A dual-color alternating calcium imaging dual-channel image extraction method

Abstract

The application relates to a double-color alternating calcium imaging double-channel image extraction method, which comprises the following steps: providing a light source assembly, a CMOS imaging assembly, a fine clock timer and an extremely fine coaxial line; when the fine clock timer outputs a high-level signal, a light source of a corresponding wavelength is triggered to light up to excite specific fluorescent probes, and in the remaining 20% of the high-level time length, fluorescent proteins are naturally attenuated; after the high-level state ends, the fine clock timer is switched to a low-level signal, a light source of another wavelength is triggered to light up to excite another kind of fluorescent probe, and in the remaining 20% of the low-level time length, fluorescent proteins are naturally attenuated; a plurality of image frames are shot under the excitation condition of different wavelength light sources through the CMOS imaging assembly; and image frame data is analyzed and processed to obtain relevant information of different fluorescent signals in a sample area to be detected. The application can obtain more accurate and reliable fluorescent signals, so that the dynamic of calcium ions in the detection sample can be accurately analyzed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical imaging, and particularly relates to a dual-color alternating calcium imaging dual-channel image extraction method. BACKGROUND

[0002] In the field of neuroscience research, in-depth exploration of neuron activity and neural network operation mechanism is of great significance for understanding brain function and conquering nervous system diseases. Calcium imaging technology, as an important means, provides researchers with cell-level neuron activity observation capability by detecting the fluorescence signal generated by calcium ion flow during neuron activity. However, traditional calcium imaging technology is mostly dependent on fixed experimental environment and is difficult to be effectively applied in freely moving animal models, which to some extent limits the research on neural activity in natural behavior state. The existing miniature calcium imaging microscope has only single-color excitation light and can only observe one type of neural activity. However, in the same observation area, there are usually two or more types of nerves, and the relationship between these neural activities is difficult to be observed by the calcium imaging microscope with only one excitation light. Therefore, integrating two calcium imaging microscopes and developing a device capable of simultaneously recording two types of neurons has become an important development trend in modern neuroscience research. Through such integration, researchers can realize real-time monitoring of different types of neurons in the same behavior in the same experimental system, or simultaneously monitor blood vessels with fluorescent markers and another type of neurons with fluorescent markers, thereby deeply analyzing the potential relationship between blood activity and neural activity, or the coordination relationship between different types of neural activities in the same behavior, thereby providing a new perspective and powerful tool for in-depth analysis of complex neural networks and central brain diseases.

[0003] However, the related devices on the market at present mostly have obvious defects. Most products only use a single LED light source. Such single light source system can only work in a specific wavelength range and cannot simultaneously carry out multi-spectral experiments. This limitation greatly limits the flexibility of experiments, so that when different fluorescent probes are operated, the equipment needs to be frequently adjusted or the light source needs to be replaced, which not only increases the complexity of experiments, but also easily introduces equipment adjustment errors, and at the same time can have a negative impact on the accuracy and stability of experimental data, and is difficult to meet the increasingly stringent requirements of neuroscience research on high-precision, multi-function and high-stability experiments.

[0004] Therefore, the application provides a dual-color alternating calcium imaging dual-channel image extraction method to solve the above technical problems. SUMMARY

[0005] In view of the above problems, the present application aims to provide a two-color alternating calcium imaging dual-channel image extraction method, which can obtain more accurate and reliable fluorescent signals, so as to accurately analyze the calcium ion dynamics in the detected sample.

[0006] The present application provides a two-color alternating calcium imaging dual-channel image extraction method, which comprises:

[0007] S1, providing a two-color alternating calcium imaging device; the two-color alternating calcium imaging device comprises a light source assembly, a CMOS imaging assembly, a fine clock timer and an ultra-fine coaxial line, the light source assembly is used for alternately emitting light of at least two different wavelengths, and different wavelength light sources of the light source assembly are respectively connected with the fine clock timer through the ultra-fine coaxial line;

[0008] S2, setting the switching period, the high-level time length and the low-level time length of the high-level signal and the low-level signal by using the fine clock timer; when the fine clock timer outputs a high-level signal, triggering the ultra-fine coaxial line to make the light source of the corresponding wavelength light up and the duration is 80% of the high-level time length, which is used for exciting specific fluorescent probes, and in the remaining 20% of the high-level time length, the excitation of the light source is stopped, so that the fluorescent protein naturally decays;

