Double-gating optical detection method and system

Abstract

The invention discloses a double-gating optical detection method and system. The method comprises the following steps: generating an acquisition signal through a signal triggering module; controlling a camera module to acquire first fluorescence information in a first time gating and acquire second fluorescence information in a second time gating according to the acquisition signal; and the signal processing module is controlled to calculate and process the first fluorescence information and the second fluorescence information to obtain a ratio image. First fluorescence information is collected through first time gating, interference of autofluorescence of biological tissue on short-life fluorescence of probe molecules is avoided, then second fluorescence information is collected through second time gating, and then ratio calculation is performed according to the first fluorescence information and the second fluorescence information to obtain a ratio image. Therefore, fluorescence signals of the probe molecule and the reference molecule are distinguished, the accuracy of optical imaging in quantitative detection is improved, and the method can be widely applied to the technical field of detection.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a dual-gated optical detection method and system. Background Art

[0002] Optical detection methods have a wide range of applications in the fields of biomedicine and materials science, especially in the quantitative analysis of the concentration of analytes in samples. Currently, a widely used technology is the ratio imaging mode, which relies on a beam of excitation light to simultaneously excite two different fluorescent materials to produce two beams of fluorescence with different wavelengths. By calculating the intensity ratio of these two beams of fluorescence, the content of the analyte in the sample can be effectively evaluated. Compared with the detection method of a single fluorescence intensity signal, this ratio mode has the advantage of removing the interference of probe concentration on quantitative detection.

[0003] Studies have shown that the propagation and penetration of light in a sample are determined by the wavelength of the light and the type of sample. This causes different wavelengths of light to attenuate to different degrees when passing through the sample. Therefore, the fluorescence intensity ratio measured outside the sample may be inconsistent with the actual situation inside the sample, which indicates that the detection result may be affected by the position of the probe inside the sample. In the prior art, different wavelengths of excitation light are used to excite fluorescent probes to obtain two different fluorescence signals. The addition of the analyte may change the energy transfer process under a certain condition, thereby affecting the fluorescence intensity, while the energy transfer process under another condition is generally unaffected. The concentration of the analyte is evaluated by the intensity ratio of the two beams of fluorescence. However, this technology also has challenges because different wavelengths of excitation light have different degrees of attenuation when penetrating the sample. Therefore, under the same concentration of the analyte, the light intensity ratio will also be affected by the thickness and properties of the sample tissue, making it difficult to accurately measure in a complex sample environment. In addition, biological tissues will produce spontaneous fluorescence when irradiated with excitation light, and their fluorescence lifetime is close to the short-lived luminescence signal of the probe, which makes it difficult to distinguish between the two signals in practical applications and reduces the accuracy of quantitative detection.

[0004] Therefore, optical detection methods still need to be further optimized and developed in dealing with the complexity of biological samples in order to improve the accuracy and reliability of detection. Summary of the invention

[0005] In view of this, in order to solve at least one of the technical problems existing in the above-mentioned prior art, an object of the embodiments of the present invention is to provide a dual-gated optical detection method and system, which can improve the accuracy of optical quantitative detection.

[0006] In a first aspect, an embodiment of the present invention provides a dual-gated optical detection method, which is applied to a dual-gated optical detection system. The dual-gated optical detection system includes a signal trigger module, a camera module, and a signal processing module. The dual-gated optical detection method includes:

[0007] Generate an acquisition signal through the signal trigger module;

[0008] According to the acquisition signal, the camera module is controlled to acquire first fluorescence information of the living tissue within a first time gate and second fluorescence information of the living tissue within a second time gate, wherein the first time gate is located between a first lifetime end point of the autofluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, wherein the short-lived fluorescence represents the fluorescence of the reference molecule, and the long-lived fluorescence represents the fluorescence of the probe molecule;

[0009] The signal processing module is controlled to perform calculation processing on the first fluorescence information and the second fluorescence information to obtain a ratio image, wherein the first fluorescence information represents first image data including both short-lived fluorescence and long-lived fluorescence, and the second fluorescence information represents second image data including only long-lived fluorescence.

[0010] In some optional embodiments, generating the acquisition signal by the signal triggering module includes:

[0011] Acquiring acquisition parameters, wherein the acquisition parameters include trigger frequency, trigger time interval and synchronization information;

[0012] storing the acquisition parameters in the signal trigger module, so that the signal trigger module generates a continuous trigger signal after reading the trigger frequency;

[0013] The signal trigger module reads the trigger time interval and the synchronization information and then controls the timing of the continuous trigger signal to obtain the acquisition signal.

[0014] In some optional embodiments, controlling the camera module to collect first fluorescence information of the living tissue within a first time gate and collecting second fluorescence information of the living tissue within a second time gate according to the acquisition signal includes:

[0015] Acquiring a first lifetime of the probe molecule, wherein the first lifetime of the probe molecule represents the lifetime of the long-lived fluorescence;

[0016] Acquiring a second lifetime of the reference molecule, wherein the second lifetime of the reference molecule represents the lifetime of the short-lived fluorescence;

[0017] Determine the first time gate, the second time gate and the acquisition period according to the first life span of the probe molecule, the second life span of the reference molecule and the acquisition signal;

[0018] The camera module is controlled to collect the first fluorescence information within the first time gate of each acquisition cycle, and to collect the second fluorescence information within the second time gate of each acquisition cycle.

[0019] In some optional embodiments, controlling the camera module to collect the first fluorescence information within the first time gate of each acquisition cycle and collecting the second fluorescence information within the second time gate of each acquisition cycle includes:

[0020] In each of the acquisition cycles, the first acquisition frequency, the first acquisition duration, the second acquisition frequency and the second acquisition duration of the camera module are controlled by the acquisition signal, wherein the first acquisition frequency represents the frequency at which the camera module acquires the first fluorescence information within the first time gating, the second acquisition frequency represents the frequency at which the camera module acquires the second fluorescence information within the second time gating, the first acquisition duration represents the duration for which the camera module acquires the first fluorescence information, and the second acquisition duration represents the duration for which the camera module acquires the second fluorescence information;

[0021] When the short-lived fluorescence decays to the end point of the second lifetime and the time used to collect the long-lived fluorescence is not less than the second lifetime of the reference molecule, start stimulating the excitation light for the next collection period so that the excitation light excites the spontaneous fluorescence, the short-lived fluorescence and the long-lived fluorescence within the next collection period.

[0022] In some optional embodiments, the camera module includes an external trigger signal generator and a time-gated acquisition unit, and after the acquisition signal is generated by the signal trigger module, the camera module further includes:

[0023] Generate a TTL signal by means of the external trigger signal generator;

[0024] Modulating the laser by the TTL signal so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing to excite the fluorescent material to reach an excited state, wherein the fluorescent material includes the probe molecule and the reference molecule;

[0025] The time-gated acquisition unit acquires the first fluorescence information within a first time gate, and acquires the second fluorescence information within a second time gate.

[0026] In some optional embodiments, the dual-gated imaging system further includes an image conversion module, and the method further includes:

[0027] The signal processing module calculates the ratio of the first image data and the second image data to generate a ratio image;

[0028] The ratio image is converted into quantitative information through the image conversion module and the preset working curve.

[0029] In some optional embodiments, the generating a ratio image after calculating the ratio of the first image data to the second image data by the signal processing module includes:

[0030] Performing pixel comparison operation on the first image data and the second image data by the signal processing module to obtain first luminescence data and second luminescence data in the first image data, wherein the first luminescence data represents image data corresponding to the short-lived fluorescence, and the second luminescence data represents image data corresponding to the long-lived fluorescence;

[0031] Deleting the second light emitting data from the first image data by the signal processing module to obtain third image data, wherein the third image data only includes the first light emitting data;

[0032] The signal processing module calculates the ratio of the second image data and the third image data to generate the ratio image.

[0033] In some optional embodiments, converting the ratio image into quantitative information by using the image conversion module and a preset working curve includes:

[0034] Reading the ratio value of each pixel in the ratio image by the image conversion module;

[0035] Acquiring the preset working curve through the image conversion module, wherein the preset working curve represents a relationship curve between a preset ratio value and an actual concentration, wherein the actual concentration represents environmental information of biological tissue or concentration information of biological molecules;

[0036] The image conversion module converts the ratio value of each pixel into the actual concentration of each pixel corresponding to the preset working curve;

[0037] The image conversion module converts each pixel in the ratio image into the actual concentration of each pixel to obtain a quantitative image, and the quantitative image indicates the quantitative information.

[0038] In some optional embodiments, the probe molecule uses a first polystyrene microsphere as a matrix, the interior of the first polystyrene microsphere is coated with a rare earth complex, and the exterior of the first polystyrene microsphere is coated with silica; the reference molecule uses a second polystyrene microsphere as a matrix, the interior of the second polystyrene microsphere is coated with a transition metal complex, and the exterior of the second polystyrene microsphere is coated with silica.

[0039] In some optional embodiments, the rare earth complex is preferably one of a europium complex, an erbium complex, a thulium complex, and a terbium complex, and the transition metal complex is preferably one of an iridium complex and a platinum complex.

[0040] In some optional embodiments, the europium complex is preferably a structure as described in Formula I, and the iridium complex is preferably a structure as described in Formula II:

[0041]

[0042] In a second aspect, an embodiment of the present invention provides a dual-gated optical detection system, comprising:

[0043] A signal trigger module, used for generating an acquisition signal;

[0044] A camera module, configured to collect first fluorescence information of the living tissue within a first time gate and second fluorescence information of the living tissue within a second time gate after receiving the collection signal, wherein the first time gate is located between a first lifetime end point of the autofluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, wherein the short-lived fluorescence represents the fluorescence of the reference molecule, and the long-lived fluorescence represents the fluorescence of the probe molecule;

[0045] A signal processing module is used to calculate and process the first fluorescence information and the second fluorescence information to obtain a ratio image, wherein the first fluorescence information represents first image data including both short-lived fluorescence and long-lived fluorescence, and the second fluorescence information represents second image data including only long-lived fluorescence.

