An external fluorescence system for use in a high-power stereo microscope and its application method

Abstract

This invention discloses an external fluorescence system and its usage method for a high-magnification stereomicroscope, relating to the field of microscopy technology. It includes a light source module, a light source transmission component, a light source collimation and focusing system, a filter switching module, a multi-dimensional adjustment bracket, and a microscope connection part. One end of the light source transmission component is connected to the light source module, and the other end is connected to the light source collimation and focusing system. The light source collimation and focusing system is used to focus the excitation light emitted by the light source module into a very small light spot. The filter switching module is positioned in the optical path between the light source transmission component and the light source collimation and focusing system. The multi-dimensional adjustment bracket is connected to the light source collimation and focusing system. The light source module uses low-loss liquid fiber transmission, which, in conjunction with the light source collimation and focusing system, compresses the light spot to match the objective lens, effectively compensating for brightness loss. Through the axial fine-tuning of the multi-dimensional adjustment bracket and the light source adjustment device, precise overlap between the external light spot and the original light spot is ensured, improving imaging uniformity.

Description

Technical Field

[0001] This invention relates to the field of microscopy technology, and more specifically to an external fluorescence system for use in a high-power stereomicroscope and its method of use. Background Technology

[0002] The core structure of the high-magnification stereo microscope is a dual-objective switching design, comprising two objectives with different functions: a large-aperture dual-beam path 1X stereo objective and a single-beam path long working distance 10X objective. In practical applications, this microscope requires a fluorescence accessory to achieve fluorescence imaging. Existing fluorescence accessories employ a dual-beam path structure, which is well-suited to the dual-beam path design of the 1X objective, achieving ideal fluorescence imaging results. However, when switching to the 10X objective, because the 10X objective has a single-beam path structure, only one of the dual-beam path excitation beams from the fluorescence accessory can enter the 10X objective, resulting in a direct 50% loss of fluorescence brightness and severely impacting the imaging quality under high-magnification objectives.

[0003] To address the fluorescence brightness loss caused by a single optical path under a 10X objective, an external fluorescence excitation structure is needed to supplement the fluorescence intensity. However, the focusing spot size of a 10X objective is extremely small (only about 1 mm). If the size of the fluorescence spot output by the external excitation structure does not match the focusing spot, most of the excitation light cannot enter the optical path of the 10X objective, resulting in low excitation efficiency and difficulty in effectively supplementing brightness. In addition, there is currently no dedicated external fluorescence system for this type of high-magnification stereomicroscope. Conventional external fluorescence devices have problems such as non-adjustable spot size, insufficient excitation intensity, poor flexibility in band switching, and difficulty in optical path alignment, which cannot meet the fluorescence imaging requirements under a 10X objective. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an external fluorescence system for use in a high-magnification stereomicroscope and a method for using it.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: An external fluorescence system for a high-magnification stereomicroscope, comprising a light source module, a light source transmission component, a light source collimation and focusing system, a filter switching module, a multi-dimensional adjustment bracket, and a microscope connection part;

[0006] One end of the light source transmission component is connected to the light source module, and the other end is connected to the light source collimation and focusing system to transmit the excitation light output by the light source module to the light source collimation and focusing system;

[0007] The light source collimation and focusing system is used to focus the excitation light emitted by the light source module into a very small spot. The size of the very small spot is adapted to the focusing spot size of the high-magnification objective of the high-magnification stereomicroscope to compensate for the loss of fluorescence brightness caused by the single optical path of the high-magnification objective.

[0008] The filter switching module is located in the optical path between the light source transmission component and the light source collimation and focusing system. It is used to switch between filters of different wavelengths, and each filter of the filter switching module corresponds to the output wavelength of the light source module.

[0009] The multi-dimensional adjustment bracket is connected to the light source collimation and focusing system to drive the light source collimation and focusing system to achieve linear adjustment in at least two axes and angle adjustment in one axis, so as to ensure that the light spot of the external excitation light overlaps with the excitation light of the microscope module itself.

