Two-color alternating calcium imaging two-channel image extraction method

Abstract

The invention relates to a two-color alternating calcium imaging two-channel image extraction method. The method comprises the following steps: providing a light source assembly, a CMOS imaging assembly, a refined clock timer and a superfine coaxial line; when the refined clock timer outputs a high-level signal, a light source with a corresponding wavelength is triggered to light up so as to excite a specific fluorescent probe, and fluorescent protein is naturally attenuated within the remaining 20% of high-level duration; after the high-level state is ended, the refined clock timer is switched to a low-level signal, the light source with another wavelength is triggered to be lightened so as to excite another fluorescent probe, and fluorescent protein is naturally attenuated within the remaining 20% of low-level duration; shooting a plurality of image frames under the excitation condition of light sources with different wavelengths through the CMOS imaging assembly; and analyzing and processing the image frame data to obtain related information of different fluorescence signals in the to-be-detected sample area. According to the method, more accurate and reliable fluorescence signals can be obtained, so that the calcium ions in a detected sample can be dynamically and accurately analyzed.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical imaging, and in particular relates to a dual-color alternating calcium imaging dual-channel image extraction method. Background Art

[0002] In the field of neuroscience research, in-depth exploration of the operating mechanisms of neuronal activity and neural networks is of great significance for understanding brain function and overcoming neurological diseases. Calcium imaging technology, as an important means, provides researchers with the ability to observe neuronal activity at the cellular level by detecting the fluorescent signal generated by the flow of calcium ions during neuronal activity. However, traditional calcium imaging techniques mostly rely on fixed experimental environments and are difficult to be effectively applied in freely moving animal models, which to a certain extent limits the study of neural activity under natural behavioral conditions. Existing micro-calcium imaging microscopes only have monochromatic 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 types of neural activities is difficult to observe using a calcium imaging microscope with only one excitation light. Therefore, integrating two calcium imaging microscopes and developing devices that can simultaneously record two types of neurons has become an important development trend in modern neuroscience research. Through this integration, researchers can achieve real-time monitoring of different types of neurons in the same behavior in the same experimental system, or simultaneous real-time monitoring of fluorescently labeled blood vessels and another type of fluorescently labeled neurons, thereby deeply analyzing the potential relationship between blood activity and neural activity, or the coordinated relationship between different types of neural activities in the same behavior, thus providing a new perspective and powerful tools for in-depth analysis of complex neural networks and central brain diseases.

[0003] However, most of the related equipment on the market currently have obvious defects. Most products only use a single LED light source. This single light source system can only work within a specific wavelength range and cannot carry out multi-spectral experiments at the same time. This limitation greatly restricts the flexibility of the experiment, requiring frequent adjustments of equipment or replacement of light sources when operating different fluorescent probes. This not only increases the complexity of the experiment, but also easily introduces equipment adjustment errors. It may also have a negative impact on the accuracy and stability of the experimental data, making it difficult to meet the increasingly stringent requirements of neuroscience research for high-precision, multi-functional and high-stability experiments.

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

[0005] In view of the above problems, the present invention aims to provide a dual-color alternating calcium imaging dual-channel image extraction method, which can obtain more accurate and reliable fluorescence signals, thereby accurately analyzing the dynamics of calcium ions in the test sample.

[0006] The present invention provides a dual-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 component, a CMOS imaging component, a refined clock timer and an extremely fine coaxial line, the light source component is used to alternately emit at least two different wavelengths of light, and the different wavelength light sources of the light source component respectively establish signal connections with the refined clock timer through the extremely fine coaxial line;

[0008] 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, which is used 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, so that the fluorescent protein decays naturally;

[0009] 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 turned on through the extremely fine coaxial line for 80% of the low level duration, which is used to excite another fluorescent probe. At the same time, within the remaining 20% ​​of the low level duration, the excitation of the light source is stopped to allow the fluorescent protein to decay naturally;

[0010] S4, capturing multiple image frames under different wavelength light source excitation conditions through the frame flip mechanism of the CMOS imaging component, wherein the triggering of the frame flip is synchronized with the level signal switching of the refined clock timer, so that the fluorescence signals corresponding to the image frames captured at different times are derived from the light source excitation conditions of different wavelengths;

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

[0012] Preferably, the relevant information of the different fluorescence signals includes fluorescence signal intensity distribution and fluorescence signal variation trend.