[0009] S3, after the high-level state ends, the fine clock timer switches to a low-level signal, and the light source of another wavelength is turned on through the ultra-fine coaxial line and the duration is 80% of the low-level time length, which is used for exciting another kind of fluorescent probe, and in the remaining 20% of the low-level time length, the excitation of the light source is stopped, so that the fluorescent protein naturally decays;

[0010] S4, a plurality of image frames are shot under different wavelength light source excitation conditions by using the frame flipping mechanism of the CMOS imaging assembly, wherein the triggering of the frame flipping is synchronized with the level signal switching of the fine clock timer, so that the fluorescent signals of the image frames shot at different times are derived from different wavelength light source excitation conditions;

[0011] S5, collecting the image frame data shot by the CMOS imaging assembly, and analyzing and processing the image frame data to obtain the related information of different fluorescent signals in the sample area to be detected.

[0012] Preferably, the related information of different fluorescent signals includes fluorescent signal intensity distribution and fluorescent signal change trend.

[0013] Preferably, the analysis and processing of the image frame data in S5 specifically comprises:

[0014] S51, locating and extracting a fluorescent signal region in the image frame data by an image recognition algorithm to obtain fluorescent signal data;

[0015] S52, performing noise reduction processing on the extracted fluorescent signal data by a signal processing algorithm;

[0016] S53, performing analysis and processing on the fluorescent signal data after noise reduction processing to obtain the fluorescent signal intensity distribution and the fluorescent signal change trend.

[0017] Preferably, the light source assembly comprises a first light source unit and a second light source unit; the first light source unit comprises: a green light source for emitting light rays in a green light band; a green excitation light filter arranged on an outgoing light path of the green light source, for filtering the light rays emitted by the green light source to screen out first excitation light of a specific wavelength range; a first achromatic lens located on an outgoing light path of the green excitation light filter, for focusing and chromatic aberration correction of the filtered first excitation light; a first beam splitter for receiving the first excitation light processed by the first achromatic lens, reflecting the first excitation light to a sample area to be detected, receiving fluorescent signals generated by excitation from the sample area to be detected, and enabling the fluorescent signals to be transmitted; a first focusing lens arranged on a fluorescent signal transmission light path of the first beam splitter, for focusing the fluorescent signals; a red fluorescent filter located on an outgoing light path of the first focusing lens, for filtering the fluorescent signals to obtain fluorescent signals of a specific wavelength range; the second light source unit comprises: a blue light source for emitting light rays in a blue light band; a blue excitation light filter arranged on an outgoing light path of the blue light source, for filtering the light rays emitted by the blue light source to screen out second excitation light of a specific wavelength range; a second achromatic lens located on an outgoing light path of the blue excitation light filter, for focusing and chromatic aberration correction of the filtered second excitation light; a second beam splitter for receiving the second excitation light processed by the second achromatic lens, reflecting the second excitation light to a sample area to be detected, receiving fluorescent signals generated by excitation from the sample area to be detected, and enabling the fluorescent signals to be transmitted; a second focusing lens arranged on a fluorescent signal transmission light path of the second beam splitter, for focusing the fluorescent signals; a green fluorescent filter located on an outgoing light path of the second focusing lens, for filtering the fluorescent signals to obtain fluorescent signals of a specific wavelength range; the CMOS imaging assembly is located on the outgoing light paths of the red fluorescent filter and the green fluorescent filter, for receiving and detecting the fluorescent signals filtered by the red fluorescent filter and the green fluorescent filter, and converting the fluorescent signals into electrical signals for subsequent signal analysis and processing.

[0018] Preferably, the green light source includes two first LED lamp beads that are diagonally distributed and used to emit green light, and the blue light source includes two second LED lamp beads that are diagonally distributed and used to emit blue light.