[0010] The microscope connector is used to fix the entire external fluorescence system onto a high-power stereomicroscope.

[0011] Furthermore, the light source transmission component is a liquid optical fiber, with one end of the liquid optical fiber connected to the output end of the light source module, and the other end connected to the light source collimation and focusing system through an optical fiber interface.

[0012] Furthermore, the filter switching module includes a filter insert plate and at least four filter sheets. The filter insert plate has mounting slots that are adapted to each filter sheet, and the filter sheets are correspondingly embedded in the mounting slots.

[0013] The filter insert plate can move back and forth along a preset horizontal direction to switch different color filters to the optical path between the light source conduction component and the light source collimation and focusing system, and the wavelength of each color filter corresponds one-to-one with the four output wavelengths of the light source module.

[0014] Furthermore, the multi-dimensional adjustment bracket includes a Z-axis adjustment mechanism, a Y-axis adjustment mechanism, and a W-axis adjustment mechanism;

[0015] The Z-axis adjustment mechanism is connected to the light source collimation and focusing system, and is used to drive the light source collimation and focusing system to move linearly along the Z-axis. One end of the Y-axis adjustment mechanism is fixedly connected to the Z-axis adjustment mechanism, and the other end is used to cooperate with the fixed structure of the high-magnification stereomicroscope, and is used to drive the Z-axis adjustment mechanism and the light source collimation and focusing system to move linearly along the Y-axis. The W-axis adjustment mechanism is connected between the Y-axis adjustment mechanism and the light source collimation and focusing system, and is used to drive the light source collimation and focusing system to rotate around the preset W-axis, with a rotation angle range of 0°-90°.

[0016] Furthermore, the Z-axis adjustment mechanism includes a dovetail guide rail, a dovetail slider, a gear rod, a locking screw, and a gear;

[0017] The dovetail groove guide rail and the dovetail groove slider are slidably engaged by the dovetail groove. The dovetail groove slider can move linearly along the length of the dovetail groove guide rail. The gear rod passes horizontally through the circular groove opened on the dovetail groove slider. The gear is fixedly sleeved on the end of the gear rod. The upper end face of the dovetail groove guide rail is provided with a rack. The rack meshes with the gear. When the gear rod rotates around its own axis, it drives the gear to rotate, thereby driving the dovetail groove slider to move along the dovetail groove guide rail.

[0018] The side of the dovetail slide block has a dovetail notch. The locking screw passes horizontally through the side wall of the dovetail slide block and presses against the dovetail notch, causing the dovetail notch to undergo elastic deformation to press against the dovetail guide rail, thereby achieving relative fixation between the dovetail slide block and the dovetail guide rail.

[0019] Furthermore, the Y-axis adjustment mechanism includes an optical axis guide rail, an optical axis slider, a rotary hinge, a Z-axis connector, an adjustment screw, a first compression spring, a front optical axis retainer, a rear optical axis retainer, and a positioning screw;

[0020] The optical axis guide rail is provided in two sets. The two sets of optical axis guide rails are arranged in parallel and both pass through the round holes opened on the optical axis slider. The two ends of the optical axis guide rail are fixedly connected to the front optical axis retainer and the rear optical axis retainer, respectively.

[0021] The first compression spring is located in the groove hole on the optical axis slider. The positioning screw passes through the first compression spring and is threadedly connected to the front retainer of the optical axis. The first compression spring is in a slightly compressed state, and its restoring force pushes the optical axis slider to always be close to the front retainer of the optical axis.

[0022] The adjusting screw is screwed into the threaded hole on the front retainer of the optical axis. By adjusting the screwing depth, the distance between the optical axis slider and the front retainer of the optical axis is changed. The Z-axis connector is fixedly connected to the Z-axis adjustment mechanism. One side of the rotating hinge is fixed to the Z-axis connector, and the other side is fixed to the optical axis slider.

