Three-wavelength beam-combining galvanometer point scanning confocal microscope and control method

Abstract

The present application relates to the field of optical microscopic imaging technology, and discloses a three-wavelength combined-beam galvanometer point scanning confocal microscope and a control method, comprising a three-in-one laser module, a confocal optical path box and an inverted fluorescence microscope, the three-in-one laser module internally integrates three single-mode lasers of different wavelengths, three laser beams are output through the same single-mode input optical fiber after being pre-combined in the three-in-one laser module, and the confocal optical path box is connected with the single-mode input optical fiber.The present application adopts the three-in-one laser module, pre-combines three single-mode lasers of different wavelengths in the module, and then outputs the three single-mode lasers to the confocal optical path box through the same single-mode input optical fiber, so as to avoid the spatial position deviation caused by the independent coupling of multiple single optical fibers in the scanning head, thereby improving the coaxiality of the multi-channel excitation light beam and the stability of the output optical power.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, specifically to a three-wavelength beam-combining galvanometer point scanning confocal microscope and its control method. Background Technology

[0002] Laser scanning confocal microscopy utilizes a confocal pinhole in the probe optical path to filter out stray light outside the focal plane, enabling the acquisition of high-resolution optical section images. It is widely used in multicolor fluorescence observation of biological samples. When performing multicolor fluorescence imaging, the system requires the introduction of multiple laser wavelengths as excitation sources.

[0003] Existing confocal microscopy systems typically use multiple independent optical fibers to introduce lasers of different wavelengths into the scanning head and then perform internal coupling when introducing multi-wavelength light sources. This separate optical path structure is prone to spatial position deviations during assembly and adjustment due to mechanical tolerances or changes in ambient temperature. As a result, the multiple excitation beams cannot maintain a strictly coaxial state, which not only makes the excitation light power of each wavelength projected onto the sample surface unstable, but also increases the difficulty of optical system alignment and daily debugging.

[0004] In terms of the physical layout of the core optoelectronic components of the system, conventional confocal equipment usually disperses components such as scanning galvanometers, fluorescent dichroic mirrors, confocal pinholes, and photomultiplier tubes in a large main frame or multiple independent support structures. When the equipment is in operation for a long time or subjected to minor external vibrations, relative displacement can easily occur between the optical components. The deviation of the optical axis position directly weakens the vibration resistance of the entire system, resulting in the need for frequent and complex mechanical alignment and optical path correction during factory transportation, on-site assembly, and subsequent daily maintenance.

[0005] In the equipment operation and data acquisition stages, for the scanning control of multicolor fluorescent samples, traditional systems, when executing the simultaneous excitation mode of multiple wavelengths, often receive stray fluorescence from non-target dyes due to the inherent physical overlap of the emission spectra of various fluorescent dyes. This results in signal aliasing in the output image, reducing the accuracy of quantitative fluorescence analysis. The lack of an automated multi-channel scheduling mechanism requires operators to frequently manually adjust the state of laser channels and optical path components, which is cumbersome and makes it difficult to ensure the accuracy of multicolor imaging parameter configuration. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a three-wavelength combined beam galvanometer point scanning confocal microscope and its control method. This solves the problems of spatial relative displacement caused by independent coupling of multiple optical fibers and dispersed installation of core components in existing confocal microscopy systems, as well as cross-coloring of the detection channel and cumbersome manual operation caused by simultaneous excitation of multiple wavelengths.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first aspect of this invention provides a three-wavelength beam-combining galvanometer point scanning confocal microscope, comprising a three-in-one laser module, a confocal optical path box, and an inverted fluorescence microscope. The three-in-one laser module integrates three single-mode lasers of different wavelengths. The three lasers are pre-combined within the three-in-one laser module and then output through the same single-mode input fiber. The confocal optical path box is connected to the single-mode input fiber. The confocal optical path box contains a fiber collimator, a multi-channel fluorescence dichroic mirror, a dual-axis scanning galvanometer, a scanning lens, a focusing lens, a confocal pinhole, and a single PMT detector.

