Cell microscopic image automatic acquisition and processing device and method based on multiband fluorescence excitation

Abstract

The invention belongs to the technical field of cell fluorescence detection, and particularly relates to an automatic cell microscopic image acquisition and processing device and method based on multiband fluorescence excitation. The device comprises a sequential fluorescence irradiation group, a microscopic fluorescence imaging group and a focusing objective table group, a main excitation light source and a back-illuminated compensation light source are matched with a spectroscope to form composite illumination light, light intensity feedback adjustment is realized by combining a light intensity sensor and a transmission-reflection curve, and stable excitation light is output. An integrated optical filter arranged in the device can switch different wavebands to realize multispectral monochromatic excitation, and a fluorescence signal is acquired and processed into image data through an imaging assembly. The method comprises the steps of sample focusing and positioning, exciting light regulation and control, image acquisition and processing and the like, has the advantages of high efficiency, automation, clear imaging and the like, and is suitable for cell multi-target fluorescence imaging analysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell fluorescence detection, and particularly relates to an automatic acquisition and processing device and method for cell microscopic images based on multi-band fluorescence excitation. Background Art

[0002] Fluorescence microscopy imaging technology is an indispensable key tool in modern biomedical research and clinical diagnosis, and has wide applications especially in aspects such as cell structure observation, subcellular component localization, protein expression analysis, and pathological detection. By fluorescently labeling specific molecules or cell structures and using fluorescent probes to emit detectable fluorescence signals at specific excitation wavelengths, researchers can achieve precise imaging and functional interpretation of complex biological samples.

[0003] However, traditional fluorescence microscopy imaging systems generally have the following limitations: First, multi-band fluorescence excitation and acquisition often rely on manual replacement of filter plates or excitation sources, and it is difficult to synchronously obtain multi-channel fluorescence signals in a short time; Second, the image acquisition process mostly relies on manual operation and offline image analysis, and it is difficult to meet the high-throughput and high-efficiency automatic processing requirements of large-scale samples; Third, problems such as crosstalk and uneven signal-to-noise ratio are prone to occur between different-band fluorescence signals, affecting the image quality and the accuracy of data analysis.

[0004] With the improvement of the demand for biological information acquisition and the development of artificial intelligence technology, automated, multi-band, and highly sensitive cell fluorescence imaging devices have gradually become a research hotspot. Therefore, there is an urgent need for a cell microscopy imaging device that can support multi-band fluorescence excitation, automatic image acquisition, multi-channel data fusion, and real-time analysis, to improve the efficiency, accuracy, and automation level of cell image acquisition and processing, and provide a more stable, efficient, and intelligent solution for scientific research and clinical applications. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to propose: An automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation, including a sequential fluorescence irradiation group 1, a microscopic fluorescence imaging group 2, and a focusing stage group 3; the sequential fluorescence irradiation group is used to provide fluorescence excitation light of different bands to a sample to be tested; the microscopic fluorescence imaging group 2 is located above the focusing stage group 3 and is used to perform microscopic fluorescence imaging on the sample to be tested; the focusing stage group 3 is located between the optical paths of the sequential fluorescence irradiation group 1 and the microscopic fluorescence imaging group 2 and is used to carry the sample to be tested.

[0006] In a preferred technical solution, the sequential fluorescence irradiation group 1 includes: a main excitation light source 11 for providing main illumination light; a compensation excitation light source 12 for providing supplementary illumination light; the main illumination light and the supplementary illumination light constitute composite illumination light; A spectroscope 13 is located between the main excitation light source 11 and the compensation excitation light source 12; the main excitation light source 11 irradiates the spectroscope 13 in a reflective manner, and the compensation excitation light source 12 irradiates the spectroscope 13 in a backlighting manner; A light intensity sensor 14 is located on the back side of the spectroscope 13 to detect the back-side light intensity of the spectroscope 13. The back-side light intensity of the spectroscope 13 includes the transmitted part of the main illumination light irradiating the front side of the spectroscope 13 and the reflected part of the supplementary illumination light irradiating the back side of the light mirror 13; the light intensity sensor 14 is electrically connected to the compensation excitation light source 12 to perform feedback regulation on the light intensity of the supplementary illumination light; A monochromatic excitation module 15 is located downstream of the optical path of the spectroscope 13; A dichroic mirror 16 is located downstream of the optical path of the monochromatic excitation module 15; an emission filter 17 is located downstream of the optical path of the dichroic mirror 16.