[0046] In some optional embodiments, the camera module includes an external trigger signal generator and a time-gated acquisition unit;

[0047] The external trigger signal generator is used to generate a TTL signal to modulate the laser, so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing to excite the fluorescent material to reach an excited state, wherein the fluorescent material includes the probe molecule and the reference molecule;

[0048] The time-gated acquisition unit is used to acquire the first fluorescence information within a first time gate and to acquire the second fluorescence information within a second time gate.

[0049] In some optional embodiments, the signal triggering module includes:

[0050] A control interface, used to input and / or adjust acquisition parameters, and store the acquisition parameters in a parameter storage unit of the signal trigger module;

[0051] A control unit, used for reading the acquisition parameters, wherein the acquisition parameters include a trigger frequency, a trigger time interval and synchronization information;

[0052] a timer and / or an oscillator, for generating a continuous trigger signal according to the trigger frequency;

[0053] The control circuit is used to control the timing of the continuous trigger signal according to the trigger time interval and the synchronization information to obtain the acquisition signal, and transmit the acquisition signal to the camera module.

[0054] In some optional embodiments, the camera module includes:

[0055] A control interface for inputting or adjusting a first lifetime of a probe molecule and a second lifetime of a reference molecule;

[0056] a calculation unit, configured to determine the first time gate, the second time gate and an acquisition period according to the first lifespan of the probe molecule, the second lifespan of the reference molecule and the acquisition signal;

[0057] a trigger controller, configured to determine the second lifetime end point of the short-life fluorescence and start a next acquisition cycle at the second lifetime end point;

[0058] An image acquisition unit, configured to acquire an image after receiving the acquisition signal;

[0059] The synchronous processing unit synchronously processes the short-life fluorescence and the long-life fluorescence in each acquisition cycle.

[0060] In some optional embodiments, the trigger controller includes:

[0061] A control unit, used to control the acquisition frequency and duration of the camera module;

[0062] a decay time determination unit, configured to start a next collection cycle when the short-lived fluorescence decays to the end point of the second lifetime and the time for collecting the long-lived fluorescence is not less than the second lifetime of the reference molecule;

[0063] The timing control unit collects the steady state of the long-life fluorescence according to the preset timing control conditions.

[0064] In some optional embodiments, the time-gated acquisition unit includes:

[0065] The time interval setting interface is used to obtain a first time interval and a second time interval of the camera module, the first time gate is located in the first time interval, and the second time gate is located in the second time interval.

[0066] In some optional embodiments, the signal processing module includes:

[0067] a data separation unit, configured to perform pixel comparison operation on the first image data and the second image data to separate the first image data into first luminescence data and second luminescence data, wherein the first luminescence data represents image data corresponding to the short-lived fluorescence, and the second luminescence data represents image data corresponding to the long-lived fluorescence;

[0068] a difference calculation unit, configured to obtain third image data by deleting the second light emitting data from the first image data, wherein the third image data only includes the first light emitting data;

[0069] A ratio calculation unit is used to generate the ratio image after performing ratio calculation on the second image data and the third image data.

[0070] In some optional embodiments, the dual-gated optical detection system further includes an image conversion module, and the image conversion module includes:

[0071] A working curve storage unit, used to store a preset working curve pre-determined according to samples of different concentrations or concentration gradients;

[0072] A ratio reading unit, used for reading the ratio value of each pixel in the generated ratio image;

[0073] The search unit is used to search for the actual concentration in the preset working curve and then determine the corresponding quantitative information.

[0074] In some optional embodiments, the probe molecule includes a europium complex Eu(tta)3TADF, and the reference molecule includes an iridium complex Ir(tfpqz)2dbm.

[0075] In a third aspect, an embodiment of the present invention provides a controller, comprising: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the above method when executing the computer program.

[0076] In a fourth aspect, an embodiment of the present invention provides a computer storage medium, wherein the computer storage medium stores computer executable instructions, and the computer executable instructions are used to execute the above-mentioned dual-gated optical detection system.

[0077] The dual-gated optical detection method according to the embodiment of the present invention has at least the following beneficial effects: the first fluorescence information is collected by first time gating to prevent the short-lived fluorescence of the probe molecule from being interfered by the spontaneous fluorescence of the biological tissue, and then the second fluorescence information is collected by second time gating, and then a ratio image is obtained by performing ratio calculation based on the first fluorescence information and the second fluorescence information, thereby distinguishing the fluorescence signals of the probe molecule from the reference molecule, thereby improving the imaging accuracy of quantitative detection of living bodies.

[0078] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 is a flowchart of a dual-gated optical detection method provided by an embodiment of the present invention;

[0080] Figure 2 is a schematic structural diagram of a dual-gated optical detection system provided by an embodiment of the present invention;

[0081] Figure 3 is a collection schematic diagram of a camera module provided by an embodiment of the present invention;

[0082] Figure 4 is a ratio information diagram of different temperature points provided by an embodiment of the present invention;

[0083] Figure 5 This is a schematic diagram of ratios under occlusion of different biological components provided by an embodiment of the present invention:

[0084] Figure 6 This is a diagram of the imaging effects of different luminescent material combinations provided by the embodiments of the present invention under different tissue shielding. DETAILED DESCRIPTION

[0085] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0086] In the embodiments of the present invention, words such as "exemplary" or "for example" are used to indicate examples, illustrations or explanations. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present invention should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0087] In the following, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, unless otherwise specified, "plurality" means two or more.

[0088] First, refer to Figure 1 The embodiment of the present invention provides a dual-gated optical detection method, which is applied to a dual-gated optical detection system, referring to Figure 2 The dual-gated optical detection system includes a signal trigger module, a camera module and a signal processing module. The dual-gated optical detection method includes:

[0089] S100, generating a collection signal through a signal trigger module;

[0090] S200, controlling the camera module to collect first fluorescence information of the living tissue within a first time gate and second fluorescence information of the living tissue within a second time gate according to the collection signal, wherein the first time gate is located between a first lifetime end point of the autofluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, wherein the short-lived fluorescence represents the fluorescence of the reference molecule, and the long-lived fluorescence represents the fluorescence of the probe molecule;

[0091] S300, the control signal processing module calculates and processes the first fluorescence information and the second fluorescence information to obtain a ratio image, wherein the first fluorescence information represents first image data including both short-lived fluorescence and long-lived fluorescence, and the second fluorescence information represents second image data including only long-lived fluorescence.

[0092] Specifically, refer to Figure 3 , Figure 3The first end point of the life of the medium spontaneous fluorescence is T1, and the second end point of the life of the short-lived fluorescence is T2, where the acquisition period is T, the first time gate is the "long + short" acquisition interval between T1 and T2, and the second time gate is the "long" acquisition interval after T2. The dual-gated optical detection system realizes dual-gated imaging for in vivo quantitative detection. The signal trigger module in the dual-gated optical detection system generates a continuous trigger signal (i.e., an acquisition signal) with preset acquisition parameters (including acquisition time point, acquisition frequency, etc.) to the camera module; after receiving the continuous trigger signal, the camera module performs image acquisition according to the preset acquisition parameters, specifically acquiring first fluorescence information of living tissue within a first time gate, and acquiring second fluorescence information of living tissue within a second time gate, the first fluorescence information is the first image data corresponding to short-lived fluorescence and long-lived fluorescence within the first time gate, and the second fluorescence information is the second image data corresponding to long-lived fluorescence within the second time gate; the first time gate is located between the first lifetime end point of the spontaneous fluorescence of the living tissue and the second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and the third lifetime end point of the long-lived fluorescence, and the specific setting time points are not limited here. The acquired first fluorescence information and second fluorescence information are sent to the signal processing module, which performs ratio calculation on the first image data and the second image data to generate a ratio image; the ratio image is transmitted to the image conversion module for conversion, and the image conversion module converts the ratio image into the required quantitative information through a preset working curve, and the quantitative information is the actual concentration information corresponding to the ratio value.

[0093] In the above embodiments, "preset acquisition parameters" generally means that before conducting an experiment, the user can set the camera acquisition parameters, such as exposure time, acquisition speed and trigger frequency, according to the experimental objectives and conditions. These parameters will affect the clarity, brightness and frame rate of the image. The continuous signal trigger unit is responsible for generating a series of continuous trigger signals according to these preset acquisition parameters. These signals are used to control the camera module, such as the IsCOMS camera, so that it can perform image acquisition within a set time interval. The specific first time gating, second time gating, second life end point and third life end point are all parameters determined by experiments, and the specific determination method is not determined here.

[0094] In some optional embodiments, an acquisition signal is generated by a signal trigger module, including: obtaining acquisition parameters, the acquisition parameters including a trigger frequency, a trigger time interval and synchronization information; storing the acquisition parameters in the signal trigger module so that the signal trigger module generates a continuous trigger signal after reading the trigger frequency; and controlling the timing of the continuous trigger signal after reading the trigger time interval and synchronization information through the signal trigger module to obtain the acquisition signal.

[0095] Specifically, the signal trigger unit generates precise electrical signals (TTL signals) that trigger the camera module to capture images at a specific frequency and exposure time. For example, the trigger signal ensures that the camera can capture images at a speed of 20fps (frames per second), thereby achieving video-level real-time imaging.