[0009] The inverted fluorescence microscope is equipped with an objective lens, the side interface of which is coupled to the output end of the confocal optical path box via an adapter. The excitation light output from the single-mode input fiber is collimated by the fiber collimator, reflected by the multi-channel fluorescence dichroism mirror, enters the dual-axis scanning galvanometer, is deflected by the scanning lens, enters the inverted fluorescence microscope, and is focused on the sample by the objective lens. The fluorescence generated by the sample returns to the multi-channel fluorescence dichroism mirror through the original optical path, is transmitted through the focusing lens, and the focused light enters the confocal pinhole and is received by the single PMT detector. The microscope also includes a system control box and a chassis, which are electrically connected to the three-in-one laser module, the confocal optical path box, and the inverted fluorescence microscope, respectively.

[0010] Preferably, the three different wavelengths in the three-in-one laser module are 405nm, 488nm and 561nm, and the three-in-one laser module is equipped with a hardware one-button main switch to control the simultaneous on and off of the three lasers.

[0011] Preferably, the confocal optical path box contains a focusing lens, a switchable emission filter, and a reflector arranged sequentially on the fluorescence transmission optical path between the multi-channel fluorescent dichroic mirror and the confocal pinhole. The confocal pinhole is configured as a manually switchable structure with apertures of 10μm, 20μm, 50μm, and 100μm.

[0012] Preferably, the multi-channel fluorescent dichroic mirror is installed in a multi-dimensional adjustment seat, which is used to adjust the deflection angle, pitch angle and position along the incident light direction of the multi-channel fluorescent dichroic mirror. The fiber collimator is connected to the multi-channel fluorescent dichroic mirror through a cage structure.

[0013] Preferably, the adapter is a mechanical structure with adjustable length and lockability, and the inverted fluorescence microscope is equipped with an electric XY-axis stage for carrying samples and a closed-loop Z-axis platform. The chassis controls the closed-loop Z-axis platform and the dual-axis scanning galvanometer to move synchronously through the system control box.

[0014] Preferably, the single PMT detector is a 16-bit image depth photomultiplier tube detector, the single PMT detector has a light-shielding structure on its outer side, and the single PMT detector is coaxially arranged with the confocal pinhole.

[0015] Preferably, the device also includes a display screen and a keyboard electrically connected to the chassis. The chassis is pre-installed with control software, which configures the time-division output sequence of each wavelength of laser in the three-in-one laser module according to the type of fluorescent dye.

[0016] Preferably, the inverted fluorescence microscope is equipped with a transmission illumination system and an epifluorescence illumination system, and the epifluorescence illumination system is configured with multiple sets of fluorescence filters to assist in wide-field observation and positioning.

[0017] The first aspect of this invention provides a control method for a three-wavelength beam-combining galvanometer point scanning confocal microscope, comprising the following steps:

[0018] The scanning command and fluorescent dye parameters input into the receiver chassis;

[0019] Based on the fluorescent dye parameters, a multi-channel time-division scanning sequence is automatically generated;

[0020] According to the time-division scanning sequence, the laser channels of the corresponding wavelengths in the three-in-one laser module are turned on in sequence, and the dual-axis scanning galvanometer in the confocal optical path box is controlled to perform point-by-point scanning and switch the emission filter of the corresponding band.

[0021] Fluorescence signals transmitted through a confocal pinhole are collected by a single PMT detector and transmitted to the chassis via the system control box.

[0022] The chassis performs image reconstruction and multicolor image overlay output on the acquired fluorescence signals from each channel.

[0023] Preferably, it also includes a 3D large image stitching step:

[0024] The dual-axis scanning galvanometer in the confocal optical path box, the motorized XY-axis stage on the inverted fluorescence microscope, and the closed-loop Z-axis platform are linked to perform synchronous scanning to acquire XYZ three-dimensional sequence image data.

[0025] The 3D image reconstruction algorithm inside the chassis is invoked to automatically scan and stitch the 3D sequence image data, outputting a spatial interactive stereoscopic image.

[0026] This invention provides a three-wavelength beam-combining galvanometer point scanning confocal microscope and its control method. It has the following beneficial effects:

[0027] 1. This invention uses a three-in-one laser module to pre-bundle three different wavelengths of single-mode lasers inside the module, and then outputs them to the confocal optical path box through the same single-mode input fiber. This avoids the spatial position deviation caused by the independent coupling of multiple separate optical fibers inside the scanning head, simplifies the optical path alignment and debugging process, and thus improves the coaxiality of the multi-channel excitation beam and the stability of the output optical power.