[0007] In a preferred technical solution, the micro-fluorescence imaging group 2 at least includes: an objective lens 21 located between the dichroic mirror 16 and the sample to be measured; an eyepiece 22 located downstream of the optical path of the emission filter 17; an imaging camera 23 located downstream of the optical path of the eyepiece 22.

[0008] In a preferred technical solution, the focusing stage group 3 includes: A stage base 31, which is a cast iron base with a shock-absorbing function; a stage 32 for carrying the sample to be measured, which can move along the horizontal direction and the height direction; a focusing member 33, which includes at least two groups of horizontal airbags 331 and at least four vertical airbags 332; the horizontal airbags 331 include an X-axis horizontal airbag and a Y-axis horizontal airbag, the vertical airbags 332 are Z-axis vertical airbags, and a return spring with a force application direction opposite to that of the vertical airbags 332 is also provided in the Z-axis direction.

[0009] In a preferred technical solution, the monochromatic excitation module 15 includes: an integrated filter, which is composed of multiple excitation filters coaxially installed, and the multiple excitation filters are arranged at an inclined angle so that the excitation light is incident on the sample to be measured at an inclination angle after exiting, for oblique-angle fluorescence excitation; a rotation driving member for driving the integrated filter to rotate into the main optical path, so that different excitation filters are respectively irradiated by the composite illumination light to emit different monochromatic excitation lights.

[0010] In a preferred technical solution, the device further includes an image processing group, specifically including: an image processor, an image memory, and a preview display; the image processor is used to process the image information captured by the imaging camera 23 into a picture file, output it to the preview display for display, and store or delete it according to the user's operation.

[0011] In a preferred technical solution, the feedback adjustment of the light intensity of the supplementary illumination light specifically includes: Retrieve the pre-stored transmission-reflection curve in the system, where the transmission-reflection curve is used to characterize the corresponding relationship between the transmitted light intensity of the main excitation light source 11 and the reflected light intensity of the compensation excitation light source 12; Collect the composite light intensity value on the back side of the beam splitter 13 through the light intensity sensor 14, and compare the composite light intensity value with the target light intensity value in the transmission-reflection curve; According to the comparison result, determine the composite light intensity value on the front side of the beam splitter 13, compare it with the required illumination light intensity value, and output a feedback signal to the compensation excitation light source 12 for negative feedback adjustment; After the light emission intensity of the compensation excitation light source 12 changes due to negative feedback adjustment, re-collect the composite light intensity value on the back side of the beam splitter 13 and loop through the comparison and adjustment steps until the absolute value of the difference between the calculated composite light intensity value on the front side of the beam splitter 13 and the target light intensity value is less than the preset threshold, then complete this feedback adjustment.

[0012] The present invention also provides an automatic acquisition and processing method for cell microscopic images based on multi-band fluorescence excitation, which is implemented based on the described device and includes the following steps: S1. Sample installation and stage focusing: Fix the sample to be measured on the focusing stage group, and achieve precise positioning of the sample in the horizontal and height directions by controlling the horizontal airbag and vertical airbag in the focusing stage group; S2. Generation and regulation of composite excitation light: Turn on the main excitation light source and the compensation excitation light source simultaneously, form the composite illumination light for irradiating the sample to be measured through the beam splitter, the light intensity sensor real-time collects the composite light intensity on the back side, and performs feedback adjustment on the compensation excitation light source through the transmission-reflection curve; S3. Output of monochromatic excitation light and optical path distribution: Regulate the rotation state of the integrated filter in the monochromatic excitation module to make different excitation filters appear in the main optical path, and realize the oblique incidence of the monochromatic excitation light of the required wavelength band to the sample.

[0013] In a preferred technical solution, it further includes the following steps: S4. Image acquisition and imaging path control: Collect the fluorescence signal through the objective lens, focus it through the dichroic mirror, emission filter and eyepiece in sequence, and obtain the image data by the imaging camera; S5. Image processing and preview: Format the image data by the image processor and output it to the preview display for display; S6. Image selection and storage: The user selects whether to store the image according to the real-time preview result, the selected image is stored in the image memory, and the remaining images can be deleted as needed.