[0013] Preferably, the step of analyzing and processing the image frame data in S5 specifically includes:

[0014] S51, locating and extracting the fluorescence signal area in the image frame data by using an image recognition algorithm to obtain fluorescence signal data;

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

[0016] 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.

[0017] Preferably, 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 a 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 after reflecting the first excitation light to the sample area to be detected, receive the fluorescence signal generated by the excitation from the sample area to be detected, and allow the fluorescence signal to pass through; a first focusing lens, which is arranged on the light 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 a blue light band; a blue excitation light filter, It 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; the second achromatic lens, which is located on the outgoing light path of the blue excitation light filter, is used to focus and correct the chromatic aberration of the filtered second excitation light; the second spectroscope is used to receive the second excitation light processed by the second achromatic lens, and after reflecting the second excitation light to the sample area to be detected, receive the fluorescence signal generated by the excitation of the sample area to be detected, and allow the fluorescence signal to pass; the second focusing lens is arranged on the light path of the second spectroscope transmitting the fluorescence signal, and is used to focus the fluorescence signal; the green fluorescence filter 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 paths of the red fluorescence filter and the green fluorescence filter, and is used to receive and detect the fluorescence signal after being filtered by the red fluorescence filter and the green fluorescence filter, and convert the fluorescence signal into an electrical signal 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 the related art, the present invention provides a dual-color alternating calcium imaging dual-channel image extraction method, comprising: providing a light source component, a CMOS imaging component, a refined clock timer and an extremely fine coaxial line, wherein the light source component is used to alternately emit at least two different wavelengths of light, and the different wavelength light sources of the light source component 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, a light source of a corresponding wavelength is triggered to light up to excite a specific fluorescent probe, and at the same time, the fluorescent protein is naturally decayed 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 a light source of another wavelength to light up to excite another fluorescent probe, and at the same time, the fluorescent protein is naturally decayed within the remaining 20% ​​of the low-level duration; a plurality of image frames are captured under the excitation conditions of light sources of different wavelengths by the CMOS imaging component; 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 limitation of a single light source is effectively avoided by alternating the operation of a dual-wavelength light source, and the alternating operation of the dual-wavelength light source improves the flexibility of the experiment; in addition, during the switching of the light source, there will be no obvious light intensity fluctuation, spectral distortion and other problems, which effectively avoids the error 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 customize the parameters of the light source (such as light intensity, pulse frequency, etc.) and the configuration of the optical path (such as adding or replacing specific optical elements) according to different experimental needs, such as requirements for different sample types and different experimental scales, further simplifying the experimental process and improving the efficiency and flexibility of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the process of a dual-color alternating calcium imaging dual-channel image extraction method of the present invention;

[0021] Figure 2 is a schematic diagram of a flow chart for analyzing and processing the image frame data in the present invention;

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

[0023] Figure 4 It is a 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 5It is a schematic diagram of the structure of the green light source and the blue light source in the present invention. DETAILED DESCRIPTION

[0025] The present invention provides a dual-color alternating calcium imaging dual-channel image extraction method, aiming to solve the problems that the existing calcium imaging technology uses a single LED light source and has significant limitations, and the accuracy and stability of experimental data are poor.

[0026] 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.

