Cell micro-image automatic acquisition and processing device and method based on multi-band fluorescence excitation

Abstract

The present application belongs to the technical field of cell fluorescence detection, and particularly relates to a device and method for automatic acquisition and processing of cell microscopic images based on multi-waveband fluorescence excitation. The device comprises a sequential fluorescence irradiation group, a microscopic fluorescence imaging group and a focusing object table group. A compound illumination light is formed by a main excitation light source cooperating with a back-illumination compensation light source and a beam splitter, light intensity feedback regulation is realized by combining a light intensity sensor and a transmission-reflection curve, and stable excitation light is output. The integrated filter provided in the device can switch different wavebands to realize multi-spectral monochromatic excitation, and the fluorescence signal is collected and processed into image data by the imaging assembly. The method comprises steps such as sample focusing positioning, excitation light regulation, image acquisition and processing, and has advantages such as high efficiency, automation and clear imaging, 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 in particular relates to a device and method for automatically collecting and processing cell microscopic images based on multi-band fluorescence excitation. Background Art

[0002] Fluorescence microscopy is an essential tool in modern biomedical research and clinical diagnosis, with particular applications in cell structure observation, subcellular component localization, protein expression analysis, and pathology detection. By fluorescently labeling specific molecules or cellular 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 systems generally have the following limitations: First, multi-band fluorescence excitation and acquisition often rely on manual replacement of filters or excitation sources, making it difficult to achieve synchronous acquisition of multi-channel fluorescence signals in a short period of time; second, the image acquisition process often relies on manual operation and offline image analysis, which is difficult to meet the needs of high-throughput and high-efficiency automatic processing of large-scale samples; third, crosstalk and uneven signal-to-noise ratio are prone to occur between fluorescence signals of different bands, affecting image quality and the accuracy of data analysis.

[0004] With the increasing demand for bioinformation 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. This can improve the efficiency, accuracy, and automation 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 response to the above problems, the purpose of the present invention is to propose: an automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation, comprising 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 tested; the microscopic fluorescence imaging group 2 is located above the focusing stage group 3, and is used to perform microscopic fluorescence imaging of 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 a composite illumination light;

[0007] The beam splitter 13 is located between the main excitation light source 11 and the compensation excitation light source 12; the main excitation light source 11 performs reflective illumination on the beam splitter 13, and the compensation excitation light source 12 performs back illumination on the beam splitter 13;

[0008] A light intensity sensor 14 is located on the back side of the spectroscope 13 and detects the back side light intensity of the spectroscope 13. The back side light intensity of the spectroscope 13 includes the transmitted portion of the main illumination light irradiating the front side of the spectroscope 13 and the reflected portion of the supplementary illumination light irradiating the back side of the spectroscope 13. The light intensity sensor 14 is electrically connected to the compensation excitation light source 12 and performs feedback adjustment on the light intensity of the supplementary illumination light.

[0009] A monochromatic excitation module 15 is located downstream of the beam splitter 13;

[0010] The dichroic mirror 16 is located downstream of the optical path of the monochromatic excitation module 15 ; the emission filter 17 is located downstream of the optical path of the dichroic mirror 16 .

[0011] In a preferred technical solution, the microscopic fluorescence imaging group 2 includes at least: 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.

[0012] In a preferred technical solution, the focusing stage group 3 includes:

[0013] The object base 31 is a cast iron base with a shock-absorbing effect; the object stage 32 is used to carry the sample to be tested and can move in the horizontal and vertical directions; the focusing component 33 includes at least two groups of horizontal airbags 331 and at least four vertical airbags 332; the horizontal airbags 331 include X-axis horizontal airbags and Y-axis horizontal airbags, and the vertical airbags 332 are Z-axis vertical airbags. A return spring is also provided in the Z-axis direction in the opposite direction to the force applied by the vertical airbags 332.

[0014] In a preferred technical solution, the monochromatic excitation module 15 includes: an integrated filter, which is composed of multiple coaxially mounted excitation filters, and 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 tested at an inclined angle after being emitted, for oblique-angle fluorescence excitation; a rotating drive member, which is used to drive the integrated filter to rotate into the main light path, so that different excitation filters are respectively irradiated by the composite illumination light and emit different monochromatic excitation lights.