[0014] In a preferred technical solution, the feedback regulation in the step S2 specifically includes: S21. Call the transmission-reflection curve built in the system and set the target light intensity range; S22. Collect the dorsal light intensity value in real time through a light intensity sensor and calculate the estimated positive-side irradiation intensity; S23. Compare the estimated value with the target light intensity to generate a feedback regulation signal to control the output intensity of the compensation excitation light source; S24. Repeat the collection and adjustment process until the difference between the calculated positive-side light intensity and the target light intensity is less than the set threshold, and this round of light intensity calibration is completed.

[0015] Beneficial effects The automatic acquisition and processing device and method for cell microscopic images based on multi-band fluorescence excitation provided by the present invention can realize efficient and continuous imaging of the same cell sample under multi-band excitation conditions, and improve the image acquisition efficiency and the resolution accuracy of fluorescence signals.

[0016] Through the cooperative illumination of the main excitation light source and the compensation excitation light source, the present invention forms a composite excitation light, and combines with the spectroscopic mirror structure setting to realize the optical path fusion of main light reflection and supplementary light backlighting. Among them, the backlighting compensation mechanism can dynamically compensate for the transmission attenuation of the main excitation light source, and only performs feedback regulation on the compensation light source, reducing the system fluctuation error, so as to stabilize the illumination intensity during different band switches. According to the preset transmission-reflection curve, the light intensity on the back side of the spectroscopic mirror is obtained in real time and the compensation excitation light source is feedback-regulated to ensure that the composite excitation light intensity received by the sample is constant and reliable, significantly improving the image quality.

[0017] The present invention also adopts a multi-piece integrated filter mounted obliquely and a rotation drive structure to realize the rapid switching of multi-band excitation lights, avoiding frequent replacement of filter elements, with convenient operation, and is applicable to high-throughput cell screening and automatic fluorescence image processing application scenarios. Brief description of the drawings

[0018] Figure 1 It is a schematic diagram of the device relationship hierarchy of the present invention; Figure 2 It is an optical path diagram of the main components of the device of the present invention; Figure 3 It is a front view of the device of the present invention; Figure 4 It is a rear view of the device of the present invention; Figure 5 It is a schematic flowchart of the method of the present invention. Detailed embodiments

[0019] To deepen the understanding of the present invention, the following will further elaborate on the present invention in conjunction with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0020] Embodiment 1 According to Figure 1 , Figure 3 and Figure 4 As shown, the automatic acquisition and processing device for cell microscopic images based on multi - band fluorescence excitation includes a sequential fluorescence irradiation group 1, a microscopic fluorescence imaging group 2, and a focusing stage group 3.

[0021] The sequential fluorescence irradiation group 1 is used to provide fluorescence excitation light of different bands to the sample to be measured; the microscopic fluorescence imaging group 2 is located above the focusing stage group 3 and is used to perform microscopic fluorescence imaging on the sample to be measured; the focusing stage group 3 is located between the optical paths of the sequential fluorescence irradiation group 1 and the microscopic fluorescence imaging group 2 and is used to carry the sample to be measured.

[0022] According to Figure 2 As shown, the sequential fluorescence irradiation group 1 includes: a main excitation light source 11 for providing main illumination light; a compensation excitation light source 12 for providing supplementary illumination light; the main illumination light and the supplementary illumination light constitute composite illumination light; a beam splitter 13 located between the main excitation light source 11 and the compensation excitation light source 12.

[0023] The main excitation light source 11 irradiates the beam splitter 13 in a reflective manner, and the compensation excitation light source 12 irradiates the beam splitter 13 in a back - illumination manner; a light intensity sensor 14 is located on the back side of the beam splitter 13 to detect the back - side light intensity of the beam splitter 13. The back - side light intensity of the beam splitter 13 includes the transmitted part of the main illumination light irradiated on the front side of the beam splitter 13 and the reflected part of the supplementary illumination light irradiated on the back side of the beam splitter 13; the light intensity sensor 14 is electrically connected to the compensation excitation light source 12 to perform feedback adjustment on the light intensity of the supplementary illumination light.

[0024] The monochromatic excitation module 15 is located downstream of the optical path of the beam splitter 13 and includes an integrated filter. The integrated filter is composed of multiple excitation filters coaxially installed. The multiple excitation filters are arranged to be installed at an inclined angle so that the excitation light is incident on the sample to be measured at an inclination angle after exiting, for oblique - angle fluorescence excitation; a rotation driving member is used to drive the integrated filter to rotate into the main optical path, so that different excitation filters are respectively irradiated by the composite illumination light to emit different monochromatic excitation lights.