[0096] It is understood that the signal trigger module is responsible for coordinating the synchronous operation of the camera module and other parts of the imaging system in a specific implementation. The signal trigger module generally includes a signal generator, a control computer or controller, a trigger signal distributor, and a camera interface. The signal generator is used to generate a trigger signal, and the signal generator can be configured by the control computer to set the frequency, pulse width, and duty cycle of the signal. It is usually connected to the control computer via USB, Ethernet, or a dedicated data line, so that the user can set parameters through the software interface. The control computer is also responsible for providing a user interface for setting acquisition parameters and controlling the entire signal trigger module. It communicates with the signal generator through a data bus (such as USB or PCIe), and can also coordinate with the camera and other system components to ensure the synchronous operation of the entire system. The trigger signal distributor receives the signal from the signal generator and distributes it to multiple devices, such as cameras and lasers. The distributor is usually connected to the signal generator and the controlled device by a cable to ensure accurate transmission of the signal. The camera interface is the output part of the signal trigger module, and the camera module receives the trigger signal through the camera interface to start image acquisition.

[0097] When the camera module receives a continuous trigger signal, it starts the image acquisition process according to the pre-set acquisition parameters. The camera module is usually connected to the trigger signal distributor of the signal trigger module and receives the timing trigger signal from the signal generator. Once the trigger signal is passed to the camera module, the camera will immediately start image acquisition. The camera's acquisition parameters usually include exposure time, frame rate, resolution, and image format. During the acquisition process, the camera will continuously receive trigger signals to ensure that the start and end of each exposure can be synchronized with other components in the system.

[0098] In some optional embodiments, the dual-gated imaging system for quantitative detection of living organisms also includes an image conversion module, and the method also includes: generating a ratio image after calculating the ratio of the first image data and the second image data through the signal processing module; and converting the ratio image into quantitative information through the image conversion module and a preset working curve.

[0099] Specifically, after completing image acquisition, the camera module will transmit the acquired image data (specifically, the first image data and the second image data) to the signal processing module for further data processing. The image data is usually transmitted through a high-speed data interface (such as USB 3.0 or optical fiber). After receiving the image data, the signal processing module starts to perform a specific image processing task. In this case, the signal processing module performs ratio calculation on the image data to generate a ratio image. The ratio calculation is achieved by performing mathematical operations on the pixel values ​​in the image, and this operation usually involves ratio processing of image data acquired under two or more different conditions. Specifically, the signal processing module performs pixel-level ratio operations on the received multiple images. For example, two images under different fluorescence conditions are acquired, and the signal processing module performs ratio calculations on the corresponding pixel values ​​of these images one by one to generate a new ratio image. The image conversion module converts the ratio image into quantitative information according to a preset working curve.

[0100] In some optional embodiments, a ratio image is generated after a signal processing module performs a ratio calculation on the first image data and the second image data, including: performing a pixel comparison operation on the first image data and the second image data by the signal processing module to obtain first luminescence data and second luminescence data in the first image data, the first luminescence data representing image data corresponding to short-lived fluorescence, and the second luminescence data representing image data corresponding to long-lived fluorescence; deleting the second luminescence data in the first image data by the signal processing module to obtain third image data, the third image data only including the first luminescence data; and generating a ratio image after a signal processing module performs a ratio calculation on the second image data and the third image data.

[0101] Specifically, the collected image data is first separated into luminescence signals including long-lifetime and short-lifetime (i.e., the first image data), and luminescence signals only including long-lifetime (i.e., the second image data); the separated first image data and second image data are calculated to respectively obtain the first luminescence data and the second luminescence data in the first image data, wherein the first luminescence data is the image data corresponding to the short-lifetime fluorescence in the first image data, and the second luminescence data is the image data corresponding to the long-lifetime fluorescence in the first image data; the image data of the long-lifetime luminescence signal in the first image data is subtracted and deleted to obtain the image data of the short-lifetime luminescence signal (i.e., the third image data); the ratio of the image data of the short-lifetime luminescence signal to the image data of the long-lifetime luminescence signal (the second image data) is calculated; and the calculation result is converted into a ratio image.

[0102] In some optional embodiments, a time gating window is set by a camera module to collect fluorescence image data containing different lifespan luminescence signals. Using the two set time gating windows (first time gating and second time gating), first separate the image data containing long-life and short-life luminescence signals (first image data collected by the first time gating), and the image data containing only long-life luminescence signals (second image data collected by the second time gating), the long-life luminescence signal is the signal of long-life fluorescence, and the short-life luminescence signal is the signal of short-life fluorescence. The separated image data is calculated by an image processing algorithm. Specifically, from the image data (first image data) containing long-life and short-life luminescence signals, the image data (second image data or second luminescence data in the first image data) containing only long-life luminescence signals is subtracted. Through the above-mentioned subtraction operation, image data (first luminescence data in the first image data) containing only short-life luminescence signals is obtained, which is based on the difference in signal lifetimes, ensuring that the influence of long-life luminescence signals is effectively removed. The image data of the short-lived luminescence signal and the image data of the long-lived luminescence signal are calculated pixel by pixel; that is, for each pixel of the image, the ratio of the intensity of the short-lived luminescence signal to the intensity of the long-lived luminescence signal is calculated. Through the ratio calculation, an image (ratio image) that emphasizes the contrast of luminescence signals with different lifetimes can be obtained. The ratio reflects the relative intensity of different signal components. The above ratio results are converted into a ratio image through appropriate color mapping and image enhancement technology. For example, the ratio image can be presented through different pseudo-color coding for easy observation and analysis.

[0103] In some optional embodiments, the ratio image is converted into quantitative information through an image conversion module and a preset working curve, including: reading the ratio value of each pixel in the ratio image through the image conversion module; obtaining the preset working curve through the image conversion module, the preset working curve representing a relationship curve between a preset ratio value and an actual concentration, the actual concentration representing environmental information of biological tissue or concentration information of biological molecules; converting the ratio value of each pixel into the actual concentration of each pixel corresponding to the preset working curve through the image conversion module; and obtaining a quantitative image after converting each pixel in the ratio image into the actual concentration of each pixel through the image conversion module, the quantitative image indicating quantitative information.

[0104] Specifically, according to samples of different concentrations or concentration gradients, their corresponding ratio values ​​are measured in advance, and a working curve between the ratio value and the actual concentration is generated, which is a preset working curve; the ratio value of each pixel in the generated ratio image is read, and the corresponding quantitative information is determined according to the preset working curve.

[0105] For the establishment of the preset working curve, a series of samples with known different concentrations or concentration gradients are prepared. The same imaging system is used to image and process these samples with known concentrations, generate their ratio images, and record the ratio value of each sample. The measured ratio value is paired with the corresponding actual concentration data and drawn into a working curve. This is a standard curve that reflects the relationship between the ratio value and the actual concentration, which can usually be obtained by linear or nonlinear fitting. For the generated ratio image, the ratio value is read pixel by pixel. This requires the use of image processing software or algorithms to obtain the ratio value data of each pixel point one by one. Using the pre-established working curve (preset working curve), the ratio value of each pixel is converted into corresponding quantitative information. Specifically, for each ratio value, the actual concentration corresponding to it is found by searching the working curve. The concentration information obtained in the above steps is assigned to each pixel position in the ratio image; in this way, the original ratio image is converted into a quantitative image reflecting the actual concentration distribution, and pseudo-color coding can be applied to the generated quantitative image to facilitate the intuitive presentation of the concentration distribution; similarly, different concentration ranges can be displayed by color scales or color scales.

[0106] The ratio image generated by the signal processing module is input into the image conversion module. The image conversion module uses a pre-established working curve, that is, a preset working curve, to convert the ratio information of the image into quantitative data / information. The "preset working curve" is a tool used to describe the relationship between the input signal (such as optical density, fluorescence intensity, etc.) and the target quantitative value (such as concentration, activity level, etc.). The working curve is usually obtained through experimental calibration. Standard samples with known concentrations or known parameters are used for imaging. By measuring the signal response of these standard samples, the relationship between the signal intensity and the standard parameters is plotted. Then, a suitable mathematical model (such as linear, logarithmic, exponential or polynomial model) is selected to fit the data points; the model parameters are optimized by the least squares method or other statistical methods to ensure that the model can accurately describe the experimental data. In the image conversion process, the ratio value of each pixel is used as input, and these ratio values ​​are converted into corresponding quantitative values ​​(quantitative information) by searching or calculating using the preset working curve; in this way, accurate conversion of image data to quantitative data can be achieved, thereby supporting further analysis and application. The quantitative data or quantitative information here can be physiological environmental parameters such as temperature, oxygen concentration, viscosity, pH, etc., or concentration information of small molecules, ions or biomacromolecules, etc.

[0107] In some optional embodiments, an acquisition signal is generated by a signal trigger module, including: obtaining acquisition parameters, the acquisition parameters including a trigger frequency, a trigger time interval and synchronization information; storing the acquisition parameters in the signal trigger module so that the signal trigger module generates a continuous trigger signal after reading the trigger frequency; and controlling the timing of the continuous trigger signal after reading the trigger time interval and synchronization information through the signal trigger module to obtain the acquisition signal.