[0028] 2. This invention centrally houses core optoelectronic components such as a dual-axis scanning galvanometer, a multi-channel fluorescence dichroic mirror, a confocal pinhole, and a single PMT detector within a confocal optical path box. These components are directly coupled to the side interface of an inverted fluorescence microscope via an adapter, reducing the risk of relative displacement caused by the dispersed installation of components, improving the overall vibration resistance of the system, and simultaneously reducing the mechanical alignment costs of the equipment during transportation, on-site installation, and subsequent maintenance.

[0029] 3. The control method of the present invention receives the input fluorescent dye parameters, automatically generates a multi-channel time-division scanning sequence, and controls the opening of the corresponding wavelength laser channel, the switching of the emission filter, and the movement of the dual-axis scanning galvanometer in a coordinated manner. This avoids the problem of cross-color in the detection channel caused by simultaneous excitation of multiple wavelength lasers, reduces the steps of operators manually and repeatedly adjusting laser parameters and component states, and improves the accuracy of multicolor sample imaging. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the overall structure of the three-wavelength beam-combining galvanometer point scanning confocal microscope of the present invention;

[0031] Figure 2 This is a block diagram illustrating the optical path principle of the three-wavelength beam combining galvanometer point scanning confocal microscope of the present invention;

[0032] Figure 3 This is a schematic diagram of the control method for the three-wavelength beam combining galvanometer point scanning confocal microscope of the present invention.

[0033] The components include: 1. Three-in-one laser module; 2. Confocal optical path box; 3. System control box; 4. Inverted fluorescence microscope; 5. Display screen; 6. Keyboard; and 7. Chassis. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] See attached document Figure 1 -Appendix Figure 2 A three-wavelength beam-combining galvanometer point scanning confocal microscope is characterized by comprising a three-in-one laser module 1, a confocal optical path box 2, and an inverted fluorescence microscope 4. The three-in-one laser module 1 integrates three different wavelengths of single-mode lasers. The three lasers are pre-combined within the three-in-one laser module 1 and then output through the same single-mode input fiber. The confocal optical path box 2 is connected to the single-mode input fiber. The confocal optical path box 2 contains a fiber collimator, a multi-channel fluorescence dichroic mirror, a dual-axis scanning galvanometer, a scanning lens, a focusing lens, a confocal pinhole, and a single PMT detector.

[0036] The inverted fluorescence microscope 4 is equipped with an objective lens. Its side interface is coupled to the output end of the confocal optical path box 2 through an adapter. The excitation light output from the single-mode input fiber is collimated by the fiber collimator and reflected by the multi-channel fluorescence dichroism mirror into the dual-axis scanning galvanometer. After deflection, it enters the inverted fluorescence microscope 4 through the scanning lens and is focused on the sample by the objective lens. The fluorescence generated by the sample returns to the multi-channel fluorescence dichroism mirror through the original optical path. After transmission, it passes through the focusing lens and the focused light enters the confocal pinhole and is received by the single PMT detector. The microscope also includes a system control box 3 and a chassis 7. The system control box 3 and the chassis 7 are electrically connected to the three-in-one laser module 1, the confocal optical path box 2 and the inverted fluorescence microscope 4, respectively.

[0037] The three-in-one laser module 1 contains three different wavelengths: 405nm, 488nm, and 561nm. The three-in-one laser module 1 is equipped with a hardware one-button main switch to control the simultaneous on / off of the three lasers. The single PMT detector is a 16-bit image depth photomultiplier tube detector. The single PMT detector has a light-shielding structure on its outside and is coaxially set with the confocal pinhole. It also includes a display screen 5 and a keyboard 6 electrically connected to the chassis 7. The chassis 7 has pre-installed control software, which configures the time-division output sequence of each wavelength of laser in the three-in-one laser module 1 according to the type of fluorescent dye.

