 includes a synchronization control logic unit 202, which is responsible for issuing a global trigger signal to lock the pulses of the dual-channel excitation light source 103, the exposure window of the back-illuminated complementary metal-oxide-semiconductor camera array 104, and the sampling clock of the synchronous data acquisition front-end 205. Through timing locking in a unified clock domain, the main control and analysis module 201 extracts the action potential curve, calcium transient curve, and mechanical contraction curve of the tested specimen along the time axis, and then calculates the electro-calcium-mechanical coupling delay parameters of the cells based on these curves. Based on this structural composition, it can synchronously capture deformation and light intensity changes in the optical dimension and potential fluctuations in the electrical dimension in situ in real time, thereby providing comprehensive data support for drug efficacy and safety evaluation.

[0027] In the specific implementation details of the optical mapping and microscopy module 101, a similar closed-loop optical architecture is adopted as the configuration reference. To simultaneously acquire mechanical shrinkage images and multi-channel fluorescence images, the multi-channel beam splitter prism 102 is specifically configured as a three-channel beam splitter prism. An infrared bright-field light source 111 with an emission wavelength greater than 700 nm is configured in the region below the sample recording bath 105. The infrared bright-field light source 111 generates infrared bright-field illumination light, which penetrates the sample within the sample recording bath 105 from bottom to top. A dual-channel excitation light source 103 is located on the side of the optical mapping and microscopy module 101, with an emission wavelength range covering 300 nm to 800 nm, a maximum light load of up to 1 A, and a constant or feedback light control mechanism with a feedback response time of no more than 60 μs. This ensures extremely high stability of the excitation light intensity and output wavelength. After being irradiated by the excitation light, the sample simultaneously emits calcium fluorescence and voltage-sensitive dye fluorescence, both containing specific wavelength bands. Infrared bright-field illumination, calcium fluorescence, and voltage-sensitive dye fluorescence all enter the three-channel beam splitter from bottom to top.

[0028] The three-channel beam-splitter is configured to receive incoming infrared bright-field illumination, calcium fluorescence, and voltage-sensitive dye fluorescence, and separate these three components, guiding the separated light signals to their respective back-illuminated complementary metal-oxide-semiconductor (CMOS) camera arrays 104. Specifically, the CMOS camera array 104 is an independent imaging unit composed of three back-illuminated scientific CMOS cameras. The three-channel beam-splitter contains a first beam-splitting surface and a second beam-splitting surface. When the infrared bright-field illumination reaches the first beam-splitting surface, this surface reflects the infrared bright-field illumination with wavelengths greater than 700 nm to the third back-illuminated camera, i.e., the bright-field camera, for capturing bright-field images of cell contraction; the remaining light penetrates the first beam-splitting surface to reach the second beam-splitting surface. The second beam-splitting surface is configured for wavelength division multiplexing isolation of specific wavelength bands, transmitting the separated 520 nm wavelength calcium fluorescence to the first back-illuminated camera, while simultaneously reflecting the 680 nm wavelength voltage-sensitive dye fluorescence to the second back-illuminated camera. The dual-channel excitation source 103 and the multi-channel beam splitter 102 employ a strictly coaxial optical architecture. The wavelengths of the infrared bright-field illumination light, calcium fluorescence, and voltage-sensitive dye fluorescence do not overlap in the frequency domain. By adopting this structure with a three-channel beam splitter prism and coaxial design based on different wavelengths, it is possible to achieve three-way coaxial high-resolution imaging of "dual fluorescence superposition and one bright field" without generating spectral crosstalk, thereby improving the clarity and signal-to-noise ratio of the microscopic field of view.

[0029] The back-illuminated complementary metal-oxide-semiconductor (CMOS) camera array 104 exhibits extremely high photosensitivity, with a quantum efficiency of 95%. The camera's pixel size distribution ranges from 6.5 μm × 6.5 μm to 11 μm × 11 μm, and it supports a dynamic range of up to 50,000:1 (94 dB), with a bit depth of 12 bits per pixel. At a full-frame size of 2048 × 2048 pixels, the camera's maximum sampling rate reaches 5 kHz. In the set region of interest mode, the maximum frame rate can be stably maintained at 3.5 kHz. Through the camera's high-speed recording characteristics, it is possible to accurately capture dynamic changes in myocardial cell contraction and fluorescence intensity occurring within milliseconds. It should be noted that the specific numerical parameters of the camera described above are merely preferred examples; those skilled in the art can also implement this application using other image sensors with equivalent high-speed and high-sensitivity characteristics.