[0015] In a preferred technical solution, the device also 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 it in the image memory or delete it according to the user's operation.

[0016] In a preferred technical solution, the feedback adjustment of the light intensity of the supplementary lighting light specifically includes:

[0017] Retrieving a transmission-reflection curve pre-stored in the system, wherein 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;

[0018] The light intensity sensor 14 collects the composite light intensity value on the back side of the spectroscope 13, and compares the composite light intensity value with the target light intensity value in the transmission-reflection curve;

[0019] According to the comparison result, the composite light intensity value on the positive side of the spectrometer 13 is determined, compared with the required illumination light intensity value, and a feedback signal is output to the compensation excitation light source 12 for negative feedback adjustment;

[0020] After compensating for the change in the luminous intensity of the excitation light source 12 due to negative feedback adjustment, the composite light intensity value on the back side of the spectrometer 13 is collected again and the comparison and adjustment steps are performed cyclically until the absolute value of the difference between the calculated composite light intensity value on the front side of the spectrometer 13 and the target light intensity value is less than the preset threshold value, and this feedback adjustment is completed.

[0021] The present invention also 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:

[0022] S1. Sample installation and stage focusing: Fix the sample to be tested on the focusing stage assembly, and achieve precise positioning of the sample in the horizontal and height directions by controlling the horizontal and vertical airbags in the focusing stage assembly;

[0023] 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 is formed by the beam splitter to illuminate the sample to be tested. The light intensity sensor collects the back-side composite light intensity in real time and uses the transmission-reflection curve to feedback and adjust the compensation excitation light source;

[0024] 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 light path, achieving oblique angle incidence of monochromatic excitation light of the required wavelength band on the sample.

[0025] In a preferred technical solution, the following steps are also included:

[0026] S4, image acquisition and imaging path control: The fluorescence signal is collected by the objective lens, focused through the dichroic mirror, emission filter and eyepiece in sequence, and then the image data is acquired by the imaging camera;

[0027] S5, image processing and preview: the image data is formatted by the image processor and output to the preview display for display;

[0028] S6. Image selection and storage: The user selects whether to store the image based on the real-time preview result. The selected image is stored in the image memory, and the remaining images can be deleted as needed.

[0029] In a preferred technical solution, the feedback adjustment in step S2 specifically includes:

[0030] S21. Call the built-in transmission-reflection curve of the system and set the target light intensity range;

[0031] S22, collecting the back side light intensity value in real time through the light intensity sensor and calculating the estimated front side illumination intensity;

[0032] S23, comparing the estimated value with the target light intensity, and generating a feedback adjustment signal to control the output intensity of the compensation excitation light source;

[0033] 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, completing this round of light intensity calibration.

[0034] Beneficial effects

[0035] The present invention provides an automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation and a method thereof, which can realize efficient and continuous imaging of the same cell sample under multi-band excitation conditions, thereby improving image acquisition efficiency and resolution accuracy of fluorescence signals.

[0036] The present invention forms composite excitation light through the coordinated illumination of the main excitation light source and the compensation excitation light source, and combines the structure of the spectrometer to realize the fusion of the optical paths of the main light reflection and the supplementary light back illumination. The back illumination compensation mechanism can dynamically compensate for the transmission attenuation of the main excitation light source, and only feedback adjust the compensation light source, reducing the system fluctuation error, thereby stabilizing the illumination intensity when switching between different bands. According to the preset transmission-reflection curve, the light intensity on the back side of the spectrometer is obtained in real time and the compensation excitation light source is feedback-adjusted to ensure that the composite excitation light intensity received by the sample is constant and reliable, significantly improving the image quality.