[0023] Furthermore, the W-axis adjustment mechanism includes a light source module connector, a light source module rotating shaft, a first limit screw, a second compression spring, a rotating shaft pressure plate, and a second limit screw;

[0024] The light source module shaft passes horizontally through the circular hole opened on the rear retainer of the optical axis, and the second limiting screw is screwed into the through hole on the right side of the rear retainer of the optical axis and abuts against the light source module shaft.

[0025] The second compression spring is located inside the rear retainer of the optical axis, and its two ends abut against the first limit screw and the second limit screw respectively. The second compression spring is in a compressed state. The first limit screw is screwed into the through-hole threaded hole opened on the rear retainer of the optical axis. The rotation angle of the light source module shaft is adjusted by tightening or loosening the first limit screw.

[0026] The rotating shaft pressure plate covers the end of the optical axis rear retainer to prevent dust from entering; one end of the light source module connector is threaded to the light source module rotating shaft, and the other end is fixedly connected to the light source collimation and focusing system to realize the synchronous rotation of the light source module rotating shaft and the light source collimation and focusing system.

[0027] Furthermore, it also includes a light source adjustment device, which includes a lens barrel adapter ring, a fiber optic positioning component, a fiber optic tightening component, a fiber optic screw tightening component, a fiber optic output interface, a lens barrel, and fixing screws.

[0028] The optical fiber output interface passes through the optical fiber tightening component and the optical fiber positioning component in sequence. The end of the optical fiber tightening component is chamfered and three slots are evenly distributed along its central axis.

[0029] The fiber optic tightening component is threadedly connected to the threaded hole on the fiber optic positioning component. When the fiber optic tightening component is screwed in, it pushes the fiber optic tightening component to be subjected to force along the chamfered slope, causing the slot of the fiber optic tightening component to shrink and press the fiber optic output interface.

[0030] The lens barrel adapter ring is fixedly connected to the fiber positioning component. The lens barrel adapter ring has a slot that matches the color filter insert plate. The color filter insert plate is inserted into the slot. The front end of the lens barrel is threadedly connected to the lens barrel adapter ring for secondary focusing of the excitation light.

[0031] The light source adjustment device is inserted into the circular hole opened on the light source module connector and can move along the axial direction of the circular hole. After adjustment, it is fixed by tightening the fixing screw.

[0032] Furthermore, the microscope connection part is an adapter plate. One side of the adapter plate is fixedly connected to the multi-dimensional adjustment bracket, and the other side is used to fixally connect to the arm bracket of the high-magnification stereomicroscope, so as to realize the fixation of the entire external fluorescence system and the microscope.

[0033] The present invention also provides a method for using an external fluorescence system applied to a high-magnification stereomicroscope, comprising the following steps:

[0034] S1: System Installation and Fixing:

[0035] The entire external fluorescence system is fixedly connected to the arm support of the high-power stereomicroscope via the adapter plate of the microscope connector, ensuring that the system is stable and secure.

[0036] S2: Connection between light source and optical path:

[0037] One end of the liquid optical fiber of the light source transmission component is connected to the output end of the light source module, and the other end is connected to the light source collimation and focusing system through the optical fiber interface to establish a transmission path for the excitation light from the light source to the light source collimation and focusing system.

[0038] S3: Excitation band selection:

[0039] Based on the fluorescent label type of the sample to be observed, the filter insertion plate of the filter switching module is pushed to move the filter that matches the target output band of the light source module into the optical path between the light source transmission component and the light source collimation and focusing system; wherein, the light source module is a four-channel LED light source, and the filter insertion plate is provided with at least four filters, and the wavelength of each filter corresponds one-to-one with the four output bands of the four-channel LED light source.