[0108] Specifically, the acquisition parameters are input or adjusted through the control interface. These parameters may include but are not limited to exposure time, gain, frame rate, resolution, trigger frequency, trigger time interval and synchronization information, etc. The system receives and stores these parameters in the parameter storage unit of the signal trigger module. The control unit reads the acquisition parameters from the parameter storage unit, and these parameters will be used to set the generation conditions of the continuous trigger signal. The control unit generates a continuous trigger signal through a timer and / or an oscillator according to the read acquisition parameters. According to the preset trigger time interval and the synchronization requirements of the synchronization information, the control unit manages and adjusts the timing of the continuous trigger signal to ensure that the time interval of each trigger signal is consistent and synchronized with other system modules (such as a light source or a processing unit). The control unit then transmits the generated continuous trigger signal to the camera module. After the camera module receives the trigger signal, it performs an image acquisition operation according to the set acquisition parameters. After that, the camera module immediately performs image acquisition after receiving each trigger signal, and transmits the acquired image data to the subsequent image processing unit for processing.

[0109] In some optional embodiments, the camera module is controlled to collect first fluorescence information of living tissue within a first time gate and second fluorescence information of living tissue within a second time gate according to an acquisition signal, including: acquiring a first lifetime of the probe molecule, the first lifetime of the probe molecule characterizing the lifetime of long-lived fluorescence; acquiring a second lifetime of the reference molecule, the second lifetime of the reference molecule characterizing the lifetime of short-lived fluorescence; determining a first time gate, a second time gate and an acquisition period according to the first lifetime of the probe molecule, the second lifetime of the reference molecule and the acquisition signal; and controlling the camera module to collect the first fluorescence information within the first time gate of each acquisition period and to collect the second fluorescence information within the second time gate of each acquisition period.

[0110] Specifically, the lifetime parameters of the probe molecule and the reference molecule are input or adjusted through the control interface, and the system automatically sets the acquisition period according to these parameters; the system calculates the time interval between the maximum signal and the minimum background noise as the acquisition period according to the lifetime difference between the probe and the reference molecule, and the specific acquisition period is equal to the excitation time of the excitation light + the first lifetime of the spontaneous fluorescence + the second lifetime of the short-lived fluorescence + the time for collecting the long-lived fluorescence; the first time gate is located between the end point of the first lifetime of the spontaneous fluorescence and the end point of the second lifetime of the short-lived fluorescence, and the first fluorescence information collected by the camera module within the first time gate includes both the short-lived fluorescence and the long-lived fluorescence; the second time gate is located between the end point of the second lifetime of the short-lived fluorescence and the end point of the third lifetime of the long-lived fluorescence, and the second fluorescence information collected by the camera module within the second time gate includes only the long-lived fluorescence. Specifically, the second time gating is set after the end point of the second lifetime of the short-lived fluorescence. The next excitation and acquisition cycle begins after the second time gating ends, so there is no need to wait for the long-lived fluorescence to decay, thereby improving the acquisition efficiency; the camera module immediately starts a new image acquisition after receiving the next excitation trigger signal; the camera module synchronously processes the short-lived and long-lived luminescence signals in each acquisition cycle, so that the ratio calculation is performed within the set acquisition time.

[0111] It should be noted that the "lifetime parameter" mentioned in the embodiment mainly refers to the fluorescence lifetime of the probe molecule or reference molecule, that is, the time it takes for them to return to the ground state after excitation. The fluorescence lifetime of different molecules may change due to their chemical structure, environmental changes or interactions. The "probe molecule" mentioned in the embodiment refers to a fluorescent molecule used to detect a specific target or environmental condition, and its lifespan parameter affects the intensity and duration of the fluorescence signal emitted under the action of the excitation light source. The "reference molecule" mentioned in the embodiment generally refers to a fluorescent molecule used as a reference or control standard in an experiment or in a detection, and is usually a fluorescent molecule with a known and stable fluorescence lifetime parameter for calibrating or comparing probe molecules. It is understood that the lifespan parameter of the reference molecule is usually not affected by external conditions. By measuring the fluorescence lifetime of the probe molecule and the reference molecule, multi-parameter analysis can be performed, such as distinguishing different biochemical environments or identifying specific biomolecules. In this embodiment, the lifespan parameters of the probe molecule and the reference molecule are used to automatically calculate the acquisition cycle, and determine the first time gating and the second time gating to ensure that the maximum signal intensity and the minimized background noise are obtained in the image acquisition.

[0112] It should be noted that the "long life" and "short life" mentioned in the embodiments are relative. Generally speaking, the lifespans of the probe molecules and the reference molecules are quite different, for example, the difference is not less than 10 times. Short-lived luminescence signals refer to situations where the fluorescence lifetime of the probe molecules or reference molecules is relatively short, usually on a time scale of a few nanoseconds to tens of nanoseconds. These signals decay rapidly and need to be quickly collected before the signal decays completely to avoid information loss. Long-lived luminescence signals refer to situations where the fluorescence lifetime of the probe molecules or reference molecules is relatively long. Usually on a time scale of hundreds of nanoseconds to microseconds, these signals take longer to decay completely, so they can be collected within a longer time interval; therefore, when the fluorescence of the probe molecule is a long-lived luminescence signal, the fluorescence of the reference molecule is a short-lived luminescence signal.

[0113] In some optional embodiments, the camera module is controlled to collect the first fluorescence information within the first time gate of each acquisition cycle and to collect the second fluorescence information within the second time gate of each acquisition cycle, including: in each acquisition cycle, the first acquisition frequency, the first acquisition duration, the second acquisition frequency and the second acquisition duration of the camera module are controlled by the acquisition signal, the first acquisition frequency represents the frequency of the camera module collecting the first fluorescence information within the first time gate, the second acquisition frequency represents the frequency of the camera module collecting the second fluorescence information within the second time gate, the first acquisition duration represents the duration of the camera module collecting the first fluorescence information, and the second acquisition duration represents the duration of the camera module collecting the second fluorescence information; when the short-lived fluorescence decays to the end point of the second lifetime and the time for collecting the long-lived fluorescence is not less than the second lifetime of the reference molecule, the excitation light of the next acquisition cycle is started to be excited, so that the excitation light excites the spontaneous fluorescence, the short-lived fluorescence and the long-lived fluorescence in the next acquisition cycle.

[0114] Specifically, within each acquisition cycle, a continuous trigger signal is used to control the acquisition frequency and duration of the camera module, and the first frequency and first duration for acquiring the first fluorescence information within the first time gate may be equal to or unequal to the second frequency and second duration for acquiring the second fluorescence information within the second time gate, and are specifically set according to the acquisition requirements; when the short-lived luminescence signal decays to a preset threshold (that is, decays to the end point of the second lifetime) and a lifetime time not less than that of the short-lived luminescence signal is left to acquire the long-lived luminescence signal, the next excitation and acquisition cycle is started; the system acquires the steady-state signal of the long-lived luminescence signal in the second time gate according to the preset timing control conditions.

[0115] It should be noted that the "short-life luminescent signal decays to a preset threshold value" in the embodiment refers to a signal strength level set by the system. When the intensity of the short-life luminescent signal drops to this level, it is considered that the signal has basically decayed. The preset threshold is determined to end the collection of the short-life luminescent signal when the signal intensity is weak enough and no longer significantly affects the subsequent collection, which helps to distinguish between short-life and long-life luminescent signals and avoid the interference of short-life luminescent signals on the collection of long-life luminescent signals. The "reservation of a life time not less than the short-life luminescent signal to collect the long-life luminescent signal" in the embodiment means that in order to ensure the full collection of the long-life luminescent signal, the collection time reserved by the system is at least the life time of the short-life luminescent signal. That is, the second life. This setting ensures that the long-life luminescent signal can be fully collected during its steady state, and also ensures that the long-life luminescent signal will not be interfered by the former after the short-life luminescent signal is completely attenuated. That is, after the system detects that the short-life luminescent signal decays to the preset threshold, it starts a timer to ensure that the long-life luminescent signal is collected within the next time (at least equal to twice the life of the long-life luminescent signal). During this period, the system synchronously collects and processes the long-lived luminescent signal according to the preset timing control conditions. The "steady-state signal" mentioned in the embodiment is accurately understood as an approximate steady state. The approximate steady-state period refers to the period during which the rate of change of the signal intensity is significantly reduced during the attenuation of the long-lived luminescent signal, showing a relatively stable state. Although the signal may still be slowly decaying, its amplitude of change can be ignored in a short period of time, so it is close to a steady state. By collecting data on the steady-state signal during the approximate steady-state period, the impact of short-lived luminescent signals and noise can be minimized, and the signal-to-noise ratio of the signal can be improved. In a specific implementation, the system monitors the signal attenuation curve, identifies the time period when the rate of change is significantly reduced, and sets it as the approximate steady-state period.

[0116] When the lifetime difference between the probe molecule and the reference molecule is large (usually >10 times), it is not necessary to allow the long-lived luminescence signal to decay completely before proceeding to the next acquisition cycle in one acquisition cycle. When the short-lived luminescence signal has decayed completely and there is enough time (not less than the second lifetime) to collect the long-lived luminescence signal, the next excitation and acquisition cycle can be started. At this time, the long-lived luminescence signal will be in an approximate steady state. However, when all timing control conditions are fixed, this approximate steady-state acquisition will not affect the accuracy of the dual-gated same-wavelength ratiometric signal. Moreover, this transient-approximate steady-state acquisition method can greatly improve the time utilization rate in the acquisition cycle, thereby efficiently collecting weak signals and improving imaging speed, which is conducive to real-time processing of quantitative signals.

[0117] In some optional embodiments, the camera module includes an external trigger signal generator and a time-gated acquisition unit. After the acquisition signal is generated by the signal trigger module, it also includes: generating a TTL signal by the external trigger signal generator; modulating the laser by the TTL signal so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing, and excites the fluorescent material to reach an excited state, and the fluorescent material includes a probe molecule and a reference molecule; and collecting first fluorescence information within a first time gate and collecting second fluorescence information within a second time gate by the time-gated acquisition unit.