[0030] To facilitate high-resolution optical mapping and eliminate optical path obstruction, the internal structure of the multi-channel electrophysiological detection module 106 incorporates special material modifications. The transparent conductive glass 108 used as the substrate for the semi-transparent microelectrode array 107 is indium tin oxide (ITO) conductive glass. ITO possesses excellent light transmittance and conductivity. The nanoring electrode 109 is attached to the surface of the ITO substrate. Made of gold and processed using an ultra-thin deposition process, it exhibits a centrally hollowed-out nanoring structure, reducing the light absorption of traditional solid metal electrodes. Fine-diameter conductive wires 110 are etched along the surface of the ITO substrate, with an insulating layer formed by photolithography covering the outer region. This insulating layer is preferably made of a negative photoresist insulating material. The wire diameter of the fine-diameter conductive wires 110 on the ITO substrate surface is configured to be less than 5 μm. Nanoring electrodes 109 and fine-diameter wires 110 are distributed in an 8×8 or 6×6 array pattern above the transparent conductive glass 108, with a distance of 0.5 mm between electrodes, a diameter of 0.05 mm for each electrode, and an overall array size of 3.5 mm × 3.5 mm. Based on this combination of nanoscale gold ring deposition and ultra-fine wire photolithography, the translucent microelectrode array 107 achieves an overall transmittance of greater than 85% for both ambient and measurement light. During system assembly, the translucent microelectrode array 107 and the multi-channel beam splitter prism 102 are aligned coaxially in the vertical dimension. Using this high-transmittance electrode substrate ensures unobstructed transmission of the infrared bright-field light source 111 emitted from the bottom and the fluorescence excitation light penetrating cells, completely solving the problems of weak calcium transient fluorescence signals being blocked and absorbed, and the presence of dark spots in the bright-field field of view caused by traditional opaque electrodes.

[0031] The environment maintenance and drug delivery module provides a stable environment for cell culture and drug efficacy assays. The environment and drug delivery control module 207 includes a temperature-controlled heating unit 208 and an eight-channel perfusion system 209. The temperature-controlled heating unit 208 regulates the temperature of the sample recording bath 105 using internal or external feedback loops, with a temperature control range set from room temperature to 70°C and a temperature control accuracy of ±0.1°C. The eight-channel perfusion system 209 is used for quantitative drug intervention, including a multi-channel rapid micro-programmable drug delivery system and an eight-in-one manifold, providing both manual and programmed control modes. The system features overflow alarms and power-off operating mode memory functions to effectively ensure cell viability and maintain the safety of long-term drug delivery assays.

[0032] To address the concurrent processing of such dense electrical, optical, and thermal multimodal signals, this application introduces a low-level bridging module to improve the hardware link. Specifically, the system also includes a power supply module 210 and a high-speed data transmission bus module 212. The power supply module 210 has a built-in isolation drive circuit, responsible for providing low-noise isolated power to the weak signal measurement front-end such as the multi-channel electrophysiological detection module 106, cutting off ground-conducted interference from the subsequent digital circuits. The main control and analysis module 201 is equipped with a computer data processing host 211 internally or externally. Multiple back-illuminated complementary metal-oxide-semiconductor camera arrays 104 and synchronous data acquisition front-ends 205 are connected to the computer data processing host 211 through the high-speed data transmission bus module 212, such as the peripheral component interconnection bus channel, to carry and preserve concurrent high-frequency multimodal data from the cameras and acquisition front-ends without loss. Through the above-mentioned low-noise power supply and high-bandwidth transmission architecture, the purity of the underlying weak signals and the reliable flow of high-throughput image data are ensured.

[0033] The synchronization control logic unit 202 can specifically employ a fluorescence mapping system controller as the timing master node responsible for global scheduling. This controller supports up to eight pulse output channels, with a maximum output frequency of 30kHz. Simultaneously, it supports four channels of analog data input, with a 16-bit resolution and a sampling rate of 12.5kHz. The synchronization control logic unit 202 integrates a field-programmable gate array 203. The field-programmable gate array 203 is configured to emit a global trigger signal, and this global trigger signal is a transistor-to-transistor logic level trigger signal. Under this synchronization mechanism, the exposure window of the back-illuminated complementary metal-oxide-semiconductor camera array 104, the strobe pulse of the dual-channel excitation light source 103, and the high-frequency sampling clock of the semi-transparent microelectrode array 107 are strictly locked. The time difference between the opening time of the exposure window and the rising edge of the clock is configured to be less than 100μs.