[0040] S4: Multi-dimensional optical path alignment:

[0041] S41: Z-axis direction adjustment:

[0042] Rotating the gear rod of the Z-axis adjustment mechanism drives the gear to mesh with the rack on the dovetail groove guide rail, which in turn drives the dovetail groove slider and the light source collimation and focusing system connected to it to move linearly along the Z-axis direction, so that the excitation light spot initially falls on the observation area of ​​the sample.

[0043] S42: Y-axis direction adjustment:

[0044] Turn the adjusting screw of the Y-axis adjustment mechanism to change the distance between the optical axis slider and the optical axis front holder, drive the optical axis slider and the light source collimation and focusing system linked with it to move along the Y-axis direction, fine adjust the front and back position of the light spot on the horizontal plane, so that the light spot gradually approaches the original excitation light spot area of ​​the microscope.

[0045] S43: W-axis angle adjustment:

[0046] The first limit screw of the W-axis adjustment mechanism is tightened and loosened. The change in the elastic force of the second compression spring drives the rotating shaft of the light source module to rotate, which in turn drives the collimating and focusing system of the light source connected to the rotating shaft to rotate around the W-axis until the external excitation light spot completely overlaps with the original excitation light spot of the high-magnification stereomicroscope.

[0047] S5: Brightness Adjustment and Imaging Observation

[0048] Turn on the light source module and adjust the channel power of the corresponding output band to make the excitation light brightness match the sample imaging requirements; switch the objective lens of the high-power stereomicroscope to the 10X objective lens, start the microscope imaging system, and complete the fluorescence imaging observation of the sample.

[0049] S6: Multi-band switching observation:

[0050] If it is necessary to observe samples with different fluorescent labels, repeat steps S3 to S5: push the filter insert plate to switch to the filter with the wavelength matching the new fluorescent label, and after completing the multi-dimensional optical path alignment and brightness adjustment, start the imaging system for observation.

[0051] The present invention has the following beneficial effects: The external fluorescence system provided by the present invention is applied to a high-magnification stereomicroscope. The light source module is transmitted through liquid optical fiber with low loss. With the light source collimation and focusing system, the light spot is compressed to match the light spot of the 10X objective lens, effectively compensating for the brightness loss. Through the axial fine adjustment of the multi-dimensional adjustment bracket and the light source adjustment device, the external light spot is precisely overlapped with the original light spot, improving the imaging uniformity. In addition, the filter plate can quickly switch between 4 bands to adapt to the needs of multiple fluorescence markings. Moreover, the adapter plate is compatible with target microscopes and conventional stereofluorescence microscopes, reducing equipment costs and adapting to multiple scenarios such as biological observation and industrial inspection. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0053] Figure 2 This is a schematic diagram of the optical path principle in this invention;

[0054] Figure 3 This is a schematic diagram of the Z-axis adjustment mechanism in this invention;

[0055] Figure 4 This is a schematic diagram of the layout structure of the Z-axis adjustment mechanism in this invention;

[0056] Figure 5 This is a partial cross-sectional view of the Z-axis adjustment mechanism in this invention;

[0057] Figure 6 This is a schematic diagram of the Y-axis adjustment mechanism in this invention. Figure 1 ;

[0058] Figure 7 This is a schematic diagram of the Y-axis adjustment mechanism in this invention. Figure 2 ;

[0059] Figure 8 This is a schematic diagram of the W-axis adjustment mechanism in this invention;

[0060] Figure 9 This is a schematic diagram of the light source adjustment device in this invention;

[0061] Figure 10 This is a cross-sectional view of the light source adjustment device in this invention;