[0118] Specifically, the external trigger signal generator of the camera module generates a TTL signal, and the TTL signal modulates the laser to emit a pulsed laser to excite the fluorescent material (probe molecule and reference molecule) to an excited state; the first fluorescent signal within the first time gate in the first interval and the second fluorescent signal within the second time gate in the second interval are continuously collected through the time-gated acquisition unit of the camera module, the first fluorescent signal within the first time gate in the first interval contains both long-life and short-life fluorescent signals, and the second fluorescent signal within the second time gate in the second interval contains only long-life fluorescent signals. The time-gated acquisition unit of the camera module sets the length and start time of the two time intervals through the camera module control interface, the first interval is set as the time period from the end of excitation to the complete attenuation of the short-life luminescent signal, and the second interval is set as the time period containing only the long-life luminescent signal after the short-life luminescent signal is completely attenuated.

[0119] In the specific implementation, the spontaneous fluorescence of biological tissues usually has a very short life span (nanosecond level). In contrast, rare earth complex probe molecules and transition metal complex reference molecules have a luminescence life span of more than microseconds. Setting a time gating window is to set the first time gating, which starts shortly after excitation to avoid the strong signal time period of spontaneous fluorescence. Through this first time gating, the fluorescence signals of the probe molecules and reference molecules are mainly collected, and the spontaneous fluorescence signals have decayed within the first time gating, thereby effectively removing their interference. Although the probe molecules (rare earth complexes) and reference molecules (transition metal complexes) are both at the microsecond level, their life spans are significantly different. Setting a second time gating window is the second time gating, which is separated according to the difference in life spans of the probe and reference molecules. In the second time gating, not only is the spontaneous fluorescence excluded, but the signals of the probe molecules and the reference molecules can also be further distinguished according to the difference in life spans, that is, the fluorescence signal of the second time gating is only a long-lived fluorescence signal, that is, the fluorescence signal of the probe molecule. The first image data and the second image data are acquired by first time gating and second time gating, and the signal intensity ratio of the probe molecule and the reference molecule is calculated to construct a ratio probe. Since the probe molecule and the reference molecule have the same emission wavelength, the influence of wavelength difference on quantitative imaging is eliminated. Double gating ensures that the influence of autofluorescence is effectively removed in the final imaging.

[0120] In some optional embodiments, the camera module includes: a control interface for inputting or adjusting the lifetime parameters of the probe and reference molecules and automatically setting the acquisition period; a calculation unit for calculating the optimal acquisition period based on the lifetime difference between the probe molecules and the reference molecules and the acquisition parameters; a trigger controller for determining the time required for the short-lived luminescence signal to completely decay and determining the next excitation and acquisition period; an image acquisition unit for starting image acquisition after receiving the excitation trigger signal; and a synchronization processing unit for synchronously processing the short-lived and long-lived luminescence signals in each acquisition period so as to perform ratio calculation within the set acquisition time.

[0121] It is understood that the control interface allows the user to input or adjust the lifetime parameters of the probe and reference molecules. These parameters include typical lifetime values ​​of short-lived and long-lived luminescent signals. Then, based on the input lifetime parameters, the calculation unit automatically calculates and sets the appropriate acquisition cycle to ensure that short-lived and long-lived luminescent signals can be fully captured in each cycle. Then, using the lifetime parameters of the probe molecule and the reference molecule, the system calculates the difference in lifetime between the two. This difference determines the time window setting of the acquisition cycle. Based on the difference in lifetime, the optimal acquisition cycle is calculated. This cycle needs to be long enough to ensure that the short-lived luminescent signal is completely attenuated while allowing for sufficient acquisition of the long-lived luminescent signal. The trigger controller is responsible for determining the time required for the short-lived luminescent signal to completely decay, that is, the time point when the signal drops to the background noise level. After the long-lived luminescent signal is acquired, the trigger controller sends a signal to start the next excitation and acquisition cycle. This trigger mechanism ensures the accuracy and synchronization of periodic acquisition. After receiving a new excitation trigger signal, the image acquisition unit can immediately start a new image acquisition to ensure that the capture of the short-lived luminescent signal is not missed. In each acquisition cycle, the acquisition unit records the complete fluorescence signal data for subsequent processing. In each acquisition cycle, the synchronous processing unit processes the short-life and long-life luminescence signals in real time. Synchronous processing ensures that the ratio calculation of the two types of signals is performed in the same time frame. Within the set acquisition time, the ratio calculation is performed in real time using the collected signal data to generate a ratio image synchronized with the cycle.

[0122] In some optional embodiments, the trigger controller further includes: a control unit, which uses a continuous trigger signal to control the acquisition frequency and acquisition duration of the camera module; an attenuation duration determination unit, which is used to start the next excitation and acquisition cycle when the short-lived luminescence signal is completely decayed and there is sufficient time to acquire the long-lived luminescence signal; and a timing control unit, which acquires the approximate steady state of the long-lived luminescence signal according to preset timing control conditions.

[0123] Specifically, the continuous trigger signal unit is responsible for generating a series of continuous trigger signals, which are used to control the acquisition frequency and acquisition duration of the camera module. According to the experimental requirements and signal characteristics, an appropriate acquisition frequency is set. The frequency setting needs to provide efficient data acquisition capabilities under the premise of ensuring that the short-lived luminescence signal is fully collected. The acquisition duration is set to ensure that the complete acquisition of short-lived and long-lived luminescence signals can be completed in each cycle. The adjustment of the duration needs to be optimized according to the signal attenuation characteristics. The attenuation duration determination unit monitors the attenuation process of the short-lived luminescence signal in real time, and determines when the short-lived luminescence signal has been fully attenuated by analyzing the changes in signal intensity. After the short-lived luminescence signal is completely attenuated and ensures that there is enough time to collect the long-lived luminescence signal, the unit immediately starts the next excitation and acquisition cycle. This process is achieved through the precise timing of the trigger signal. It can be understood that a series of timing control conditions can be set according to the experimental design and needs. These conditions may include the time points of the start and end of the acquisition and the duration of each stage. Under the preset timing conditions, the long-life luminescence signal is collected in an approximately steady state, which usually includes acquiring data in the time period after the short-life luminescence signal decays and before the long-life luminescence signal reaches a steady state, that is, the second time gating is set in the interval when the long-life luminescence signal reaches a steady state. The entire trigger controller realizes the accurate collection of short-life and long-life luminescence signals through the coordinated work of the control of the acquisition frequency and acquisition time, the determination of the decay time, and the timing control.

[0124] In some optional embodiments, the camera module includes: an external trigger signal generator, used to generate a TTL signal, modulate the laser to emit a pulsed laser to excite the fluorescent material to reach an excited state; a time-gated acquisition unit, used to continuously acquire first time-gated and second time-gated luminescence signals, the first time-gated luminescence signal contains both long-life and short-life luminescence signals, and the second time-gated luminescence signal contains only long-life luminescence signals.

[0125] Specifically, the external trigger signal generator integrated in the camera module is responsible for generating TTL signals, which are used to accurately control the pulse emission of the laser. The TTL signal modulates the laser so that it emits pulsed lasers according to the set frequency and timing. It is understood that the frequency and duration of the laser pulses can be adjusted according to experimental requirements. Through the irradiation of the pulsed laser, the fluorescent material is excited to an excited state, generating specific luminescent signals. These signals contain short-lived and long-lived components related to the characteristics of the fluorescent material.

[0126] In some optional embodiments, the time-gated acquisition unit includes: a time interval setting interface, which sets the length and start time of two time intervals through the camera module control interface, the first interval is set to the time period from the end of excitation to the complete decay of the short-lived luminescence signal, and the second interval is set to the time period that only contains the long-lived luminescence signal after the short-lived luminescence signal is completely decayed.

[0127] Specifically, the time-gated acquisition unit performs signal acquisition within a predetermined time interval by setting a time window. The selection of the time window (first time gating and second time gating) determines the signal components collected. In the first time gating of the first interval after the laser pulse is emitted, the acquisition unit collects the luminescence signal. In this interval, due to the short time, the luminescence signal includes both short-lived luminescence signal and long-lived luminescence signal components. In a longer time interval after the excitation (i.e., the second time gating), the signal acquisition continues. During this period, since the short-lived luminescence signal has decayed, the collected signal is mainly a long-lived luminescence signal component. By synchronizing with the TTL signal, it is ensured that the time-gated acquisition unit is synchronized with the laser pulse emission to achieve accurate time window selection. By comparing and analyzing the first image data collected by the first time gating in the first interval and the second image data collected by the second time gating in the second interval, the intensity and characteristics of the short-lived luminescence signal and the long-lived luminescence signal can be separated.

[0128] In some optional embodiments, the signal processing module includes: a data separation unit, which is used to perform pixel comparison operation on the first image data and the second image data to separate the first image data into first luminescence data and second luminescence data, the first luminescence data representing the image data corresponding to the short-lived fluorescence, and the second luminescence data representing the image data corresponding to the long-lived fluorescence; a difference calculation unit, which is used to delete the second luminescence data in the first image data to obtain third image data, the third image data only includes the first luminescence data, that is, the image data of the short-lived fluorescence; a ratio calculation unit, which is used to perform ratio calculation on the image data of the short-lived luminescence signal (luminescence signal of short-lived fluorescence) and the image data of the long-lived luminescence signal (luminescence signal of long-lived fluorescence) to generate a ratio image.