[0019] Compared with related technologies, the present invention provides a dual-color alternating calcium imaging dual-channel image extraction method, comprising: providing a light source assembly, a CMOS imaging assembly, a refined clock timer, and an extremely fine coaxial line, wherein the light source assembly is used to alternately emit light of at least two different wavelengths, and the different wavelength light sources of the light source assembly respectively establish signal connections with the refined clock timer through the extremely fine coaxial line; when the refined clock timer outputs a high-level signal, it triggers the light source of the corresponding wavelength to light up to excite a specific fluorescent probe, and at the same time, the fluorescent protein is naturally attenuated within the remaining 20% ​​of the high-level duration; after the high-level state ends, the refined clock timer switches to a low-level signal, triggering the light source of another wavelength to light up to excite another fluorescent probe, and at the same time, the fluorescent protein is naturally attenuated within the remaining 20% ​​of the low-level duration; multiple image frames are captured by the CMOS imaging assembly under excitation conditions of light sources of different wavelengths; and the image frame data is analyzed and processed to obtain relevant information of different fluorescent signals in the sample area to be detected. In the above method, the limitations of a single light source are effectively avoided by alternating the operation of the dual-wavelength light source, and the alternating operation of the dual-wavelength light source improves the flexibility of the experiment. In addition, during the light source switching process, there will be no obvious light intensity fluctuations, spectral distortion and other problems, which effectively avoids the errors caused by equipment adjustment, thereby significantly improving the accuracy and repeatability of the experimental data. At the same time, the dual-wavelength light source of the present invention can also be customized according to different experimental requirements, such as the requirements of different sample types and different experimental scales, for light source parameters (such as light intensity, pulse frequency, etc.) and optical path configuration (such as adding or replacing specific optical components), further simplifying the experimental process and improving the efficiency and flexibility of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic flow chart of a dual-channel image extraction method for dual-color alternating calcium imaging according to the present invention;

[0021] Figure 2 Schematic diagram of the process of analyzing and processing the image frame data in the present invention;

[0022] Figure 3 It is a structural block diagram of the dual-color alternating calcium imaging device of the present invention;

[0023] Figure 4 Schematic diagram of the structure of the first light source unit, the second light source unit and the CMOS imaging component in the present invention;

[0024] Figure 5The structure diagram of the green light source and the blue light source in the present application. DETAILED DESCRIPTION

[0025] The present application provides a double-color alternating calcium imaging double-channel image extraction method, aiming at solving the problem that the existing calcium imaging technology uses a single LED light source, which has significant limitations, and the accuracy and stability of experimental data are poor.

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] Please refer to the accompanying Figures 1-5 The present application provides a double-color alternating calcium imaging double-channel image extraction method, which comprises:

[0028] S1, providing a double-color alternating calcium imaging device; the double-color alternating calcium imaging device 10 comprises a light source assembly 1, a CMOS imaging assembly 2, a refined clock timer 3 and an extremely fine coaxial line 4, the light source assembly 1 is used to alternately emit light of at least two different wavelengths, and different wavelength light sources of the light source assembly 2 are respectively connected with the refined clock timer 3 through the extremely fine coaxial line 4;

[0029] S2, using the refined clock timer 3 to set the switching period, the high-level time length and the low-level time length of high-level signal and low-level signal, when the refined clock timer 3 outputs a high-level signal, triggering the extremely fine coaxial line 4 to make the light source of the corresponding wavelength light up and the duration is 80% of the high-level time length, for exciting a specific fluorescent probe, at the same time in the remaining 20% of the high-level time length, stopping the excitation of the light source, making the fluorescent protein naturally attenuate;

[0030] S3, after the high-level state ends, the refined clock timer 3 switches to a low-level signal, through the extremely fine coaxial line 4, the light source of another wavelength is turned on and the duration is 80% of the low-level time length, for exciting another fluorescent probe, at the same time in the remaining 20% of the low-level time length, stopping the excitation of the light source, making the fluorescent protein naturally attenuate;

[0031] S4, under the excitation condition of different wavelength light sources, a plurality of image frames are shot through the frame flipping mechanism of the CMOS imaging assembly 2, wherein the trigger of frame flipping is synchronized with the level signal switching of the refined clock timer, so that the fluorescent signals of the image frames shot at different times are derived from the excitation conditions of different wavelength light sources;

[0032] S5, collecting image frame data obtained by the CMOS imaging assembly 2, and analyzing and processing the image frame data to obtain relevant information of different fluorescent signals in the sample region to be detected.