[0062] Figures 1 to 10 The reference numerals in the accompanying drawings represent: 10-light source module, 11-light source transmission component, 1-light source collimation and focusing system, 2-color filter switching module, 3-multi-dimensional adjustment bracket, 4-microscope connection part, 20-color filter, 21-color filter insert plate, 30-Z-axis adjustment mechanism, 31-Y-axis adjustment mechanism, 32-W-axis adjustment mechanism, 301-dovetail groove guide rail, 302-dovetail groove slider, 303-gear rod, 304-locking screw, 305-gear, 310-optical axis guide rail, 311-optical axis slider, 312-rotating hinge, 31 3-Z-axis connector, 314-adjusting screw, 315-first compression spring, 316-front optical axis retainer, 317-rear optical axis retainer, 318-positioning screw, 320-light source module connector, 321-light source module shaft, 322-first limit screw, 323-second compression spring, 324-shaft pressure plate, 325-second limit screw, 40-lens adapter ring, 41-fiber positioning component, 42-fiber tightening component, 43-fiber tightening component, 44-fiber output interface, 45-lens, 46-fixing screw, 5-arm bracket. Detailed Implementation

[0063] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0064] like Figures 1 to 2 As shown, an external fluorescence system for a high-power stereomicroscope includes a light source module 10, a light source transmission component 11, a light source collimation and focusing system 1, a filter switching module 2, a multi-dimensional adjustment bracket 3, and a microscope connection part 4. The microscope connection part 4 is used to fix the entire external fluorescence system to the high-power stereomicroscope. One end of the light source transmission component 11 is connected to the light source module 10, and the other end is connected to the light source collimation and focusing system 1 to conduct the excitation light output from the light source module 10 to the light source collimation and focusing system 1. The microscope connection part 4 is an adapter plate. One side of the adapter plate is fixedly connected to the multi-dimensional adjustment bracket 3, and the other side is used to fixally connect to the arm bracket 5 of the high-power stereomicroscope to achieve the fixation of the entire external fluorescence system to the microscope.

[0065] The light source module 10, as the core source of excitation light, provides the basic excitation light required for fluorescence imaging, and provides the optical signal basis for subsequent brightness supplementation and multi-band imaging. Preferably, the light source module 10 is a four-channel LED light source, with each LED having an electrical power of 15-20W. The light source transmission component 11 is a liquid optical fiber, with one end connected to the output end of the four-channel LED light source and the other end connected to the light source collimation and focusing system 1 via an optical fiber interface. The light source transmission component 11 accurately transmits the excitation light output from the light source module 10 to the light source collimation and focusing system 1 via the liquid optical fiber, avoiding light intensity attenuation during transmission and ensuring sufficient excitation light intensity reaching the subsequent optical path, thus laying the foundation for brightness supplementation.

[0066] The light source collimation and focusing system 1 is used to focus the excitation light emitted by the light source module 10 into a very small spot. The size of the very small spot is adapted to the focusing spot size of the high-power objective lens of the high-power stereo microscope to compensate for the loss of fluorescence brightness caused by the single optical path of the high-power objective lens.

[0067] The filter switching module 2 is located in the optical path between the light source transmission component 11 and the light source collimation and focusing system 1. It is used to switch between different wavelength filters 20. Each filter 20 of the filter switching module 2 corresponds to the output wavelength of the light source module 10, which avoids non-target wavelength light from interfering with imaging and improves the specificity of fluorescence excitation. At the same time, it meets the imaging requirements of different fluorescently labeled samples without replacing the entire light source system, which greatly expands the applicable scenarios of the microscope.

[0068] Specifically, the color filter switching module 2 includes a color filter insert plate 21 and at least four color filters 20. The color filter insert plate 21 has mounting slots that are adapted to each color filter 20. The color filters 20 are correspondingly embedded in the mounting slots to physically position each color filter 20, preventing the color filter 20 from shifting or tilting due to vibration, operation, etc., and ensuring that the color filter 20 is always aligned with the center of the optical path. This avoids the leakage of excitation light or the mixing of stray light due to the offset of the color filter 20. At the same time, it can unify the installation height and angle of the color filters 20, so that when different color filters 20 are switched, the same optical path position reference can be maintained, eliminating the need to recalibrate the optical axis after each switch and reducing the cost of optical path debugging.