[0038] Specifically, after the system completes the startup process, the operator inputs the fluorescent dye parameters of the current sample to be tested into the control software of the chassis 7 via the keyboard 6 and the display screen 5. The control unit of the chassis 7 analyzes this information and generates corresponding drive commands, which are then sent to control the three-in-one laser module 1 to enter the working state. During this process, three different wavelengths of single-mode lasers will complete high-precision optical pre-beaming inside the three-in-one laser module 1, and then be transmitted directly to the confocal optical path box 2 via a single single-mode input fiber.

[0039] By effectively avoiding spatial position deviations and mechanical alignment drifts that are prone to occur when multiple individual optical fibers are independently coupled inside the scanning head in traditional equipment at the hardware physical connection level, the optical assembly and adjustment difficulty of the entire equipment is greatly reduced. Furthermore, it ensures from the source that the multi-channel excitation beam entering the core optical path system always has high optical coaxiality and long-term power output stability, thus providing a reliable light source guarantee for the subsequent acquisition of high-quality confocal images.

[0040] See attached document Figure 1-2 The confocal optical path box 2 contains a focusing lens, a switchable emission filter, and a reflector arranged sequentially on the fluorescence transmission optical path between the multi-channel fluorescent dichroic mirror and the confocal pinhole. The confocal pinhole is configured with a manually switchable structure that includes apertures of 10μm, 20μm, 50μm, and 100μm. The multi-channel fluorescent dichroic mirror is installed in a multi-dimensional adjustment seat, which is used to adjust the deflection angle, pitch angle, and position along the incident light direction of the multi-channel fluorescent dichroic mirror. The fiber collimator is connected to the multi-channel fluorescent dichroic mirror through a cage structure.

[0041] The adapter is a mechanical structure with adjustable length and lockability. The inverted fluorescence microscope 4 is equipped with an electric XY-axis stage for carrying samples and a closed-loop Z-axis platform. The chassis 7 controls the closed-loop Z-axis platform and the dual-axis scanning galvanometer to move synchronously through the system control box 3. The inverted fluorescence microscope 4 is equipped with a transmission illumination system and an epifluorescence illumination system. The epifluorescence illumination system is equipped with multiple sets of fluorescence filters to assist in wide-field observation and positioning.

[0042] Specifically, after the excitation beam enters the confocal optical path box 2 from the single-mode input fiber, it is first processed by the fiber collimating mirror and shaped into a parallel collimated beam. This collimated beam is then projected onto the surface of the multi-channel fluorescence dichroic mirror and reflected to change its direction of travel, accurately incident on the reflecting surface of the dual-axis scanning galvanometer. Driven by the control system, the dual-axis scanning galvanometer performs high-speed angular deflection of the X and Y axes according to the preset voltage waveform, guiding the beam through the scanning lens and directly coupled into the optical path system of the inverted fluorescence microscope 4 through a tight physical adapter. Finally, the microscope objective is precisely focused on the focal plane of the sample to be tested, performing a two-dimensional point-by-point scan of the set area.

[0043] When the fluorescent material at the focal plane of the sample is excited, it emits a fluorescent signal carrying biological information. This signal is collected by the same objective lens and returns strictly in reverse along the original excitation light path. The returning dynamic fluorescence beam is restored to a stationary spatial beam after the descanning action of the dual-axis scanning galvanometer. It is then directly transmitted through a multi-channel fluorescence dichroic mirror to achieve wavelength separation of the excitation and emission light. The separated pure fluorescence signal continues to travel along the detection light path, passing sequentially through a switchable emission filter of a specific wavelength band used to filter out stray light and a focusing lens group that acts as a convergent lens. It is focused and passes through a confocal pinhole with a defined aperture. The spatial filtering characteristics of the pinhole effectively block stray background light outside the focal plane. Finally, it is received by a single PMT detector and converted into a voltage signal.

[0044] All the core optoelectronic components involved in beam deflection, wavelength separation, and spatial filtering are concentrated in the rigid structure of the confocal optical path box 2, achieving a high degree of modular integration of the optical path. This eliminates the small relative displacements that are easily generated during long-term operation when multiple components are dispersed and installed in the traditional way, greatly improving the optical alignment accuracy and overall mechanical vibration resistance of the system, and ensuring the long-term positional stability between the micron-level pinhole and the detection optical axis.