[0037] The present invention also uses tilted multi-piece integrated filters and a rotary drive structure to achieve rapid switching of multi-band excitation light, avoiding frequent replacement of filter elements. It is easy to operate and suitable for high-throughput cell screening and automatic fluorescence image processing application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the device relationship hierarchy of the present invention;

[0039] Figure 2 A light path diagram of the main components of the device of the present invention;

[0040] Figure 3 is a forward view of the device of the present invention;

[0041] Figure 4 A back view of the device of the present invention;

[0042] Figure 5 Schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0043] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0044] Example 1

[0045] according to Figure 1 、 Figure 3 and Figure 4 As shown, the automatic cell microscopic image acquisition and processing device 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.

[0046] The sequential fluorescence irradiation group 1 is used to provide fluorescence excitation light of different bands to the 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 of 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.

[0047] according to Figure 2 The sequential fluorescence irradiation group 1 shown 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; and a spectrometer 13 located between the main excitation light source 11 and the compensation excitation light source 12.

[0048] The main excitation light source 11 performs reflective illumination on the spectroscope 13, and the compensation excitation light source 12 performs back illumination on the spectroscope 13; the light intensity sensor 14 is located on the back side of the spectroscope 13, and detects 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 spectroscope 13; the light intensity sensor 14 is electrically connected to the compensation excitation light source 12, and performs feedback adjustment on the light intensity of the supplementary illumination light.

[0049] The monochromatic excitation module 15 is located downstream of the optical path of the spectroscope 13 and includes an integrated filter. The integrated filter is composed of multiple coaxially mounted excitation filters. 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 tested at an inclined angle after being emitted, which is used for oblique-angle fluorescence excitation; a rotating drive 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 and emit different monochromatic excitation lights.

[0050] The dichroic mirror 16 is located downstream of the optical path of the monochromatic excitation module 15 ; the emission filter 17 is located downstream of the optical path of the dichroic mirror 16 .

[0051] 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; and an imaging camera 23 located downstream of the optical path of the eyepiece 22.

[0052] The focusing stage assembly 3 includes: a stage base 31, which is a cast iron base with a shock-absorbing effect; a stage 32, which is used to carry the sample to be tested and can move in the horizontal and vertical directions; 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 X-axis horizontal airbags and Y-axis horizontal airbags, and the vertical airbags 332 are Z-axis vertical airbags. A return spring is also provided in the Z-axis direction in the opposite direction to the force applied by the vertical airbags 332.

[0053] The system also 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 it in the image memory or delete it according to the user's operation.

[0054] The feedback regulation process of the light intensity of the supplementary illumination light includes: calling the pre-stored transmission-reflection curve of the system, the transmission-reflection curve being used to characterize the corresponding relationship between the transmission light intensity of the main excitation light source 11 and the reflection light intensity of the compensation excitation light source 12; collecting the composite light intensity value on the back side of the beam splitter 13 by the light intensity sensor 14, and comparing the composite light intensity value with the target light intensity value in the transmission-reflection curve.

[0055] According to the comparison result, the composite light intensity value on the front side of the beam splitter 13 is determined, which is compared with the required illumination light intensity value, and a feedback signal is output to the compensation excitation light source 12 for negative feedback regulation; after the light intensity of the compensation excitation light source 12 changes due to negative feedback regulation, the composite light intensity value on the back side of the beam splitter 13 is re-collected and the comparison and regulation steps are repeatedly performed 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 a pre-set threshold value, and the feedback regulation is completed.

[0056] Embodiment Two

[0057] As shown in Figure 5 The embodiment provides a cell microscopic image automatic acquisition and processing method based on multi-band fluorescence excitation, which is implemented based on the above device and includes the following steps:

[0058] S1, sample installation and focusing of the objective table: the sample to be measured is fixed on a glass slide and installed on the objective table of the focusing objective table group; by adjusting the X-axis horizontal air bag, the Y-axis horizontal air bag and the Z-axis vertical air bag arranged in the focusing objective table group, and the auxiliary action of the return spring, the fine movement of the sample in the horizontal plane and the accurate focusing in the vertical direction are realized, so that the sample area is in the center of the imaging field and has a suitable focal depth.

[0059] S2, composite excitation light generation and regulation: the main excitation light source and the compensation excitation light source are started synchronously, the main excitation light source emits main illumination light to the front side of the beam splitter, and the compensation excitation light source implements back illumination from the back side of the beam splitter, and the two form composite illumination light together. The composite illumination light forms superimposed output after passing through the beam splitter and irradiates the sample area to excite different fluorescence responses.