[0069] The filter plate 21 can reciprocate along a preset horizontal direction to switch different filters 20 to the optical path between the light source conduction component 11 and the light source collimation and focusing system 1. The wavelength of each filter 20 corresponds one-to-one with the four output wavelengths of the light source module 10, so that each filter 20 only allows the excitation light of the corresponding light source wavelength to pass through, while filtering out stray light or non-target wavelength light in the light source. This prevents non-target wavelength light from exciting other fluorescent substances in the sample, which would cause the imaging background to become brighter and the fluorescence signal to become blurred. Furthermore, precise wavelength matching allows the excitation light energy to be more concentrated on the target fluorescent label, improving the fluorescence excitation efficiency and indirectly assisting in supplementing the effective fluorescence brightness under the high-magnification objective lens, thus forming a synergy with the system to supplement the brightness loss of a single optical path.

[0070] The multi-dimensional adjustment bracket 3 is connected to the light source collimation and focusing system 1, and is used to drive the light source collimation and focusing system 1 to achieve linear adjustment of at least two axes and angle adjustment of one axis, so as to ensure that the light spot of the external excitation light overlaps with the excitation light of the microscope module itself.

[0071] Specifically, the multi-dimensional adjustment bracket 3 includes a Z-axis adjustment mechanism 30, a Y-axis adjustment mechanism 31, and a W-axis adjustment mechanism 32. The Z-axis adjustment mechanism 30 is connected to the light source collimation and focusing system 1 and is used to drive the light source collimation and focusing system 1 to move linearly along the Z-axis direction (vertical direction), adjust the height of the light source collimation and focusing system 1, and ensure that the focusing plane of the external excitation light is completely coincident with the sample surface and the focusing plane of the original excitation light of the microscope.

[0072] One end of the Y-axis adjustment mechanism 31 is fixedly connected to the Z-axis adjustment mechanism 30, and the other end is used to cooperate with the fixed structure of the high-magnification stereomicroscope. It is used to drive the Z-axis adjustment mechanism 30 and the light source collimation and focusing system 1 to move linearly along the Y-axis (along the horizontal front-back direction) to adjust the front-back position of the external light spot on the horizontal plane, ensuring that it is aligned with the original excitation light spot of the microscope in the horizontal direction and covers the same sample observation area. When the microscope stage moves back and forth (adjusting the sample observation position), the original excitation light spot will move synchronously with the sample. The Y-axis adjustment can drive the light source collimation and focusing system 1 to move back and forth synchronously, ensuring that the external light spot always covers the current observation area and avoiding brightness loss caused by the excitation light leaving the sample.

[0073] The W-axis adjustment mechanism 32 is connected between the Y-axis adjustment mechanism 31 and the light source collimation and focusing system 1. It is used to drive the light source collimation and focusing system 1 to rotate around a preset W-axis, with a rotation angle range of 0°-90°. By rotating the light source collimation and focusing system 1, the W-axis adjustment can correct the incident angle deviation, allowing the two beams of light to be incident in approximately the same direction, with the light spots completely overlapping, thus avoiding the excitation light from failing to enter the effective field of view of the objective lens due to angle discrepancies.

[0074] like Figures 3 to 5 As shown, specifically, the Z-axis adjustment mechanism 30 includes a dovetail guide rail 301, a dovetail slider 302, a gear rod 303, a locking screw 304, and a gear 305. The dovetail guide rail 301 and the dovetail slider 302 are slidably engaged through the dovetail groove. The dovetail slider 302 can move linearly along the length of the dovetail guide rail 301. The gear rod 303 passes horizontally through the circular groove opened on the dovetail slider 302. The gear 305 is fixedly sleeved on the end of the gear rod 303. The upper end face of the dovetail guide rail 301 is provided with a rack. The rack meshes with the gear 305. When the gear rod 303 rotates around its own axis, it drives the gear 305 to rotate, thereby driving the dovetail slider 302 to move along the dovetail guide rail 301.