[0025] A dichroic mirror 16 is located downstream of the optical path of the monochromatic excitation module 15; an emission filter 17 is located downstream of the optical path of the dichroic mirror 16.

[0026] The micro-fluorescence imaging group 2 at least includes: an objective lens 21, located between the dichroic mirror 16 and the sample to be measured; an eyepiece 22, located downstream of the optical path of the emission filter 17; and an imaging camera 23, located downstream of the optical path of the eyepiece 22.

[0027] The focusing stage group 3 includes: a stage base 31, which is a cast iron base with a shock-absorbing function; a stage 32, used to carry the sample to be measured and can move in the horizontal and height directions; a focusing member 33, including at least two groups of horizontal airbags 331 and at least four vertical airbags 332; the horizontal airbags 331 include an X-axis horizontal airbag and a Y-axis horizontal airbag, the vertical airbags 332 are Z-axis vertical airbags, and a return spring with a force application direction opposite to that of the vertical airbags 332 is also provided in the Z-axis direction.

[0028] The system further includes an image processor, an image memory, and a preview display; the image processor is used to process the image information captured by the imaging camera 23 into a picture file, output it to the preview display for display, and store or delete it according to the user's operation.

[0029] The process of feedback adjustment of the light intensity of the supplementary illumination light includes: retrieving the pre-stored transmission-reflection curve of the system, which is used to characterize the corresponding relationship between the transmitted light intensity of the main excitation light source 11 and the reflected light intensity of the compensation excitation light source 12; collecting the composite light intensity value on the back side of the beam splitter 13 through the light intensity sensor 14, and comparing the composite light intensity value with the target light intensity value in the transmission-reflection curve.

[0030] According to the comparison result, determine the composite light intensity value on the front side of the beam splitter 13, compare it with the required irradiation light intensity value, and output a feedback signal to the compensation excitation light source 12 for negative feedback adjustment; after the light emission intensity of the compensation excitation light source 12 changes due to negative feedback adjustment, re-collect the composite light intensity value on the back side of the beam splitter 13 and loop through the comparison and adjustment steps until the absolute value of the difference between the calculated composite light intensity value on the front side of the beam splitter 13 and the target light intensity value is less than the preset threshold, then complete this feedback adjustment.

[0031] Embodiment 2 As Figure 5 shown, this embodiment provides a method for automatically collecting and processing cell microscopic images based on multi-band fluorescence excitation, which is implemented based on the above device and includes the following steps: S1. Sample installation and stage focusing: Place the sample to be measured on a glass slide for fixation and install it on the stage of the focusing stage group. By adjusting the X-axis horizontal airbag, Y-axis horizontal airbag, and Z-axis vertical airbag set in the focusing stage group, and with the assistance of the return spring, fine-tuning movement of the sample in the horizontal plane and precise focusing in the vertical direction are achieved, ensuring that the sample area is at the center of the imaging field of view and has an appropriate depth of focus.

[0032] S2. Generation and regulation of composite excitation light: The main excitation light source and the compensation excitation light source are started synchronously. The main excitation light source emits main illumination light towards the front of the beam splitter, and the compensation excitation light source performs back-illumination irradiation from the back side of the beam splitter. The two together form composite illumination light. The composite illumination light forms a superimposed output after passing through the beam splitter and irradiates the sample area to excite different fluorescence responses.

[0033] The light intensity sensor is installed on the back side of the beam splitter and is used to detect in real time the combined light intensity of the transmitted main light and the reflected complementary light passing through the beam splitter. It performs comparison and analysis according to the preset transmission-reflection curve of the system, and automatically adjusts the output power of the compensation excitation light source so that the final composite light intensity irradiating the sample is stabilized within the target range.

[0034] S3. Output of monochromatic excitation light and optical path distribution: Regulate the integrated filter assembly set in the monochromatic excitation module. The driver controls it to rotate around the optical axis so that the excitation filter of the selected wavelength band cuts into the main optical path. When the excitation filter is in the main optical path, the composite illumination light is filtered into monochromatic excitation light of a specific wavelength band and is incident on the sample area to be measured at an inclination angle to complete the specific spectral excitation under the condition of multi-band fluorescence excitation.