[0027] Please refer to the attached Figure 1-5 As shown, the present invention provides a dual-color alternating calcium imaging dual-channel image extraction method, which comprises:

[0028] S1, providing a two-color alternating calcium imaging device; the two-color alternating calcium imaging device 10 comprises a light source component 1, a CMOS imaging component 2, a refined clock timer 3 and an extremely fine coaxial line 4, the light source component 1 is used to alternately emit at least two different wavelengths of light, and the different wavelength light sources of the light source component 2 respectively establish signal connections 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 cycle, high level duration and low level duration of the high level signal and the low level signal, when the refined clock timer 3 outputs a high level signal, triggering the extremely fine coaxial line 4 to light up the light source of the corresponding wavelength and lasting for 80% of the high level duration, which is used 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, so that the fluorescent protein decays naturally;

[0030] S3, after the high level state ends, the refined clock timer 3 switches to a low level signal, and a light source of another wavelength is turned on through the extremely fine coaxial line 4 for 80% of the low level duration, which is used to excite another fluorescent probe. At the same time, within the remaining 20% ​​of the low level duration, the excitation of the light source is stopped to allow the fluorescent protein to decay naturally;

[0031] S4, capturing multiple image frames under different wavelength light source excitation conditions through the frame flip mechanism of the CMOS imaging component 2, wherein the triggering of the frame flip is synchronized with the level signal switching of the refined clock timer, so that the fluorescence signals corresponding to the image frames captured at different times are derived from the light source excitation conditions of different wavelengths;

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

[0033] It should be noted that the frame flip mechanism uses the ultra-fine coaxial line 4 as a signal transmission link, carrying the key "frame" signal, that is, the control instruction of alternating high and low levels, and its transmission frequency is accurately adapted to the frame rate of imaging to ensure that the entire imaging process is orderly; when the high-level signal is transmitted to the light source component 1 along the ultra-fine coaxial line 4, it drives an LED of a specific wavelength to emit light, and the light beam irradiates the detection sample to stimulate a specific fluorescent signal. At the same time, the CMOS imaging component 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 the acquisition of the corresponding image frame; and when the level flips to a low level, the power supply to the current LED is stopped, and another wavelength LED is driven to emit light, and the imaging parameters are also changed accordingly. In this way, through the frame flip mechanism, efficient synchronization of light source and imaging is achieved, which greatly reduces background noise and image distortion, and improves imaging quality and data acquisition efficiency. In addition, the above method uses the alternating working mode of the light source, which can be customized according to the experimental needs. Researchers can choose the appropriate wavelength combination according to specific scientific problems to meet the diverse needs of different research fields, which not only reduces the complexity of operation, but also reduces the potential cost, enabling more laboratories to participate in high-precision neuroscience research, which provides stronger support for scientific exploration and the possibility of 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, and researchers can monitor neuronal activity more accurately, 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 of 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, and explore new directions for improving human cognitive ability and treating neurological diseases.

[0034] In the above method, the limitation of a single light source is effectively avoided by the alternating operation of the dual-wavelength light source, and the alternating operation of the dual-wavelength light source improves the flexibility of the experiment, and during the switching process of the dual-wavelength light source, there will be no obvious light intensity fluctuations, spectral distortion and other problems, effectively avoiding the error caused by equipment adjustment, thereby significantly improving the accuracy and repeatability of the experimental data. In addition, the refined clock timer 3 can realize the accurate management of two light sources with different wavelengths. This accurate timing control method can maximize the signal intensity when the light sources with different wavelengths are quickly switched, and effectively avoid the overlapping or blurring of the fluorescence signals caused by the untimely switching of the excitation light source. At the same time, by shooting multiple image frames under different wavelength light source excitation conditions through the frame flip mechanism of the CMOS imaging component 2, the influence of background noise and image distortion can be minimized, and this efficient switching capability enables researchers to distinguish and record the different fluorescence signals present in the detection sample (cell or tissue).