[0060] The light intensity sensor is installed on the back side of the beam splitter, which is used to detect the composite light intensity of the transmission main light and the reflection compensation light passing through the beam splitter in real time, and compare and analyze according to the pre-set transmission-reflection curve of the system, automatically adjust the output power of the compensation excitation light source, so that the composite light intensity of the sample irradiated finally is stabilized in the target range.

[0061] S3. Monochromatic Excitation Light Output and Optical Path Allocation: The integrated filter assembly within the monochromatic excitation module is controlled by a driver, which rotates it around the optical axis, causing the excitation filter of the selected wavelength band to intersect the main optical path. When the excitation filter is positioned within the main optical path, the composite illumination light is filtered into monochromatic excitation light of a specific wavelength band, which is incident on the sample area under test at an oblique angle, achieving specific spectral excitation under multi-band fluorescence excitation conditions.

[0062] S4. Image Acquisition and Imaging Path Control: The excited fluorescence signal is collected by the objective lens and optically processed along a predetermined optical path through a dichroic mirror, emission filter, and eyepiece system. The processed light signal is ultimately focused onto the photosensitive element of the imaging camera, which captures the current fluorescence state of the sample, generating high-resolution microscopic image data.

[0063] S5. Image processing and preview: The acquired raw image data is format converted, color balanced, and enhanced by the image processor, and the processed image is displayed in real time on the preview monitor, allowing the operator to instantly observe the image quality and fluorescence response status of the current field of view.

[0064] S6. Image selection and storage: The user decides whether to keep the current image based on the preview display effect. If so, the system automatically stores the image in the image memory. If the image has problems such as drifting focus, light spots, or an unsatisfactory signal-to-noise ratio, the system control interface can be used to delete it to avoid the accumulation of redundant images.

[0065] The feedback regulation process in step S2 includes:

[0066] S21. Calling a transmission-reflection curve preset in the system, wherein the curve reflects the ratio between the transmitted light of the main excitation light source and the reflected light of the compensation excitation light source, and setting an allowable range of the target illumination light intensity;

[0067] S22, using a light intensity sensor to collect the composite light intensity value on the back side of the spectroscope in real time, and calculating the equivalent illumination intensity of the current composite illumination light on the front side of the spectroscope through an algorithm;

[0068] S23, comparing and analyzing the estimated illumination intensity with the target light intensity value, generating a feedback control signal based on the deviation, and sending the signal to the compensation excitation light source controller to adjust its output light intensity;

[0069] S24. Repeat the acquisition and adjustment process to gradually approach the target illumination intensity through multiple cycles. When the absolute value of the difference between the calculated illumination intensity on the positive side of the spectrometer and the set target value is lower than the tolerance threshold set by the system, the feedback adjustment is terminated and the next round of image acquisition process is entered.

[0070] This method ensures sufficient excitation of fluorescence signals under different excitation bands while avoiding overexposure or underexposure, improving the balance of image data and analysis stability, and is suitable for the standardized acquisition process of multi-target cell fluorescence imaging analysis.