[0045] See attached document Figure 3 A control method for a three-wavelength beam-combining galvanometer point scanning confocal microscope includes the following steps:

[0046] The scanning command and fluorescent dye parameters are input into receiver box 7;

[0047] Based on the fluorescent dye parameters, the multi-channel time-division scanning sequence is automatically generated;

[0048] According to the time-division scanning sequence, the laser channels of the corresponding wavelengths in the three-in-one laser module 1 are turned on in sequence, and the dual-axis scanning galvanometer in the confocal optical path box 2 is controlled to perform point-by-point scanning and switch the emission filter of the corresponding band.

[0049] Fluorescence signals transmitted through the confocal pinhole are collected by a single PMT detector and transmitted to the chassis 7 via the system control box 3.

[0050] Chassis 7 performs image reconstruction and multicolor image overlay output on the acquired fluorescence signals from each channel.

[0051] It also includes the steps for stitching together 3D large images:

[0052] The dual-axis scanning galvanometer in the confocal optical path box 2, the motorized XY-axis stage on the inverted fluorescence microscope 4, and the closed-loop Z-axis platform are synchronously scanned to acquire XYZ three-dimensional sequence image data.

[0053] The 3D image reconstruction algorithm inside chassis 7 is invoked to automatically scan and stitch together the 3D sequence image data, and output a spatial interactive stereoscopic image.

[0054] Specifically, this invention provides a control method for a three-wavelength beam-combining galvanometer point scanning confocal microscope, comprising the following steps:

[0055] The control unit receives scanning commands from the operator and the fluorescent dye parameters corresponding to the sample to be tested. The control software inside the unit analyzes the received fluorescent dye parameters and extracts the excitation and emission band information for each dye. Based on the extracted band characteristics, the control software calculates and generates a multi-channel time-division scanning sequence. This time-division scanning sequence sets the activation timing of different wavelength laser channels, allocating fluorescent dyes with overlapping excitation spectra to different time periods for independent excitation and detection.

[0056] The system control box outputs a synchronous trigger signal according to the generated multi-channel time-division scanning sequence. Under the action of this trigger signal, each single-wavelength laser channel inside the three-in-one laser module turns on and off sequentially according to a prescribed timing sequence.

[0057] During the period when the laser channel at a specific wavelength is open, the system control box synchronously outputs a driving voltage to the dual-axis scanning mirror inside the confocal optical path box, driving the dual-axis scanning mirror to perform a point-by-point deflection scanning action in a two-dimensional plane. At the same time, the control mechanism switches the switchable emission filter on the transmission optical path side of the confocal optical path box according to the currently activated laser band, so that the passband range of the emission filter matches the fluorescence emission band of the currently stimulated dye.

[0058] After spatial filtering, the fluorescence signal passing through the confocal pinhole enters the single PMT detector. The single PMT detector continuously converts the received optical signal into an electrical signal, which is then processed into a digital signal sequence by the analog-to-digital converter circuit inside the system control box, and subsequently transmitted to the chassis.

[0059] The chassis extracts real-time deflection coordinate data from the dual-axis scanning galvanometer, which is sent by the system control box. The received digital signal sequence is then matched with the corresponding spatial coordinates. For the data acquired within each time period, the chassis performs two-dimensional image reconstruction, generating a monochrome image matrix corresponding to a single laser wavelength channel. After data acquisition for each channel is complete, the chassis control software performs pixel registration and multi-color channel overlay operations on the generated monochrome image matrices, outputting a multi-channel confocal composite image on the display screen.

[0060] When the control method involves large-scale scanning or 3D imaging processes, the chassis generates multi-dimensional scanning tasks based on the set spatial range. The system control box, while controlling the dual-axis scanning galvanometer to perform planar scanning within a single field of view, outputs stepping pulse signals to drive the electric XY-axis stage and closed-loop Z-axis platform on the inverted fluorescence microscope to produce displacement.

[0061] The motorized XY-axis stage performs planar translation between adjacent fields of view, while the closed-loop Z-axis platform performs axial layer-by-layer switching between different focal planes. Through the synchronous linkage of the stage and Z-axis platform movements with galvanometer scanning, the system acquires spatial sequence image data containing X, Y, and Z directions.