[0129] Specifically, the data separation unit collects image data of two time intervals from the first time gate and the second time gate, the data of the first interval contains long-life and short-life luminescence signals, and the data of the second interval contains only long-life luminescence signals. The collected image data is input into the data separation unit. The unit uses an algorithm (such as time domain filtering or Fourier transform) to analyze the data of different time intervals, identify and separate the long-life and short-life luminescence signals. The difference calculation unit receives two image data sets output from the data separation unit: one contains image data of long-life and short-life luminescence signals, and the other contains only long-life luminescence signals. Then perform pixel-level subtraction operations to subtract image data containing only long-life luminescence signals from image data containing long-life and short-life luminescence signals. Through this calculation, image data containing only short-life luminescence signals is obtained. It can be understood that, considering possible noise or acquisition errors, filtering or other signal processing techniques are applied to correct and optimize the difference results to ensure accurate extraction of short-life luminescence signals. The ratio calculation unit receives the short-life luminescence signal image data output by the difference calculation unit and the image data containing only long-life luminescence signals. Then, a pixel-level ratio operation is performed to calculate the ratio of the image data of the short-lived luminescence signal to the image data of the long-lived luminescence signal. The ratio calculation result is used to generate a ratio image. The ratio data is used to generate a ratio image, which can highlight the distribution and intensity of the short-lived luminescence signal relative to the long-lived luminescence signal, facilitating subsequent analysis and visualization. According to the characteristics of the ratio image, the contrast and brightness are appropriately adjusted and enhanced to improve the visibility and analytical value of the image.

[0130] In some optional embodiments, the image conversion module includes: a working curve storage unit, used to store the ratio values ​​pre-determined according to samples of different concentrations or concentration gradients, and generate a preset working curve between the ratio value and the actual concentration; a ratio reading unit, used to read the ratio value of each pixel in the generated ratio image; and a search unit, which determines the corresponding quantitative information by searching a pre-established working curve.

[0131] Through the above units and steps, the image conversion module can accurately convert the ratio information in the ratio image into the quantitative concentration information of the sample or other physiological environment related parameter information.

[0132] It is understandable that the dual-gated imaging system for quantitative detection of living organisms in the embodiment of the present invention may actually include other components, such as IsCOMS camera (scientific complementary metal oxide semiconductor camera), lens, filter, laser, dark box, lifting system, etc., and also includes a set of python programs in the data processing process, which can perform signal processing and ratio calculation on the collected pictures, and restore the optical intensity information to the measured quantitative information. In the process of real-time imaging, the signal triggering program continuously triggers the IsCOMS camera to repeatedly collect signals of two intervals in a sequence and save them to the hard disk. Then, under the monitoring of the real-time signal processing system, the two newly appeared pictures in the hard disk are calculated to obtain a ratio image, and the working curve between the ratio and the information to be measured is used to restore it to the information to be measured, and then it is output in real time, and finally the real-time quantitative imaging of the video level is realized, and the imaging speed is expected to reach 20fps.

[0133] In some optional embodiments, the probe molecule uses the first polystyrene microsphere as a matrix, the interior of the first polystyrene microsphere is coated with a rare earth complex, and the exterior of the first polystyrene microsphere is coated with silica; the reference molecule uses the second polystyrene microsphere as a matrix, the interior of the second polystyrene microsphere is coated with a transition metal complex, and the exterior of the second polystyrene microsphere is coated with silica.

[0134] In some optional embodiments, the rare earth complex is preferably one of a europium complex, an erbium complex, a thulium complex, and a terbium complex, and the transition metal complex is preferably one of an iridium complex and a platinum complex.

[0135] In some optional embodiments, the europium complex is preferably a structure as described in Formula I, and the iridium complex is preferably a structure as described in Formula II:

[0136]

[0137] The dual-gated optical detection method according to the embodiment of the present invention has at least the following beneficial effects: an acquisition signal is generated by a signal trigger module; a camera module is controlled according to the acquisition signal to acquire first fluorescence information of living tissue within a first time gate and second fluorescence information of living tissue within a second time gate, the first time gate is located between a first lifetime end point of the spontaneous fluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, the short-lived fluorescence characterizes the fluorescence of the reference molecule, and the long-lived fluorescence characterizes the fluorescence of the probe molecule; a signal processing module is controlled to perform calculations on the first fluorescence information and the second fluorescence information to obtain a ratio image, the first fluorescence information characterizes first image data including both short-lived fluorescence and long-lived fluorescence, and the second fluorescence information characterizes second image data including only long-lived fluorescence. The first fluorescence information is collected through the first time gating to prevent the short-lived fluorescence of the probe molecule from being interfered by the spontaneous fluorescence of the biological tissue. The second fluorescence information is then collected through the second time gating. The ratio of the first fluorescence information and the second fluorescence information is calculated to obtain a ratio image, thereby distinguishing the fluorescence signals of the probe molecule from the reference molecule, thereby improving the accuracy of dual-gated imaging for quantitative detection of living organisms.

[0138] In a second aspect, an embodiment of the present invention provides a dual-gated optical detection system, which uses a dual-gated optical detection method, wherein the camera module includes an external trigger signal generator and a time-gated acquisition unit;

[0139] The external trigger signal generator is used to generate a TTL signal to modulate the laser, so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing, and excites the fluorescent material to reach an excited state, and the fluorescent material includes a probe molecule and a reference molecule;

[0140] The time-gated acquisition unit is used to acquire first fluorescence information within a first time gate and to acquire second fluorescence information within a second time gate.

[0141] In some optional embodiments, the signal triggering module includes:

[0142] A control interface, used to input and / or adjust acquisition parameters, and store the acquisition parameters in a parameter storage unit of the signal trigger module;

[0143] A control unit, used for reading acquisition parameters, including trigger frequency, trigger time interval and synchronization information;

[0144] a timer and / or an oscillator for generating a continuous trigger signal according to a trigger frequency;

[0145] The control circuit is used to control the timing of continuous trigger signals according to the trigger time interval and synchronization information to obtain acquisition signals, and transmit the acquisition signals to the camera module.

[0146] In some optional embodiments, the camera module includes:

[0147] A control interface for inputting or adjusting a first lifetime of a probe molecule and a second lifetime of a reference molecule;

[0148] A calculation unit, used for determining a first time gate, a second time gate and an acquisition period according to a first life span of the probe molecule, a second life span of the reference molecule and an acquisition signal;

[0149] A trigger controller, used to determine a second lifetime end point of the short-life fluorescence and start a next acquisition cycle at the second lifetime end point;

[0150] An image acquisition unit, used for acquiring images after receiving an acquisition signal;

[0151] The synchronous processing unit synchronously processes the short-life fluorescence and the long-life fluorescence in each acquisition cycle.

[0152] In some optional embodiments, the trigger controller includes:

[0153] A control unit, used to control the acquisition frequency and duration of the camera module;

[0154] a decay time determination unit, used to start the next collection cycle when the short-lived fluorescence decays to the end point of the second lifetime and the time for collecting the long-lived fluorescence is not less than the second lifetime of the reference molecule;

[0155] The timing control unit collects the steady state of the long-life fluorescence according to the preset timing control conditions.

[0156] In some optional embodiments, the time-gated acquisition unit includes:

[0157] The time interval setting interface is used to obtain the first time interval and the second time interval of the camera module, the first time gate is located in the first time interval, and the second time gate is located in the second time interval.

[0158] It should be noted that the wavelength of the next target wavelength excitation light is determined according to the sample to be tested or the test requirements, and is not specifically limited. By controlling the emission time of the next target wavelength excitation light, the next target wavelength excitation light is prevented from interfering with the generation and detection of the feedback light signal.

[0159] In some optional embodiments, the signal processing module includes:

[0160] a data separation unit, configured to perform pixel comparison operation on the first image data and the second image data to separate the first image data into first luminescence data and second luminescence data, wherein the first luminescence data represents image data corresponding to the short-lived fluorescence and the second luminescence data represents image data corresponding to the long-lived fluorescence;

[0161] a difference calculation unit, used for deleting the second light emitting data in the first image data to obtain third image data, where the third image data only includes the first light emitting data;

[0162] The ratio calculation unit is used to generate a ratio image after performing ratio calculation on the second image data and the third image data.

[0163] In some optional embodiments, the dual-gated optical detection system further includes an image conversion module, and the image conversion module includes:

[0164] A working curve storage unit, used to store a preset working curve pre-determined according to samples of different concentrations or concentration gradients;

[0165] A ratio reading unit, used for reading the ratio value of each pixel in the generated ratio image;

[0166] The search unit is used to search for the actual concentration in the preset working curve and then determine the corresponding quantitative information.

[0167] In some optional embodiments, the probe molecule includes the europium complex Eu(tta)3TADF (see Formula I in the above method embodiment), and the reference molecule includes the iridium complex Ir(tfpqz)2dbm (see Formula II in the above method embodiment).

[0168] It can be seen that the contents of the above method embodiments are all applicable to the present system embodiments, the functions specifically implemented by the present system embodiments are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0169] In a third aspect, an embodiment of the present invention provides a controller, comprising: a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the above method is implemented when the processor executes the computer program.

[0170] It can be seen that the contents of the above system embodiments are all applicable to the present controller embodiment, the functions specifically implemented by the present controller embodiment are the same as those of the above system embodiments, and the beneficial effects achieved are also the same as those achieved by the above system embodiments.

[0171] The dual-gated optical detection method and system provided in the embodiments of the present invention can realize quantitative imaging of various physiological environment parameters, reactive small molecules, ions and biomacromolecules of living bodies, including temperature, viscosity, oxygen, pH, HClO, etc. This mainly depends on the structural design of the luminescent material.