[0033] It should be noted that the above frame flipping mechanism carries the key "frame" signal, i.e., the control instruction of high and low level alternation, through the extremely thin coaxial line 4 as the signal transmission link. The transmission frequency is accurately adapted to the frame rate of imaging, ensuring that the entire imaging process is orderly. When the high-level signal is transmitted along the extremely thin coaxial line 4 to the light source assembly 1, it drives a LED of a specific wavelength to emit light. The light beam irradiates the detection sample and excites a specific fluorescent signal. At the same time, the CMOS imaging assembly 2 detects the high level and quickly adjusts the imaging parameters such as exposure time and gain according to the preset program to prepare for collecting the corresponding image frame. When the level flips to low, the power supply to the current LED is stopped, and another wavelength LED is driven to emit light. The imaging parameters are also changed. In this way, through the frame flipping mechanism, efficient synchronization of light source and imaging is achieved, greatly reducing background noise and image distortion, and improving imaging quality and data acquisition efficiency. In addition, the above method adopts an alternating working mode of the light source, which can be customized according to experimental needs. Researchers can select suitable wavelength combinations according to specific scientific problems to meet the diversified needs of different research fields, not only reducing the complexity of operation, but also reducing potential costs, enabling more laboratories to participate in high-precision neuroscience research. This provides stronger support for scientific exploration and also provides the possibility for developing new neural intervention strategies. At the same time, the dual-color alternating calcium imaging dual-channel image extraction method provides higher sensitivity and contrast, allowing researchers to more accurately monitor neuron activity and greatly improving experimental efficiency. In addition, this method also reduces the complexity of equipment and operation, enabling more laboratories to conduct high-precision neuroscience research, which not only provides strong support for scientific exploration, but also provides the possibility for developing new neural intervention strategies. Through this "all-optical" technology, scientists can reveal the functions of different neural pathways in real-time monitoring and manipulation, providing new directions for improving human cognitive ability and treating neurological diseases.

[0034] In the above method, the limitations of a single light source are effectively avoided by alternating operation of the dual-wavelength light source, and the alternating operation of the dual-wavelength light source improves the flexibility of the experiment. During the switching process of the dual-wavelength light source, there are no obvious problems such as light intensity fluctuation and spectral distortion, which effectively avoids errors caused by equipment adjustment, thereby significantly improving the accuracy and repeatability of experimental data. In addition, the fine clock timer 3 can realize accurate management of two different wavelength light sources. This accurate timing control method can maximize signal strength when rapidly switching different wavelength light sources, while effectively avoiding overlapping or blurring of fluorescent signals caused by delayed switching of the excitation light source. At the same time, by using the frame flipping mechanism of the CMOS imaging assembly 2 to capture multiple image frames under different wavelength light source excitation conditions, the influence of background noise and image distortion can be minimized. This efficient switching capability enables researchers to distinguish and record different fluorescent signals present in the detected sample (cells or tissues).

[0035] Specifically, the related information of the different fluorescent signals includes fluorescent signal intensity distribution and fluorescent signal change trend. The analysis and processing of the image frame data in S5 specifically includes: S51, positioning and extracting the fluorescent signal region in the image frame data by an image recognition algorithm to obtain fluorescent signal data; S52, performing noise reduction processing on the extracted fluorescent signal data by a signal processing algorithm; S53, analyzing and processing the fluorescent signal data after noise reduction processing to obtain the fluorescent signal intensity distribution and the fluorescent signal change trend.

[0036] In the embodiment, the light source assembly 1 comprises a first light source unit 11 and a second light source unit 12; the first light source unit 11 comprises a green light source 111 for emitting light rays in a green light band; a green excitation light filter 112 arranged on an outgoing light path of the green light source 111 for filtering the light rays emitted by the green light source 111 to screen out first excitation light of a specific wavelength range; a first achromatic lens 113 located on an outgoing light path of the green excitation light filter 112 for focusing and chromatic aberration correction of the filtered first excitation light; a first beam splitter 114 for receiving the first excitation light processed by the first achromatic lens 113 and reflecting the first excitation light to a sample area to be detected and receiving fluorescent signals generated by excitation from the sample area to be detected and capable of transmitting the fluorescent signals; a first focusing lens 115 arranged on a light path of the fluorescent signals transmitted by the first beam splitter 114 for focusing the fluorescent signals; a red fluorescent filter 116 located on an outgoing light path of the first focusing lens 115 for filtering the fluorescent signals to obtain fluorescent signals of a specific wavelength range; the second light source unit 12 comprises a blue light source 121 for emitting light rays in a blue light band; a blue excitation light filter 122 arranged on an outgoing light path of the blue light source 121 for filtering the light rays emitted by the blue light source 121 to screen out second excitation light of a specific wavelength range; a second achromatic lens 123 located on an outgoing light path of the blue excitation light filter 122 for focusing and chromatic aberration correction of the filtered second excitation light; a second beam splitter 124 for receiving the second excitation light processed by the second achromatic lens 123 and reflecting the second excitation light to a sample area to be detected and receiving fluorescent signals generated by excitation from the sample area to be detected and capable of transmitting the fluorescent signals; a second focusing lens 125 arranged on a light path of the fluorescent signals transmitted by the second beam splitter 124 for focusing the fluorescent signals; a green fluorescent filter 126 located on an outgoing light path of the second focusing lens 125 for filtering the fluorescent signals to obtain fluorescent signals of a specific wavelength range; the CMOS imaging assembly 2 is located on outgoing light paths of the red fluorescent filter 116 and the green fluorescent filter 126 for receiving and detecting the fluorescent signals filtered by the red fluorescent filter 116 and the green fluorescent filter 126 and converting the fluorescent signals into electrical signals for subsequent signal analysis and processing.