[0034] Furthermore, to address the background noise generated at the electrode tip during intense light exposure, i.e., the photoelectric coupling artifact problem, the synchronous data acquisition front-end 205 internally includes an amplifier 206. The field-programmable gate array 203 is configured to send a forced hold command to the control pin of amplifier 206 during the microsecond-level time period when the camera's exposure window is open. This command instructs amplifier 206 to suspend and maintain its current level until the exposure pulse ends, at which point sampling updates will resume. By employing this anti-artifact logic, the spike interference caused by drastic light intensity switching on localized minute potentials can be strongly shielded at the hardware level, ensuring a high signal-to-noise ratio in the electrophysiological curve.

[0035] To ensure more precise pulse transmission, the dual-channel excitation light source 103 is configured as a stroboscopic light source with stroboscopic characteristics. The stroboscopic frequency of this light source is equal to the frame rate of the multiple back-illuminated complementary metal-oxide-semiconductor camera arrays 104, and the start pin of the dual-channel excitation light source 103 is controlled by a global trigger signal output by the synchronization control logic unit 202. Upon receiving a system start command, the synchronization control logic unit 202 triggers the timing array generator 204. The timing array generator 204 generates a timing array pulse containing multiple square waves according to a preset frequency. The first branch of the timing array pulse is transmitted via hardwire to the dual-channel excitation light source 103 as the lighting pulse, while the second branch is transmitted via hardwire to the synchronization data acquisition front-end 205 as the sampling reference pulse. This purely hardware-level branching and distribution mechanism fundamentally eliminates timer jitter deviations in the software operating system, achieving sub-millisecond-level tight synchronization of multiple devices.

[0036] The main control and analysis module 201 is responsible not only for hardware scheduling but also for multimodal data alignment and quantification index calculation. After receiving real-time data buffered by the computer data processing host 211, the main control and analysis module 201 calls an online digital filtering algorithm to purify the signal. Specifically, the online digital filtering uses a Butterworth filter, Gaussian smoothing, or zero-phase filtering to remove data baseline drift. In this embodiment, for low-frequency electromyographic drift signals, a fourth-order Butterworth low-pass filter is used to effectively filter out slowly changing baseline fluctuations while retaining the high-frequency peaks of the action potential, obtaining a smooth and denoised reference signal. For the denoised cell membrane potential signal, the main control and analysis module 201 extracts the action potential conduction mode, conduction velocity, depolarization time, dispersion, and action potential duration. For the calculation of the action potential duration variable, a specific difference operation logic is executed, that is, the potential recovery time span is obtained by subtracting parameters.

[0037] In a preferred embodiment, the calculation logic for the above-mentioned action potential time history is implemented through the following formula:

[0038]

[0039] Among them, APD 10-90 t represents the action potential time-history variable; 90% This indicates the time point at which the action potential falls back to 90% from its peak repolarization; t 10% This represents the time point at which the action potential falls back to 10% from its peak repolarization. Quantifying this action potential duration index provides a direct reflection of the delayed effect of drugs on the closure of ion channels in cardiac cells.

[0040] Similarly, for the calcium transient curve generated above, the main control and analysis module 201 is configured to extract characteristic indicators such as intracellular calcium concentration amplitude and calcium transient duration, so as to comprehensively quantify the characteristics of intracellular calcium ion activity.

[0041] In terms of mechanical deformation assessment, the main control and analysis module 201 is configured to perform pixel-level optical flow analysis and adaptive edge contour tracking on the infrared bright-field image sequence. Based on the analysis results, it outputs a displacement curve and maps the displacement curve to a mechanical contraction curve representing shrinkage deformation. Specifically, the main control and analysis module 201 extracts the optical flow field vector between adjacent frames in the infrared bright-field image sequence and, combined with the gray-level gradient change features of pixels in the infrared bright-field image sequence, locates the adaptive edge contour. The deformation field is then converted into a displacement curve through normal direction extraction. The main control and analysis module 201 extracts the normal direction of the adaptive edge contour, integrates the optical flow field vector along the normal direction to generate the displacement curve, and registers the time axis of the displacement curve to the reference clock domain of the sampling clock.

[0042] In a preferred embodiment, specifically, the system performs the optical flow displacement calculation logic through the following formula:

[0043]

[0044] Where D(t) represents the displacement amplitude on the mechanical contraction displacement curve calculated at time t; This represents the optical flow vector extracted at time t' between adjacent image frames; This represents the normal direction vector of the adaptive edge contour determined during edge contour tracking. An optical flow method based on bright-field images is used for calculation, avoiding the potential cytotoxicity of adding additional fluorescent dyes. One-dimensional cell geometric deformation displacement is reconstructed with high fidelity using unlabeled bright-field images combined with an integral algorithm.