[0172] For example, the luminescence intensity or luminescence lifetime mostly has a responsive characteristic to temperature. Taking advantage of this characteristic, metal complexes can be used to measure or image temperature. In addition, since the luminescence of metal complexes usually involves the triplet energy level of metal ions, their luminescence is easily quenched by oxygen. As a classic oxygen-responsive molecule, metal luminescent complexes can use this characteristic to achieve quantitative detection of oxygen. In addition, the movement and aggregation state of the ligand will also change the luminescence properties of the complex, so metal complexes can indicate changes in microenvironment viscosity caused by protein aggregation in vivo.

[0173] For another example, organic macrocyclic ligands with specific response functions can be synthesized through the responsiveness of organic ligands to reactive small molecules. For example, modifying active reaction chains and active reaction groups outside the macrocyclic ring will cause changes in the ligand structure or rearrangement of charges when in contact with reactive small molecules, thereby changing the overall luminescence intensity, wavelength or lifetime of the complex. Through this responsive ligand design, HClO, ONOO - In addition, some metal ions in the body have a competitive effect on the coordination of the central ion of the metal complex. The concentration of metal ions will affect the coordination balance of the complex, thereby changing the luminescence intensity. 2+ 、Zn 2+ , Ca 2+ , Cu 2+ Quantitative detection probe for specific metal ions.

[0174] In addition, biological proteins and biological enzymes have a shearing effect on specific functional groups. For example, by modifying the luminescence quenching group on the ligand molecule, the metal complex is in a dark state under normal conditions. However, when it comes into contact with the responding protein and biological enzyme, the catalytic action of the enzyme can shear off the luminescence quenching group, thereby restoring the luminescence of the metal complex. Based on this, an "on-off" type probe that responds to biological macromolecules can be constructed. By constructing a working curve based on the degree of change in luminescence intensity, quantitative detection of specific biological macromolecules can be achieved.

[0175] The following uses the dual-gated same-wavelength ratio method to quantitatively detect the temperature of a living body as an example to illustrate the application of the dual-gated optical detection method and system in the embodiments of the present invention.

[0176] In this study, the europium complex Eu(tta)3TADF and the iridium complex Ir(tfpqz)2dbm were selected as probe molecules and reference molecules, respectively. The Eu complex is sensitive to temperature, and its luminescence intensity decreases rapidly with increasing temperature; while the luminescence of the Ir complex is insensitive to temperature and shows thermal inertness. The two molecules were respectively encapsulated in polystyrene (PS) microspheres with a diameter of about 160nm, and a layer of SiO2 was coated on the outer layer of the PS microspheres to form PS@SiO2 microspheres with an overall diameter of about 220nm. This design is intended to maintain the morphology of the PS spheres at high temperatures and prevent external reactive molecules from entering the PS spheres. The main emission peak of the Eu complex is in the range of 600-630nm, and the luminescence lifetime is about 549 microseconds, while the main emission peak of the Ir complex is in the range of 580-800nm, and the luminescence lifetime is about 688 nanoseconds. The two completely overlap in the spectrum and cannot be directly distinguished, but can be distinguished by time gating methods.

[0177] In the IsCOMS time-resolved camera, the ratiometric probe is excited by an externally triggered 450nm laser, and the excitation time is set to 10 microseconds. During this process, the Eu and Ir complexes reach an excited state. When the excitation light is turned off, the Eu and Ir complexes begin to return from the excited state to the ground state and emit photons. Since the signal within 10 to 10.8 microseconds contains the incompletely attenuated excitation light and biological autofluorescence, it is not collected. Subsequently, the signals within 10.8 to 12.8 microseconds and 12.8 to 14.8 microseconds are collected respectively, of which the first time interval contains the signal of the Eu and Ir complexes (S Ir+Eu ), while the second time interval contains only the signal of the Eu complex (S Eu ). The entire excitation and acquisition process can be completed within 15 microseconds, so more than 48,000 cycles can be acquired in 1 second; however, if the traditional transient excitation acquisition method is used, each cycle will be extended to 5 milliseconds, and only 200 cycles can be acquired in 1 second. If the gate width of the acquisition signal is the same, the signal strength difference between the two will reach 240 times. Therefore, this transient-near-steady-state excitation acquisition method greatly improves time utilization and signal strength. Finally, by Ir+Eu Subtract S from Eu To obtain I r The signal of the complex (S Ir ), and S Eu / S Ir As the final ratio signal.

[0178] Due to S Eu and S Ir All of them change with the ambient temperature. By changing the ambient temperature through temperature control equipment, the specific degree at a specific temperature can be obtained, thereby constructing the temperature response curve of the material. Figure 4, the ambient temperature was raised from 30 degrees Celsius to 90 degrees Celsius, and the ratio information of a series of temperature points was collected during the process, and finally the equation that conforms to the Boltzmann fitting curve was obtained:

[0179]

[0180] According to the inverse function of this formula, the ratio can also be restored back to temperature, thereby achieving quantitative measurement of ambient temperature.

[0181] This dual-gated wavelength ratio method can effectively eliminate the effects of biological autofluorescence, biological tissue shielding, and small molecules, ions, and biological macromolecules. Figure 5 The selected materials can maintain a stable ratio in a 2-cm thick 1% fat emulsion shield, a 5-mm thick biological tissue shield, PBS solution, fetal bovine serum, whole blood, pH=2-12 range, and 100 mM NaClO. The ratio only changes by 10% under a 5-mm biological tissue shield, thereby verifying the ability of this method to achieve accurate quantitative detection in a biological environment.

[0182] In addition, this method was compared with the traditional dual-wavelength ratiometry and single-gated same-wavelength ratiometry methods. Based on this, the luminescent material combinations of BODIPY (Boron DiPyromethene, fluoroboron dipyrrole compound) + Eu complex and Nile Red + Eu complex were constructed, and they were also coated in PS@SiO2 microspheres. The ratiometric signals of the two systems were collected under the condition of biological tissue shielding using dual-wavelength ratiometry and single-gated same-wavelength ratiometry methods. Figure 6 The results showed that both methods could not completely eliminate the influence of biological tissue occlusion. The traditional dual-wavelength ratio method changed the ratio by 100% under 1mm tissue occlusion, while the single-gated same-wavelength ratio method could not completely eliminate the influence of tissue autofluorescence, so the ratio changed by 50% under 5mm tissue occlusion.

[0183] The entire imaging system in this embodiment includes a real-time acquisition and image processing system, in which the camera will continuously acquire signals in two gates, and save two pictures to the hard disk, and then repeat the process in a loop. At the same time, the system also includes a real-time image processing program algorithm, which can monitor the generation of new pictures in real time. Whenever it detects that two new pictures are generated, it will calculate the pixel-to-pixel ratio of the two pictures, and then restore the ratio of each pixel point to the temperature information according to the inverse function of the temperature response curve, and write the temperature calculation result into a new picture, so as to obtain the temperature distribution information in space, and display it in the window interface. During the calculation process, the temperature value of the corresponding position on the picture can also be read by the mouse pointer position. The image processing process theoretically takes no more than 0.01s, plus the time for image acquisition and storage, and finally can achieve a temperature imaging speed of 20fps, the temperature error is no more than 1 degree Celsius compared with the thermal imager, and the imaging pixels reach 1.6 million, realizing real-time and accurate temperature imaging.

[0184] In a fourth aspect, an embodiment of the present invention provides a computer storage medium, wherein the computer storage medium stores computer executable instructions, and the computer executable instructions are used to execute the above-mentioned dual-gated optical detection method.

[0185] It can be seen that the contents of the above-mentioned dual-gated optical detection method embodiment are all applicable to the present storage medium embodiment, the functions specifically implemented by the present storage medium embodiment are the same as those of the above-mentioned dual-gated optical detection method embodiment, and the beneficial effects achieved are also the same as those achieved by the above-mentioned dual-gated optical detection method embodiment.

[0186] It is understood that all or some steps and systems in the disclosed method above can be implemented as software, firmware, hardware and appropriate combinations thereof. Some physical components or all physical components can be implemented as processors, such as software executed by a central processing unit, a digital information processor or a microprocessor, or implemented as hardware, or implemented as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, and the computer-readable medium can include a computer storage medium (or a non-transitory medium) and a communication medium (or a temporary medium). As known to those of ordinary skill in the art, the term computer storage medium includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules or other data). Computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tapes, disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, it is well known to those skilled in the art that communication media generally contain computer readable instructions, data structures, program modules, or other data in modulated data information such as carrier waves or other transport mechanisms, and may include any information delivery media.

[0187] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the technical field without departing from the purpose of the present invention.

Claims

1. A dual-gated optical detection method, characterized in that: Applied to a dual-gated optical detection system, the dual-gated optical detection system includes a signal trigger module, a camera module and a signal processing module, and the dual-gated optical detection method includes: Generate an acquisition signal through the signal trigger module; Control the camera module to collect first fluorescence information of the living tissue within a first time gate and collect second fluorescence information of the living tissue within a second time gate according to the acquisition signal; Controlling the signal processing module to perform calculation processing on the first fluorescence information and the second fluorescence information to obtain a ratio image; Wherein, the first time gate is located between a first lifetime end point of the autofluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, the short-lived fluorescence characterizes the fluorescence of the reference molecule, and the long-lived fluorescence characterizes the fluorescence of the probe molecule; The first fluorescence information representation includes first image data of both short-life fluorescence and long-life fluorescence, and the second fluorescence information representation includes second image data of only long-life fluorescence.