[0037] It is worth mentioning that the green light source 111 includes two first LED lamp beads arranged in a diagonal direction for emitting green light, the blue light source 121 includes two second LED lamp beads arranged in a diagonal direction for emitting blue light, and the first LED lamp bead and the second LED lamp bead are arranged on the same plane of the substrate. The structure has the following advantages: 1. The light output efficiency is improved, the light loss is reduced, and the excitation light source is stronger. 2. It can help to achieve more uniform color mixing, thereby reducing color gradient or shadow; and it can achieve more uniform illumination, not only making the response of the fluorescent probe to different wavelengths more consistent, reducing signal fluctuation, but also eliminating the color difference problem that may be caused by color distribution difference, improving the color consistency of the image, so that more accurate calcium imaging data and more accurate and reliable fluorescent signals can be obtained, and the dynamic of calcium ions in the detected sample (cells or tissues) can be accurately analyzed; 3. The resolution of the image is improved, and more detailed intracellular structure observation is supported; 4. There is a slight difference in heat distribution between different types of LED lamp beads. Through the diagonal layout, better heat distribution is brought, and the heat is more evenly distributed on the substrate, which helps to prolong the service life of the LED lamp bead and improve the reliability. From some angles, the diagonal arrangement can reduce the phenomenon of color dominance. In visual perception, this diagonal arrangement reduces the influence of different color lamp wicks on the viewing angle, improves the display effect, and makes the color performance more consistent when viewed from most angles.

[0038] Compared with the related art, the application provides a double-color alternating calcium imaging double-channel image extraction method, which comprises the following steps: providing a light source assembly, a CMOS imaging assembly, a fine clock timer and an extremely thin coaxial line, the light source assembly is used for alternately emitting light of at least two different wavelengths, and different wavelength light sources of the light source assembly are respectively connected with the fine clock timer through the extremely thin coaxial line; when the fine clock timer outputs a high-level signal, the light source of the corresponding wavelength is triggered to light up to excite a specific fluorescent probe, and the fluorescent protein is naturally attenuated in the remaining 20% of the high-level time; after the high-level state ends, the fine clock timer switches to a low-level signal, triggers the light source of another wavelength to light up to excite another fluorescent probe, and the fluorescent protein is naturally attenuated in the remaining 20% of the low-level time; a plurality of image frames are shot under the excitation condition of different wavelength light sources by the CMOS imaging assembly; and the image frame data is analyzed and processed to obtain related information of different fluorescent signals in the sample area to be detected. In the above method, the limitations of a single light source are effectively avoided by the alternating operation of the double-wavelength light source, and the alternating operation of the double-wavelength light source improves the flexibility of the experiment. In addition, during the light source switching process, there are no obvious problems such as light intensity fluctuation and spectral distortion, which effectively avoids the error caused by equipment adjustment, thereby significantly improving the accuracy and repeatability of experimental data. At the same time, the double-wavelength light source of the application can also be customized and adjusted according to different experimental requirements, such as the requirements of different sample types, different experimental scales and the like, the parameters of the light source (such as light intensity, pulse frequency and the like), the configuration of the light path (such as adding or replacing specific optical elements), further simplifying the experimental process and improving the efficiency and flexibility of the experiment.

[0039] The above-mentioned embodiments should be understood as illustrative rather than limiting the scope of the application, and the scope of the application is defined by the claims. Some non-essential improvements and adjustments made to the application by those skilled in the art without departing from the spirit and scope of the application still fall within the scope of the application.