[0071] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation, characterized by: 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 wavelength bands to the 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; The focusing stage assembly (3) comprises: a stage base (31); The sequential fluorescence irradiation group (1) comprises: A main excitation light source (11), used for providing main illumination light; A compensating excitation light source (12) for providing supplementary illumination light; The main lighting light and the supplementary lighting light constitute a composite lighting 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) performs reflective illumination on the spectroscope (13), and the compensation excitation light source (12) performs back-illumination illumination on the spectroscope (13); A light intensity sensor (14) is located on the back side of the spectroscope (13) and detects the back side light intensity of the spectroscope (13), wherein the back side light intensity of the spectroscope (13) includes a transmission portion of the main illumination light irradiated on the front side of the spectroscope (13) and a reflection portion of the supplementary illumination light irradiated on the back side of the spectroscope (13); the light intensity sensor (14) is electrically connected to the compensation excitation light source (12) and performs feedback adjustment on the light intensity of the supplementary illumination light; The feedback adjustment of the light intensity of the supplementary lighting light specifically includes: Retrieving a transmission-reflection curve pre-stored in the system, wherein the transmission-reflection curve is used to characterize the corresponding relationship between the transmission light intensity of the main excitation light source (11) and the reflection light intensity of the compensation excitation light source (12); collecting a composite light intensity value on the back side of the spectroscope (13) through the light intensity sensor (14), and comparing the composite light intensity value with a target light intensity value in the transmission-reflection curve; According to the comparison result, the composite light intensity value on the positive side of the spectroscope (13) is determined, compared with the required irradiation light intensity value, and a feedback signal is output to the compensation excitation light source (12) for negative feedback regulation; After the luminous intensity of the compensation excitation light source (12) changes due to the negative feedback adjustment, the composite light intensity value on the back side of the spectroscope (13) is collected again and the comparison and adjustment steps are cyclically performed until the absolute value of the difference between the calculated composite light intensity value on the front side of the spectroscope (13) and the target light intensity value is less than a preset threshold value, and the feedback adjustment is completed; 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 dichroic mirror (16); The monochromatic excitation module (15) comprises an integrated filter, which is composed of multiple coaxially mounted excitation filters. The multiple excitation filters are arranged to be installed at an oblique angle so that the excitation light is incident on the sample to be tested at an oblique angle after being emitted, for oblique-angle fluorescence excitation.

2. The automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation according to claim 1, characterized in that: 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) is located downstream of the optical path of the eyepiece (22).

3. The automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation according to claim 1, characterized in that: The focusing stage assembly (3) further comprises: The loading platform (32) is used to carry the sample to be tested and can move in the horizontal direction and the height direction; A focusing member (33) comprising at least two groups of horizontal air bags (331) and at least four vertical air bags (332); The horizontal airbag (331) includes an X-axis horizontal airbag and a Y-axis horizontal airbag, and the vertical airbag (332) is a Z-axis vertical airbag. A return spring is further provided in the Z-axis direction in the opposite direction to the force applied by the vertical airbag (332).

4. The automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation according to claim 1, characterized in that: The monochromatic excitation module (15) further includes: The rotary drive member is used to drive the integrated filter to rotate into the main light path, so that different excitation filters are respectively irradiated by the composite illumination light and emit different monochromatic excitation lights.

5. The automatic cell microscopic image acquisition and processing device based on multi-band fluorescence excitation according to claim 4, characterized in that: The device also 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 it in the image memory or delete it according to the user's operation.

6. A method for automatically acquiring and processing cell microscopic images based on multi-band fluorescence excitation, implemented based on the device according to any one of claims 1 to 5, characterized in that: The steps include: S1. Sample installation and stage focusing: Fix the sample to be tested on the focusing stage assembly, and achieve precise positioning of the sample in the horizontal and height directions by controlling the horizontal and vertical airbags in the focusing stage assembly; 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 is formed by the beam splitter to illuminate the sample to be tested. The light intensity sensor collects the back-side composite light intensity in real time and uses the transmission-reflection curve to feedback and adjust the compensation excitation light source; 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 light path, achieving oblique angle incidence of monochromatic excitation light of the required wavelength band on the sample.

7. The method for automatically collecting and processing cell microscopic images based on multi-band fluorescence excitation according to claim 6, characterized in that: The following steps are also included: S4, image acquisition and imaging path control: The fluorescence signal is collected by the objective lens, focused through the dichroic mirror, emission filter and eyepiece in sequence, and then the image data is acquired 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 based on the real-time preview result. The selected image is stored in the image memory, and the remaining images are deleted as needed.

8. The method for automatically collecting and processing cell microscopic images based on multi-band fluorescence excitation according to claim 7, characterized in that: The feedback adjustment in step S2 specifically includes: S21. Call the built-in transmission-reflection curve of the system and set the target light intensity range; S22, collecting the back side light intensity value in real time through the light intensity sensor and calculating the estimated front side illumination intensity; S23, comparing the estimated value with the target light intensity, generating 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, completing this round of light intensity calibration.

Images