[0035] Specifically, the relevant information of the different fluorescent signals includes the distribution of fluorescent signal intensity and the changing trend of the fluorescent signal. The image frame data is analyzed and processed in S5, specifically including: S51, locating and extracting the fluorescent signal area 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 distribution of fluorescent signal intensity and the changing trend of the fluorescent signal.

[0036] In this embodiment, the light source assembly 1 includes a first light source unit 11 and a second light source unit 12; the first light source unit 11 includes: a green light source 111, which is used to emit light in the green light band; a green excitation light filter 112, which is arranged on the outgoing light path of the green light source 111, and is used to filter the light emitted by the green light source 111 to filter out the first excitation light in a specific wavelength range; a first achromatic lens 113, which is located on the outgoing light path of the green excitation light filter 112, and is used to focus and correct the chromatic aberration of the filtered first excitation light; a first beam splitter 114, which is used to receive the first excitation light The achromatic lens 113 processes the first excitation light, and after reflecting the first excitation light to the sample area to be detected, receives the fluorescence signal generated by the excitation from the sample area to be detected, and allows the fluorescence signal to pass through; the first focusing lens 115 is arranged on the optical path of the fluorescence signal transmitted by the first beam splitter 114, and is used to focus the fluorescence signal; the red fluorescence filter 116 is located on the outgoing optical path of the first focusing lens 115, and is used to filter the fluorescence signal to obtain the fluorescence signal in a specific wavelength range; the second light source unit 12 includes: a blue light source 121, which is used to emit light in the blue light band; a blue excitation light filter The optical sheet 122 is arranged on the outgoing optical path of the blue light source 121, and is used to filter the light emitted by the blue light source 121 to screen out the second excitation light within a specific wavelength range; the second achromatic lens 123 is located on the outgoing optical path of the blue excitation light filter 122, and is used to focus and correct the chromatic aberration of the filtered second excitation light; the second beam splitter 124 is used to receive the second excitation light processed by the second achromatic lens 123, 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; the second focusing lens 124 is used to focus and correct the chromatic aberration of the filtered second excitation light. The lens 125 is arranged on the optical path of the fluorescent signal transmitted by the second beam splitter 124, and is used for focusing the fluorescent signal; the green fluorescent filter 126 is located on the output optical path of the second focusing lens 125, and is used for filtering the fluorescent signal to obtain the fluorescent signal in a specific wavelength range; the CMOS imaging component 2 is located on the output optical path of the red fluorescent filter 116 and the green fluorescent filter 126, and is used for receiving and detecting the fluorescent signal after filtering by the red fluorescent filter 116 and the green fluorescent filter 126, and converting the fluorescent signal into an electrical signal for subsequent signal analysis and processing.