Claims

1. A dual-color alternating calcium imaging dual-channel image extraction method, characterized in that: The method comprises: S1, providing a dual-color alternating calcium imaging device; the dual-color alternating calcium imaging device includes a light source assembly, a CMOS imaging assembly, a refined clock timer, and an extremely fine coaxial line, wherein the light source assembly is configured to alternately emit light of at least two different wavelengths, and the light sources of different wavelengths of the light source assembly are respectively connected to the refined clock timer via the extremely fine coaxial line to establish a signal connection; S2, using the refined clock timer to set the switching cycle, high-level duration, and low-level duration of the high-level signal and the low-level signal, when the refined clock timer outputs a high-level signal, triggering the extremely fine coaxial line to light up the light source of the corresponding wavelength and lasting for 80% of the high-level duration to excite a specific fluorescent probe, and at the same time, stopping the excitation of the light source within the remaining 20% ​​of the high-level duration to allow the fluorescent protein to decay naturally; S3, after the high-level state ends, the refined clock timer switches to a low-level signal, and a light source of another wavelength is illuminated through the extremely fine coaxial line for 80% of the low-level duration to excite another fluorescent probe. At the same time, during the remaining 20% ​​of the low-level duration, the excitation of the light source is stopped to allow the fluorescent protein to decay naturally; S4, capturing multiple image frames under excitation conditions of light sources of different wavelengths using a frame flip mechanism of the CMOS imaging component, wherein the triggering of the frame flip is synchronized with the switching of the level signal of the refined clock timer, so that the fluorescence signals corresponding to the image frames captured at different times are derived from excitation conditions of light sources of different wavelengths; S5, collecting image frame data captured by the CMOS imaging component, and analyzing and processing the image frame data to obtain relevant information of different fluorescence signals in the sample area to be detected; The relevant information of the different fluorescence signals includes the distribution of fluorescence signal intensity and the variation trend of the fluorescence signal; The image frame data is analyzed and processed in S5, specifically including: S51, locating and extracting the fluorescence signal region in the image frame data by using an image recognition algorithm to obtain fluorescence signal data; S52, performing noise reduction processing on the extracted fluorescence signal data using a signal processing algorithm; S53 , analyzing and processing the fluorescence signal data after the noise reduction process to obtain the fluorescence signal intensity distribution and the fluorescence signal change trend.

2. A dual-color alternating calcium imaging dual-channel image extraction method according to claim 1, characterized in that: The light source assembly includes a first light source unit and a second light source unit; The first light source unit includes: a green light source for emitting light in the green light band; a green excitation light filter, which is arranged on the outgoing light path of the green light source, and is used to filter the light emitted by the green light source to screen out the first excitation light in a specific wavelength range; a first achromatic lens, which is located on the outgoing light path of the green excitation light filter, and is used to focus and correct the chromatic aberration of the filtered first excitation light; a first spectroscope, which is used to receive the first excitation light processed by the first achromatic lens, and reflect the first excitation light to the sample area to be detected, and then receive the fluorescence signal generated by the excitation from the sample area to be detected, and can allow the fluorescence signal to pass through; a first focusing lens, which is arranged on the optical path of the fluorescence signal transmitted by the first spectroscope, and is used to focus the fluorescence signal; a red fluorescence filter, which is located on the outgoing light path of the first focusing lens, and is used to filter the fluorescence signal to obtain the fluorescence signal in a specific wavelength range; The second light source unit includes: a blue light source for emitting light in the blue light band; a blue excitation light filter, which is arranged on the outgoing light path of the blue light source, and is used to filter the light emitted by the blue light source to screen out the second excitation light in a specific wavelength range; a second achromatic lens, which is located on the outgoing light path of the blue excitation light filter, and is used to focus and correct the chromatic aberration of the filtered second excitation light; a second spectroscope, which is used to receive the second excitation light processed by the second achromatic lens, and reflect the second excitation light to the sample area to be detected, and then receive the fluorescence signal generated by the excitation from the sample area to be detected, and can allow the fluorescence signal to pass through; a second focusing lens, which is arranged on the optical path of the fluorescence signal transmitted by the second spectroscope, and is used to focus the fluorescence signal; a green fluorescence filter, which is located on the outgoing light path of the second focusing lens, and is used to filter the fluorescence signal to obtain the fluorescence signal in a specific wavelength range; The CMOS imaging component is located on the outgoing light path of the red fluorescent filter and the green fluorescent filter, and is used to receive and detect the fluorescent signals filtered by the red fluorescent filter and the green fluorescent filter, and convert the fluorescent signals into electrical signals for subsequent signal analysis and processing.

3. A dual-color alternating calcium imaging dual-channel image extraction method according to claim 2, characterized in that: The green light source includes two first LED lamp beads that are diagonally distributed and used to emit green light, and the blue light source includes two second LED lamp beads that are diagonally distributed and used to emit blue light.

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