[0045] Furthermore, when performing analysis on the infrared bright-field image sequence, the main control and analysis module 201 is configured to support reducing background noise interference by setting a disabled region. Based on the mapped mechanical contraction curve, the main control and analysis module 201 incorporates a contraction kinetics algorithm to quantify and extract the mechanical contraction parameters of the cells. These mechanical contraction parameters specifically include peak height, peak interval, baseline recovery time, and peak time.

[0046] Based on the obtained three-dimensional curves, the main control and analysis module 201 further extracts the electro-calcium-mechanical coupling delay parameters. These parameters include the electro-calcium delay time and the calcium-contraction delay time. The main control and analysis module 201 extracts the depolarization start point of the action potential curve as the electrical signal start point, the rising branch start point of the calcium transient curve as the calcium signal start point, and the contraction start point of the mechanical contraction curve as the mechanical signal start point. The main control and analysis module 201 subtracts the timestamp corresponding to the electrical signal start point from the timestamp corresponding to the calcium signal start point to obtain the difference as the electro-calcium delay time. Similarly, the main control and analysis module 201 subtracts the timestamp corresponding to the calcium signal start point from the timestamp corresponding to the mechanical signal start point to obtain the difference as the calcium-contraction delay time.

[0047] In a preferred embodiment, specifically, the system performs the calculation logic for the electro-calcium-mechanical coupling delay parameters using the following formula:

[0048]

[0049]

[0050] Among them, T E-C Indicates the electro-calcium delay time; t Ca This indicates the time stamp corresponding to the start of the calcium signal, i.e., the beginning of the rising branch of the calcium transient curve; t E T represents the timestamp corresponding to the start of the electrical signal, i.e., the depolarization start of the action potential curve; C-M Indicates the calcium-contraction delay time; t M This indicates the timestamp corresponding to the starting point of the mechanical signal, i.e., the contraction start point of the mechanical contraction curve. Finally, the main control and analysis module 201 uses the timestamp as the unique primary key to reconstruct and display the action potential curve, calcium transient curve, and mechanical contraction curve on the same time axis. By extracting feature points from each dimension to calculate delay indices, the difference in the drug's impact on intracellular ion channels and myofibril contraction machinery can be directly quantified, revealing the root cause of the drug's effect on cardiovascular toxicology.

[0051] In summary, this application provides a full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system. Through a microelectrode array fabricated using a special transparent indium tin oxide substrate combined with a nano-gold ring process, it eliminates the physical obstruction of the underlying optical path imaging caused by local signal acquisition components. By using a three-channel beam splitter, infrared illumination and fluorescence signals of different wavelengths are accurately separated. Internal programmable logic gates emit hard-wired transistor-transistor logic level trigger signals to lock exposure and high-frequency sampling, while an amplifier maintains anti-artifact logic, ensuring high temporal alignment and purity of electrical signals and image recordings. Furthermore, optical flow methods are used to calculate mechanical contraction indices, ultimately enabling comprehensive, multi-dimensional quantitative calculation of the time delays generated under drug induction in different dimensions. This application not only solves the industry pain points of ineffective fusion of different detection modalities and temporal offset fragmentation but also considers the microenvironmental stability in cell experiments, achieving a system-level leap forward in research on cardiac safety pharmacology and mechanisms.

[0052] The above description is merely a preferred embodiment of this application and does not limit the scope of patent protection of this application. Any equivalent structural or procedural transformations made based on the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of this application.

Claims

1. A full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system, characterized in that, include: An optical mapping microscopy module includes a multi-channel beam splitter, a dual-channel excitation source, and a multi-back-illuminated complementary metal-oxide-semiconductor camera array. The optical mapping microscopy module is configured to acquire calcium fluorescence, voltage-sensitive dye fluorescence, and infrared bright-field image sequences. A multi-channel electrophysiological detection module is located at the bottom of a sample recording bath. The multi-channel electrophysiological detection module includes a semi-transparent microelectrode array and a synchronous data acquisition front end. The substrate of the semi-transparent microelectrode array is made of transparent conductive glass. The semi-transparent microelectrode array includes fine-diameter wires and nanoring electrodes. The main control and analysis module is communicatively connected to the optical mapping and microscopy module and the multi-channel electrophysiological detection module. The main control and analysis module includes a synchronization control logic component, which is configured to issue a global trigger signal to lock the pulse of the dual-channel excitation light source, the exposure window of the multiple back-illuminated complementary metal-oxide-semiconductor camera array, and the sampling clock of the synchronous data acquisition front end. It also extracts the action potential curve, calcium transient curve, and mechanical contraction curve of the tested specimen on the same time axis, and calculates the electro-calcium-mechanical coupling delay parameter of the cell based on the action potential curve, the calcium transient curve, and the mechanical contraction curve.