[0062] The chassis calls a pre-built 3D image reconstruction algorithm to process the acquired spatial sequence image data. For large-scale planar image stitching, the algorithm module reads the absolute coordinate system data of the motorized XY-axis stage, performs edge feature calculation and alignment registration on the image matrices of adjacent fields of view, and stitches together to generate a large-scale panoramic image that exceeds the field of view of a single objective lens.

[0063] For 3D imaging, the algorithm module performs voxel construction and coordinate transformation on the sequence of sliced ​​image data acquired by the closed-loop Z-axis platform. After 3D reconstruction, the system generates a spatial stereo data model reflecting the internal structure of the sample and outputs a 3D stereo image with interactive functions of spatial rotation and cross-sectional sectioning on the display terminal.

[0064] Working principle: After the system is started, the operator inputs the fluorescent dye parameters of the sample into the control software of the chassis 7 through the keyboard 6 and the display screen 5. The chassis 7 generates instructions accordingly and controls the three-in-one laser module 1 to work. The three different wavelengths of single-mode lasers are pre-beamed inside the three-in-one laser module 1, and then transmitted to the confocal optical path box 2 through a single single-mode input fiber. This beam combining and single-fiber transmission method directly avoids the spatial position deviation caused by the independent coupling of multiple individual optical fibers inside the scanning head, ensuring that the excitation beam entering the optical path system has high coaxiality and power stability.

[0065] The excitation light entering the confocal optical path box 2 is collimated by the fiber collimator and then reflected by the multi-channel fluorescence dichroism to the dual-axis scanning galvanometer. The dual-axis scanning galvanometer deflects the light beam according to the preset waveform, so that the beam passes through the scanning lens and enters the inverted fluorescence microscope 4 through the physical adapter. Finally, the objective lens focuses the light onto the sample surface for point-by-point scanning. The fluorescence generated by the sample returns along the original excitation light path, is de-scanned by the dual-axis scanning galvanometer, and then passes through the multi-channel fluorescence dichroism, the switchable emission filter and related lens group, and finally passes through the confocal pinhole and is received by the single PMT detector. The high integration of the core optoelectronic components in the confocal optical path box 2 effectively avoids the relative displacement caused by the dispersed installation of components, and improves the overall optical alignment accuracy and vibration resistance of the system.

[0066] During the scanning and acquisition phase, the chassis 7 synchronously schedules each hardware module through the system control box 3, strictly follows the time-division scanning mechanism to sequentially open the laser channels of the corresponding wavelengths, and synchronously switches the corresponding emission filters to avoid cross-color problems in the detection channels caused by multiple wavelength lasers irradiating the sample at the same time. After the single PMT detector converts the received fluorescence light signal into an electrical signal, it is transmitted back to the chassis 7 through the system control box 3. The chassis 7, combined with the spatial position coordinates of the motorized XYZ stage in the inverted fluorescence microscope 4, processes the fluorescence data of each channel, completes the superposition of multicolor images and three-dimensional reconstruction, and finally outputs a high-definition confocal image on the display screen 5.