[0075] When the height of the light source collimation and focusing system 1 needs to be adjusted, the operator rotates the gear rod 303 (rotating around its own axis). The gear rod 303 drives the gear 305, which is fixedly sleeved at its end, to rotate synchronously. Since the gear 305 meshes with the rack on the upper end face of the dovetail guide rail 301, the rotational motion of the gear 305 is converted into the linear motion of the dovetail slider 302 along the length direction of the dovetail guide rail 301 (i.e., up and down movement in the Z-axis direction). The light source collimation and focusing system 1 is connected to the dovetail slider 302, so it moves synchronously along the Z-axis with the slider, realizing the vertical position adjustment.

[0076] The side of the dovetail slide block 302 is provided with a dovetail notch. The locking screw 304 passes horizontally through the side wall of the dovetail slide block 302 and presses against the dovetail notch, so that the dovetail notch undergoes elastic deformation to press the dovetail guide rail 301, thereby achieving relative fixation between the dovetail slide block 302 and the dovetail guide rail 301.

[0077] After the Z-axis direction is adjusted to the target position, the slider and the guide rail are relatively fixed by the locking screw 304. Specifically, the side of the dovetail slider 302 is provided with a dovetail notch. The dovetail notch is a kind of elastic groove structure. When the locking screw 304 passes horizontally through the side wall of the slider and presses against the notch, the notch is squeezed and undergoes elastic deformation, and the width of the notch is reduced. The squeezing force generated by the deformation makes the inner wall of the dovetail groove of the slider and the outer wall of the dovetail groove of the guide rail fit tightly. By increasing the static friction between the two, the slider is prevented from moving along the guide rail, thereby stably fixing the light source collimation and focusing system 1 at the adjusted Z-axis position and avoiding position displacement caused by vibration, external force contact, etc.

[0078] like Figures 6 to 7 As shown, specifically, the Y-axis adjustment mechanism 31 includes an optical axis guide rail 310, an optical axis slider 311, a rotating hinge 312, a Z-axis connector 313, an adjusting screw 314, a first compression spring 315, a front optical axis retainer 316, a rear optical axis retainer 317, and a positioning screw 318.

[0079] Two sets of optical axis guide rails 310 are provided. The two sets of optical axis guide rails 310 are arranged in parallel and both pass through the circular holes opened on the optical axis slider 311. The two ends of the optical axis guide rails 310 are fixedly connected to the front optical axis retainer 316 and the rear optical axis retainer 317, respectively. The two sets of parallel optical axis guide rails 310 provide a linear motion track for the optical axis slider 311. The circular holes of the optical axis slider 311 are clearance-fitted with the optical axis guide rails 310. The two ends of the optical axis guide rails 310 are fixed to the front optical axis retainer 316 and the rear optical axis retainer 317, forming a rigid support structure to ensure that the optical axis slider 311 can only move linearly along the Y-axis (horizontal front-back direction) and avoid horizontal deviation.

[0080] The first compression spring 315 is located in the groove hole on the optical axis slider 311. The positioning screw 318 passes through the first compression spring 315 and is threadedly connected to the optical axis front retainer 316. The first compression spring 315 is in a slightly compressed state. Its restoring force pushes the optical axis slider 311 to always be close to the optical axis front retainer 316. The restoring force of the first compression spring 315 continuously pushes the optical axis slider 311, so that it is always close to the optical axis front retainer 316, providing an initial reference position for adjustment. In its natural state, the slider is close to the front retainer.