[0035] S4. Image acquisition and imaging path control: The excited fluorescence signal is collected by the objective lens and sequentially passes through the dichroic mirror, emission filter, and eyepiece system along the established optical path for optical processing. The processed optical signal is finally focused on the photosensitive element of the imaging camera, and the imaging camera takes pictures of the current fluorescence state of the sample to obtain high-resolution microscopic image data.

[0036] S5. Image processing and preview: The obtained original image data is processed by the image processor for format conversion, color balance, and image enhancement, and the processed image is displayed in real time on the preview monitor, facilitating the operator to immediately observe the current field image quality and fluorescence response state.

[0037] S6. Image selection and storage: The user decides whether to retain the current image according to the preview display effect. If it is necessary to retain it, the system automatically stores the image in the image memory; if there are problems such as defocusing, light spots, or unsatisfactory signal-to-noise ratio in the image, the deletion operation can be performed through the system control interface to avoid the accumulation of redundant images.

[0038] The feedback adjustment process in step S2 includes: S21. Call the preset transmission-reflection curve in the system, where the curve reflects the ratio relationship between the transmitted light of the main excitation light source and the reflected light of the compensation excitation light source, and set the allowable range of the target irradiation light intensity; S22. The composite light intensity value on the back side of the beam splitter is collected in real time by the light intensity sensor, and the equivalent irradiation intensity of the current composite illumination light on the front side of the beam splitter is calculated through an algorithm; S23. Compare and analyze the predicted irradiation intensity with the target light intensity value, generate a feedback control signal according to the deviation, and this signal will be sent to the compensation excitation light source controller to adjust its output light intensity; S24. Repeat the collection and adjustment process, gradually approach the target irradiation intensity through multiple cycles. When the absolute value of the difference between the calculated irradiation light intensity on the front side of the beam splitter and the set target value is lower than the tolerance threshold set by the system, the feedback adjustment terminates and enters the next round of image acquisition process.

[0039] While ensuring the full excitation of the fluorescence signal under different excitation wavelength conditions, this method avoids overexposure or underexposure phenomena, improves the balance and analysis stability of image data, and is applicable to the standardized acquisition process of multi-target cell fluorescence imaging analysis.

[0040] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. An automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation, characterized in that: It includes a sequential fluorescence irradiation group (1), a microscopic fluorescence imaging group (2), and a focusing stage group (3); The sequential fluorescence irradiation group is used to provide fluorescence excitation light of different bands to the sample to be measured; The microscopic fluorescence imaging group (2) is located above the focusing stage group (3) and is used to perform microscopic fluorescence imaging on the sample to be measured; The focusing stage group (3) is located between the optical paths of the sequential fluorescence irradiation group (1) and the microscopic fluorescence imaging group (2) and is used to carry the sample to be measured.

2. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 1, characterized in that: The sequential fluorescence irradiation group (1) includes: A main excitation light source (11) for providing main illumination light; A compensation excitation light source (12) for providing supplementary illumination light; The main illumination light and the supplementary illumination light constitute composite illumination light; A beam splitter (13) located between the main excitation light source (11) and the compensation excitation light source (12); the main excitation light source (11) performs reflective irradiation on the beam splitter (13), and the compensation excitation light source (12) performs back-illumination irradiation on the beam splitter (13); A light intensity sensor (14) located on the back side of the beam splitter (13) to detect the back-side light intensity of the beam splitter (13). The back-side light intensity of the beam splitter (13) includes the transmitted part of the main illumination light irradiating on the front side of the beam splitter (13) and the reflected part of the supplementary illumination light irradiating on the back side of the beam splitter (13); the light intensity sensor (14) is electrically connected to the compensation excitation light source (12) to perform feedback adjustment on the light intensity of the supplementary illumination light; A monochromatic excitation module (15) located downstream of the optical path of the beam splitter (13); A dichroic mirror (16) located downstream of the optical path of the monochromatic excitation module (15); An emission filter (17) located downstream of the optical path of the dichroic mirror (16).

3. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 1, wherein: The microscopic fluorescence imaging group (2) at least includes: An objective lens (21) located between the dichroic mirror (16) and the sample to be measured; An eyepiece (22) located downstream of the optical path of the emission filter (17); An imaging camera (23) located downstream of the optical path of the eyepiece (22).

4. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 1, wherein: The focusing stage group (3) includes: A stage base (31); A stage (32) for carrying the sample to be measured, which can move along the horizontal direction and the height direction; A focusing member (33) including at least two groups of horizontal airbags (331) and at least four vertical airbags (332); The horizontal airbags (331) include an X-axis horizontal airbag and a Y-axis horizontal airbag, and the vertical airbags (332) are Z-axis vertical airbags. A return spring with a force application direction opposite to that of the vertical airbags (332) is also provided in the Z-axis direction.

5. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 2, wherein: The monochromatic excitation module (15) includes: An integrated filter, which is composed of multiple excitation filters coaxially installed. The multiple excitation filters are arranged at an inclined angle so that the excitation light is incident on the sample to be measured at an inclined angle after exiting, for oblique-angle fluorescence excitation; A rotation driving member is used to drive the integrated filter to rotate into the main optical path, so that different excitation filters are respectively irradiated by the composite illumination light to emit different monochromatic excitation lights.

6. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 5, wherein: The device further includes an image processing group, specifically including: an image processor, an image memory, and a preview display; the image processor is configured to process the image information captured by the imaging camera (23) into a picture file, output it to the preview display for display, and store it in the image memory or delete it according to the user's operation.

7. The automatic acquisition and processing device for cell microscopic images based on multi-band fluorescence excitation according to claim 2, wherein: The feedback adjustment of the light intensity of the supplementary illumination light specifically includes: Retrieving the pre-stored transmission-reflection curve in the system, where the transmission-reflection curve is used to characterize the corresponding relationship between the transmitted light intensity of the main excitation light source (11) and the reflected light intensity of the compensation excitation light source (12); Collecting the composite light intensity value on the back side of the beam splitter (13) through the light intensity sensor (14), and comparing the composite light intensity value with the target light intensity value in the transmission-reflection curve; According to the comparison result, determining the composite light intensity value on the front side of the beam splitter (13), comparing it with the required illumination light intensity value, and outputting a feedback signal to the compensation excitation light source (12) for negative feedback adjustment; After the luminous intensity of the compensation excitation light source (12) changes due to negative feedback adjustment, re-collect the composite light intensity value on the back side of the beam splitter (13) and loop through the comparison and adjustment steps until the absolute value of the difference between the calculated composite light intensity value on the front side of the beam splitter (13) and the target light intensity value is less than the preset threshold, and this feedback adjustment is completed.

8. An automatic acquisition and processing method for cell microscopic images based on multi-band fluorescence excitation, implemented based on the device according to any one of claims 1-7, characterized in that, It includes the following steps: S1. Sample installation and stage focusing: Fix the sample to be measured on the focusing stage group, and achieve precise positioning of the sample in the horizontal and height directions by controlling the horizontal airbag and vertical airbag in the focusing stage group; S2. Composite excitation light generation and regulation: The main excitation light source and the compensation excitation light source are turned on simultaneously, and the composite illumination light for irradiating the sample to be measured is formed by the beam splitter. The light intensity sensor continuously collects the composite light intensity on the back side, and performs feedback adjustment on the compensation excitation light source through the transmission-reflection curve; S3. Monochromatic excitation light output and optical path distribution: Regulate the rotation state of the integrated filter in the monochromatic excitation module, so that different excitation filters appear in the main optical path, and the monochromatic excitation light of the required wavelength band is obliquely incident on the sample.

9. The automatic acquisition and processing method of cell microscopic images based on multi-band fluorescence excitation according to claim 8, wherein, It also includes the following steps: S4. Image acquisition and imaging path control: Collect the fluorescence signal through the objective lens, focus it through the dichroic mirror, emission filter, and eyepiece in sequence, and obtain the image data by the imaging camera; S5. Image processing and preview: The image data is formatted by the image processor and output to the preview display for display; S6. Image selection and storage: The user selects whether to store the image according to the real-time preview result. The selected image is stored in the image memory, and the remaining images can be deleted as needed.

10. The automatic acquisition and processing method of cell microscopic images based on multi-band fluorescence excitation according to claim 9, characterized in that, The feedback adjustment in step S2 specifically includes: S21. Call the built-in transmission-reflection curve in the system and set the target light intensity range; S22. Continuously collect the back-side light intensity value through the light intensity sensor and calculate the estimated front-side illumination intensity; S23. Compare the predicted value with the target light intensity to generate a feedback adjustment signal to control the output intensity of the compensation excitation light source; S24. Repeat the acquisition and adjustment process until the difference between the calculated positive side light intensity and the target light intensity is less than the set threshold, and complete the current round of light intensity calibration.

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