Claims

1. A three-wavelength beam combining galvanometer point scanning confocal microscope, characterized in that, The system includes a three-in-one laser module (1), a confocal optical path box (2), and an inverted fluorescence microscope (4). The three-in-one laser module (1) integrates three different wavelengths of single-mode lasers. The three lasers are pre-combined in the three-in-one laser module (1) and then output through the same single-mode input fiber. The confocal optical path box (2) is connected to the single-mode input fiber. The confocal optical path box (2) is equipped with a fiber collimator, a multi-channel fluorescence dichroic mirror, a dual-axis scanning galvanometer, a scanning lens, a focusing lens, a confocal pinhole, and a single PMT detector. The inverted fluorescence microscope (4) is equipped with an objective lens, and its side interface is coupled to the output end of the confocal optical path box (2) through an adapter. The excitation light output from the single-mode input fiber is collimated by the fiber collimating lens, reflected by the multi-channel fluorescence dichroic mirror and enters the dual-axis scanning galvanometer. After deflection, it enters the inverted fluorescence microscope (4) through the scanning lens and is focused on the sample by the objective lens. The fluorescence generated by the sample returns to the multi-channel fluorescence dichroic mirror through the original optical path. After transmission, it passes through the focusing lens, and the focused light enters the confocal pinhole and is received by the single PMT detector. The microscope also includes a system control box (3) and a chassis (7). The system control box (3) and the chassis (7) are electrically connected to the three-in-one laser module (1), the confocal optical path box (2) and the inverted fluorescence microscope (4), respectively. 2.The three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 1, wherein, The three different wavelengths in the three-in-one laser module (1) are 405nm, 488nm and 561nm respectively. The three-in-one laser module (1) is equipped with a hardware one-button start switch to control the simultaneous on and off of the three lasers. 3.The three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 1, wherein, The confocal optical path box (2) is provided with a focusing lens, a switchable emission filter and a reflector in sequence on the fluorescence transmission optical path between the multi-channel fluorescent dichroic mirror and the confocal pinhole. The confocal pinhole is configured as a manually switchable structure with apertures of 10μm, 20μm, 50μm and 100μm.

4. The three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 1, characterized in that, The multi-channel fluorescent dichroic mirror is installed in a multi-dimensional adjustment base, which is used to adjust the deflection angle, pitch angle and position of the multi-channel fluorescent dichroic mirror along the incident light direction. The fiber collimator is connected to the multi-channel fluorescent dichroic mirror through a cage structure.

5. The three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 1, characterized in that, The adapter is a mechanical structure with adjustable length and lockability. The inverted fluorescence microscope (4) is equipped with an electric XY-axis stage for carrying samples and a closed-loop Z-axis platform. The chassis (7) controls the closed-loop Z-axis platform and the dual-axis scanning galvanometer to move synchronously through the system control box (3).

6. The three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 1, characterized in that, The single PMT detector is a 16-bit image depth photomultiplier tube detector. The single PMT detector has a light-shielding structure on its outer side and is coaxially arranged with the confocal pinhole.

7. A three-wavelength beam-combining galvanometer point scanning confocal microscope according to claim 1, characterized in that, It also includes a display screen (5) and a keyboard (6) electrically connected to the chassis (7). The chassis (7) is pre-installed with control software, which configures the time-division output sequence of each wavelength laser in the three-in-one laser module (1) according to the type of fluorescent dye. 8.The three-wavelength combined-beam galvanometer point scanning confocal microscope of claim 1, wherein, The inverted fluorescence microscope (4) is equipped with a transmission illumination system and an epifluorescence illumination system. The epifluorescence illumination system is equipped with multiple sets of fluorescence filters to assist in wide-field observation and positioning.

9. A control method of a three-wavelength combined-beam galvanometer point scanning confocal microscope, characterized by, The application of a three-wavelength beam-combining galvanometer point scanning confocal microscope according to any one of claims 1-8 includes the following steps: The scanning command and fluorescent dye parameters input into the receiver box (7); Based on the fluorescent dye parameters, a multi-channel time-division scanning sequence is automatically generated; According to the time-division scanning sequence, the laser channels of the corresponding wavelengths in the three-in-one laser module (1) are turned on in sequence, and the dual-axis scanning galvanometer in the confocal optical path box (2) is controlled to perform point-by-point scanning and switch the emission filter of the corresponding band. Fluorescence signals transmitted through the confocal pinhole are collected by a single PMT detector and transmitted to the chassis (7) via the system control box (3). The chassis (7) performs image reconstruction and multicolor image superposition output on the collected fluorescence signals from each channel.

10. The control method of a three-wavelength combined-beam galvanometer point scanning confocal microscope according to claim 9, wherein, It also includes the steps for stitching together 3D large images: The dual-axis scanning galvanometer in the confocal optical path box (2), the electric XY-axis stage on the inverted fluorescence microscope (4), and the closed-loop Z-axis platform are linked to perform synchronous scanning to acquire XYZ three-dimensional sequence image data; The three-dimensional image reconstruction algorithm inside the chassis (7) is invoked to automatically scan and stitch the three-dimensional sequence image data, and output a spatial interactive stereoscopic image.

Images