[0037] It is worth mentioning that the green light source 111 includes two diagonally distributed first LED lamp beads for emitting green light, and the blue light source 121 includes two diagonally distributed second LED lamp beads for emitting blue light, and the first LED lamp beads and the second LED lamp beads are arranged on the same plane of the substrate. The arrangement of this structure has the following advantages: 1. It improves the light output efficiency, reduces light loss, and provides a stronger excitation light source. 2. It can help achieve more uniform color mixing, thereby reducing color gradients or shadows; and it can achieve more uniform lighting, which not only makes the fluorescent probe respond more consistently to different wavelengths and reduces signal fluctuations, but also eliminates the color difference problem that may be caused by differences in color distribution, and improves the color consistency of the image, so that higher-precision calcium imaging data and more accurate and reliable fluorescence signals can be obtained, and accurate analysis of calcium ion dynamics in the test sample (cell or tissue) can be achieved; 3. The resolution of imaging is improved, supporting more detailed observation of intracellular structures; 4. There are slight differences in thermal distribution between different types of LED lamp beads. The diagonal layout brings better thermal distribution, so that the heat is more evenly distributed on the substrate, which helps to extend the service life of the LED lamp beads and improve reliability. From some angles, the diagonal arrangement can reduce the phenomenon of a certain color dominance. In visual perception, this diagonal arrangement reduces the impact of different color 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 present invention provides a dual-color alternating calcium imaging dual-channel image extraction method, comprising: providing a light source component, a CMOS imaging component, a refined clock timer and an extremely fine coaxial line, wherein the light source component is used to alternately emit at least two different wavelengths of light, and the different wavelength light sources of the light source component 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, a light source of a corresponding wavelength is triggered to light up to excite a specific fluorescent probe, and at the same time, the fluorescent protein is naturally decayed 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 a light source of another wavelength to light up to excite another fluorescent probe, and at the same time, the fluorescent protein is naturally decayed within the remaining 20% ​​of the low-level duration; a plurality of image frames are captured under the excitation conditions of light sources of different wavelengths by the CMOS imaging component; 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 limitation of a single light source is effectively avoided by alternating the operation of a dual-wavelength light source, and the alternating operation of the dual-wavelength light source improves the flexibility of the experiment; in addition, during the switching of the light source, there will be no obvious light intensity fluctuation, spectral distortion and other problems, which effectively avoids the error 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 customize the parameters of the light source (such as light intensity, pulse frequency, etc.) and the configuration of the optical path (such as adding or replacing specific optical elements) according to different experimental needs, such as requirements for different sample types and different experimental scales, further simplifying the experimental process and improving the efficiency and flexibility of the experiment.

[0039] The above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, which shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention, without departing from the essence and scope of the present invention, still fall within the scope of protection of the present invention.

Claims

1. A dual-color alternating calcium imaging dual-channel image extraction method, characterized in that: The method comprises: S1, providing a two-color alternating calcium imaging device; the two-color alternating calcium imaging device comprises a light source component, a CMOS imaging component, a refined clock timer and an extremely fine coaxial line, the light source component is used to alternately emit at least two different wavelengths of light, and the different wavelength light sources of the light source component respectively establish signal connections with the refined clock timer through the extremely fine coaxial line; 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, which is used 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, so that the fluorescent protein decays 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 turned on through the extremely fine coaxial line for 80% of the low level duration, which is used to excite another fluorescent probe. At the same time, within 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 different wavelength light source excitation conditions through the frame flip mechanism of the CMOS imaging component, wherein the triggering of the frame flip is synchronized with the level signal switching of the refined clock timer, so that the fluorescence signals corresponding to the image frames captured at different times are derived from the light source excitation conditions of different wavelengths; S5, collecting the image frame data captured by the CMOS imaging component, and analyzing and processing the image frame data to obtain relevant information of different fluorescent signals in the sample area to be detected.

2. A dual-color alternating calcium imaging dual-channel image extraction method according to claim 1, characterized in that: The related information of the different fluorescence signals includes the fluorescence signal intensity distribution and the fluorescence signal change trend.

3. A dual-color alternating calcium imaging dual-channel image extraction method according to claim 2, characterized in that: The image frame data is analyzed and processed in S5, specifically including: S51, locating and extracting the fluorescence signal area 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 by 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.

4. 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 comprises: 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 after reflecting the first excitation light to the sample area to be detected, receive the fluorescence signal generated by the excitation from the sample area to be detected, and allow the fluorescence signal to pass through; a first focusing lens, which is arranged on the light 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 comprises: a blue light source for emitting light in a 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 perform chromatic aberration correction on the filtered second excitation light; a second spectroscope, which is used to receive the second excitation light processed by the second achromatic lens, and after reflecting the second excitation light to the sample area to be detected, receives the fluorescence signal generated by the excitation from the sample area to be detected, and allows the fluorescence signal to pass through; a second focusing lens, which is arranged on the light 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 signal after filtering by the red fluorescent filter and the green fluorescent filter, and convert the fluorescent signal into an electrical signal for subsequent signal analysis and processing.

5. A dual-color alternating calcium imaging dual-channel image extraction method according to claim 4, 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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