2. The dual-gated optical detection method according to claim 1, characterized in that: The generating of the acquisition signal by the signal triggering module includes: Acquiring acquisition parameters, wherein the acquisition parameters include trigger frequency, trigger time interval and synchronization information; Storing the acquisition parameters in the signal trigger module, so that the signal trigger module generates a continuous trigger signal after reading the trigger frequency; The signal trigger module reads the trigger time interval and the synchronization information and then controls the timing of the continuous trigger signal to obtain the acquisition signal.

3. The dual-gated optical detection method according to claim 1, characterized in that: The method of controlling the camera module to collect first fluorescence information of living tissue within a first time gate and second fluorescence information of living tissue within a second time gate according to the acquisition signal includes: Acquiring a first lifetime of the probe molecule, wherein the first lifetime of the probe molecule represents the lifetime of the long-lived fluorescence; Acquiring a second lifetime of the reference molecule, wherein the second lifetime of the reference molecule represents the lifetime of the short-lived fluorescence; Determine the first time gate, the second time gate and the acquisition period according to the first life span of the probe molecule, the second life span of the reference molecule and the acquisition signal; The camera module is controlled to collect the first fluorescence information within the first time gate of each acquisition cycle, and to collect the second fluorescence information within the second time gate of each acquisition cycle.

4. The dual-gated optical detection method according to claim 3, characterized in that: The controlling the camera module to collect the first fluorescence information within the first time gate of each acquisition cycle and to collect the second fluorescence information within the second time gate of each acquisition cycle includes: In each of the acquisition cycles, the first acquisition frequency, the first acquisition duration, the second acquisition frequency and the second acquisition duration of the camera module are controlled by the acquisition signal, wherein the first acquisition frequency represents the frequency at which the camera module acquires the first fluorescence information within the first time gating, the second acquisition frequency represents the frequency at which the camera module acquires the second fluorescence information within the second time gating, the first acquisition duration represents the duration for which the camera module acquires the first fluorescence information, and the second acquisition duration represents the duration for which the camera module acquires the second fluorescence information; When the short-lived fluorescence decays to the end point of the second lifetime and the time used to collect the long-lived fluorescence is not less than the second lifetime of the reference molecule, start stimulating the excitation light for the next collection period so that the excitation light excites the spontaneous fluorescence, the short-lived fluorescence and the long-lived fluorescence within the next collection period.

5. The dual-gated optical detection method according to claim 1, characterized in that: The camera module includes an external trigger signal generator and a time-gated acquisition unit. After the acquisition signal is generated by the signal trigger module, the camera module further includes: Generate a TTL signal by means of the external trigger signal generator; Modulating the laser by the TTL signal so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing to excite the fluorescent material to reach an excited state, wherein the fluorescent material includes the probe molecule and the reference molecule; The time-gated acquisition unit acquires the first fluorescence information within a first time gate, and acquires the second fluorescence information within a second time gate.

6. The dual-gated optical detection method according to claim 1, characterized in that: The dual-gated optical detection system further includes an image conversion module, and the method further includes: The signal processing module calculates the ratio of the first image data and the second image data to generate a ratio image; The ratio image is converted into quantitative information through the image conversion module and the preset working curve.

7. The dual-gated optical detection method according to claim 6, characterized in that: The calculating and processing the first fluorescence information and the second fluorescence information by the signal processing module to obtain a ratio image includes: Performing pixel comparison operation on the first image data and the second image data by the signal processing module to obtain first luminescence data and second luminescence data in the first image data, wherein the first luminescence data represents image data corresponding to the short-lived fluorescence, and the second luminescence data represents image data corresponding to the long-lived fluorescence; Deleting the second light emitting data from the first image data by the signal processing module to obtain third image data, wherein the third image data only includes the first light emitting data; The signal processing module calculates the ratio of the second image data and the third image data to generate the ratio image.

8. The dual-gated optical detection method according to claim 6, characterized in that: The step of converting the ratio image into quantitative information by using the image conversion module and a preset working curve includes: Reading the ratio value of each pixel in the ratio image by the image conversion module; Acquiring the preset working curve through the image conversion module, wherein the preset working curve represents a relationship curve between a preset ratio value and an actual concentration, wherein the actual concentration represents environmental information of biological tissue or concentration information of biological molecules; The image conversion module converts the ratio value of each pixel into the actual concentration of each pixel corresponding to the preset working curve; The image conversion module converts each pixel in the ratio image into the actual concentration of each pixel to obtain a quantitative image, and the quantitative image indicates the quantitative information.

9. The dual-gated optical detection method according to any one of claims 1 to 8, characterized in that: The probe molecule uses the first polystyrene microsphere as a matrix, the first polystyrene microsphere is coated with a rare earth complex inside, and the first polystyrene microsphere is coated with silicon dioxide outside. The reference molecule uses the second polystyrene microsphere as a matrix, the second polystyrene microsphere is coated with a transition metal complex inside, and the second polystyrene microsphere is coated with silicon dioxide outside.

10. The dual-gated optical detection method according to claim 9, characterized in that: The rare earth complex is preferably one of a europium complex, an erbium complex, a thulium complex and a terbium complex, and the transition metal complex is preferably one of an iridium complex and a platinum complex.

11. The dual-gated optical detection method according to claim 10, characterized in that: The europium complex is preferably a structure as described in Formula I, and the iridium complex is preferably a structure as described in Formula II:

12. A dual-gated optical detection system, characterized in that: include: A signal trigger module, used for generating an acquisition signal; A camera module, configured to collect first fluorescence information of the living tissue within a first time gate and second fluorescence information of the living tissue within a second time gate after receiving the collection signal, wherein the first time gate is located between a first lifetime end point of the autofluorescence of the living tissue and a second lifetime end point of the short-lived fluorescence, and the second time gate is located between the second lifetime end point and a third lifetime end point of the long-lived fluorescence, wherein the short-lived fluorescence represents the fluorescence of the reference molecule, and the long-lived fluorescence represents the fluorescence of the probe molecule; A signal processing module is used to calculate and process the first fluorescence information and the second fluorescence information to obtain a ratio image, wherein the first fluorescence information represents first image data including both short-lived fluorescence and long-lived fluorescence, and the second fluorescence information represents second image data including only long-lived fluorescence.

13. The dual-gated optical detection system according to claim 12, characterized in that: The camera module includes an external trigger signal generator and a time-gated acquisition unit; The external trigger signal generator is used to generate a TTL signal to modulate the laser, so that the laser emits excitation light to the biological tissue according to a preset frequency and a preset timing to excite the fluorescent material to reach an excited state, wherein the fluorescent material includes the probe molecule and the reference molecule; The time-gated acquisition unit is used to acquire the first fluorescence information within a first time gate and to acquire the second fluorescence information within a second time gate.

14. The dual-gated optical detection system according to claim 12, characterized in that: The signal triggering module comprises: A control interface, used to input and / or adjust acquisition parameters, and store the acquisition parameters in a parameter storage unit of the signal trigger module; A control unit, used for reading the acquisition parameters, wherein the acquisition parameters include a trigger frequency, a trigger time interval and synchronization information; a timer and / or an oscillator, for generating a continuous trigger signal according to the trigger frequency; The control circuit is used to control the timing of the continuous trigger signal according to the trigger time interval and the synchronization information to obtain the acquisition signal, and transmit the acquisition signal to the camera module.

15. The dual-gated optical detection system according to claim 12, characterized in that: The camera module comprises: A control interface for inputting or adjusting a first lifetime of a probe molecule and a second lifetime of a reference molecule; a calculation unit, configured to determine the first time gate, the second time gate and an acquisition period according to the first lifespan of the probe molecule, the second lifespan of the reference molecule and the acquisition signal; a trigger controller, configured to determine the second lifetime end point of the short-life fluorescence and start a next acquisition cycle at the second lifetime end point; An image acquisition unit, configured to acquire an image after receiving the acquisition signal; The synchronous processing unit synchronously processes the short-life fluorescence and the long-life fluorescence in each acquisition cycle.

16. The dual-gated optical detection system according to claim 15, characterized in that: The trigger controller comprises: A control unit, used to control the acquisition frequency and duration of the camera module; a decay time determination unit, configured to start a next collection cycle when the short-lived fluorescence decays to the end point of the second lifetime and the time for collecting the long-lived fluorescence is not less than the second lifetime of the reference molecule; The timing control unit collects the steady state of the long-life fluorescence according to the preset timing control conditions.

17. The dual-gated optical detection system according to claim 13, characterized in that: The time-gated acquisition unit comprises: The time interval setting interface is used to obtain a first time interval and a second time interval of the camera module, the first time gate is located in the first time interval, and the second time gate is located in the second time interval.

18. The dual-gated optical detection system according to claim 12, characterized in that: The signal processing module comprises: a data separation unit, configured to perform pixel comparison operation on the first image data and the second image data to separate the first image data into first luminescence data and second luminescence data, wherein the first luminescence data represents image data corresponding to the short-lived fluorescence, and the second luminescence data represents image data corresponding to the long-lived fluorescence; a difference calculation unit, configured to obtain third image data by deleting the second light emitting data from the first image data, wherein the third image data only includes the first light emitting data; A ratio calculation unit is used to generate the ratio image after performing ratio calculation on the second image data and the third image data.

19. The dual-gated optical detection system according to claim 12, characterized in that: The dual-gated optical detection system further includes an image conversion module, which includes: A working curve storage unit, used to store a preset working curve pre-determined according to samples of different concentrations or concentration gradients; A ratio reading unit, used for reading the ratio value of each pixel in the generated ratio image; The search unit is used to search for the actual concentration in the preset working curve and then determine the corresponding quantitative information.

Images