2. The full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system as described in claim 1, characterized in that, The multi-channel beam splitter is configured as a three-channel beam splitter, which is configured to receive the incoming infrared bright-field illumination light, the calcium fluorescence and the voltage-sensitive dye fluorescence, and separate the three, and guide the separated light signals to the corresponding multiple back-illuminated complementary metal-oxide-semiconductor camera arrays.

3. The full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system as described in claim 2, characterized in that, The dual-channel excitation light source and the multi-channel beam splitter are configured in a coaxial optical architecture, and the wavelengths of the infrared bright-field illumination light, the calcium fluorescence, and the voltage-sensitive dye fluorescence do not overlap in the frequency domain.

4. The full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system as described in claim 1, characterized in that, The transparent conductive glass uses an indium tin oxide substrate, the nanoring electrode is attached to the surface of the indium tin oxide substrate, and the fine-diameter wires are etched along the surface of the indium tin oxide substrate.

5. The full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system as described in claim 4, characterized in that, The fine-diameter wires on the surface of the indium tin oxide substrate are configured to have a diameter of less than 5 μm, and the nanoring electrodes and the fine-diameter wires are arrayed in the upper region of the transparent conductive glass. The overall transmittance of the semi-transparent microelectrode array for ambient light and measurement light is configured to be greater than 85%. The semi-transparent microelectrode array and the multi-channel beam splitter are coaxially aligned.

6. The full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system as described in claim 1, characterized in that, The synchronous control logic component integrates a field-programmable gate array (FPGA), which is configured to emit the global trigger signal. The level type of the global trigger signal is a transistor-to-transistor logic level trigger signal.

7. The full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system as described in claim 6, characterized in that, The field-programmable gate array (FPGA) locks the time period during which the exposure windows of the multiple back-illuminated complementary metal-oxide-semiconductor (CMOS) camera arrays are in the open state with the sampling clock of the synchronous data acquisition front-end by issuing the transistor-to-transistor logic level trigger signal. The time difference between the opening time of the exposure window and the rising edge of the clock is configured to be less than 100 μs. The synchronous data acquisition front-end contains an amplifier. The FPGA is configured to send a forced hold command to the amplifier during the time period during which the exposure window is in the open state, instructing the amplifier to maintain the current level state.

8. The full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system as described in claim 1, characterized in that, The main control and analysis module is configured to perform pixel-level optical flow analysis and adaptive edge contour tracking on the infrared bright field image sequence, output a displacement curve based on the analysis results, and map the displacement curve to the mechanical contraction curve.

9. The full-scale "electro-calcium-mechanical" coupled pharmacodynamic evaluation system as described in claim 8, characterized in that, When executing the pixel-level optical flow method, the main control and analysis module extracts the optical flow field vector between adjacent frames in the infrared bright field image sequence, and locates the adaptive edge contour by combining the gray-level gradient changes of the pixels in the infrared bright field image sequence. The main control and analysis module is configured to extract the normal direction of the adaptive edge contour, integrate the optical flow field vector along the normal direction to generate the displacement curve, and register the time axis of the displacement curve to the reference clock domain of the sampling clock.

10. The full-scale "electro-calcium-mechanism" coupled pharmacodynamic evaluation system as described in claim 9, characterized in that, The electro-calcium-mechanical coupling delay parameters include the electro-calcium delay time and the calcium-contraction delay time. The main control and analysis module is configured to extract the depolarization start point of the action potential curve as the electrical signal start point, extract the rising branch start point of the calcium transient curve as the calcium signal start point, extract the contraction start point of the mechanical contraction curve as the mechanical signal start point, subtract the timestamp corresponding to the electrical signal start point from the timestamp corresponding to the calcium signal start point to obtain the difference as the electro-calcium delay time, and subtract the timestamp corresponding to the calcium signal start point from the timestamp corresponding to the timestamp corresponding to the mechanical signal start point to obtain the difference as the calcium-contraction delay time.

11. The use of the system as described in claim 1, characterized in that, Used for in vitro drug screening of cardiomyocytes, evaluation of cardiotoxicity, or research on excitation-contraction coupling mechanisms.

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