[0081] Adjusting screw 314 is screwed into the threaded hole on the optical axis front retainer 316. By adjusting the screwing depth of adjusting screw 314, the distance between optical axis slider 311 and optical axis front retainer 316 is changed. Z-axis connector 313 is fixedly connected to Z-axis adjustment mechanism 30. One side of rotating hinge 312 is fixed to Z-axis connector 313, and the other side is fixed to optical axis slider 311, forming a transmission chain of optical axis slider 311 → rotating hinge 312 → Z-axis connector 313 → Z-axis adjustment mechanism 30 → light source collimation and focusing system 1, ensuring that the Y-axis movement of optical axis slider 311 can be synchronously transmitted to light source collimation and focusing system 1.

[0082] When it is necessary to adjust the horizontal position of the collimation and focusing system 1, precise control can be achieved by adjusting screw 314, as follows:

[0083] Forward adjustment (near the sample direction): If it is necessary to move the collimating and focusing system 1 of the light source forward, closer to the front retainer, turn the adjusting screw 314 in the opposite direction to unscrew it from the front retainer 316 of the optical axis. At this time, the restoring force of the first compression spring 315 pushes the optical axis slider 311 forward. Since the slider is no longer blocked by the adjusting screw 314, the slider will move forward until it is close to the front retainer or the end of the adjusting screw 314 again, thus realizing the position adjustment of the Y axis in the forward direction.

[0084] Rearward adjustment (away from the sample direction): If it is necessary to move the collimating and focusing system 1 of the light source backward away from the front retainer, turn the adjusting screw 314 in the forward direction and screw it into the front retainer 316 of the optical axis. The end of the adjusting screw 314 pushes the optical axis slider 311, overcoming the elastic force of the first compression spring 315, so that the slider moves backward along the optical axis guide rail 310 away from the front retainer until the target position is reached, thereby realizing the position adjustment of the Y axis in the rearward direction;

[0085] After adjustment, the elastic force of the first compression spring 315 and the tightening force of the adjusting screw 314 are balanced. If the slider is pushed to a certain position by the adjusting screw 314, the spring force and the screw force are equal, so that the optical axis slider 311 is stably kept in the target position. At the same time, the cooperation between the dual optical axis guide rail 310 and the slider further restricts the shaking of the slider, ensuring that the Y-axis position of the light source collimation and focusing system 1 does not shift under slight external vibration or touch, thus ensuring the stability of the light spot alignment.

[0086] like Figure 8As shown, specifically, the W-axis adjustment mechanism 32 includes a light source module connector 320, a light source module rotating shaft 321, a first limiting screw 322, a second compression spring 323, a rotating shaft pressure plate 324, and a second limiting screw 325. Through the coordinated design of rotating shaft rotation + spring pre-tightening + threaded fine adjustment, the light source collimation and focusing system 1 is driven to rotate around the preset W-axis (horizontal axis), accurately correcting the incident angle of the external excitation light, ensuring that it is consistent with the optical path direction of the original excitation light of the microscope, and realizing the coaxial overlap of the two light spots. At the same time, the structural constraints ensure the stability of the adjusted angle, providing directional dimension accuracy for the overlap of the light spots.

[0087] The light source module shaft 321 passes horizontally through the round hole opened on the optical axis rear retainer 317. The limiting screw is screwed into the through hole on the right side of the optical axis rear retainer 317 and abuts against the light source module shaft 321. The light source module shaft 321 passes horizontally through the round hole of the optical axis rear retainer 317. The optical axis rear retainer 317 provides rigid support for the shaft, ensuring that the shaft can only rotate around its own axis (without radial offset), providing a stable rotation center for angle adjustment.

[0088] The second compression spring 323 is located inside the optical axis rear retainer 317, and its two ends abut against the first limiting screw 322 and the second limiting screw 325 respectively. The second compression spring 323 is in a compressed state. The first limiting screw 322 is screwed into the through-hole threaded hole on the optical axis rear retainer 317. The rotation angle of the light source module shaft 321 is adjusted by tightening or loosening the first limiting screw 322. The spring's restoring force is transmitted to the light source module shaft 321 through the second limiting screw 325, so that the light source module shaft 321 is tightly fitted with the inner wall of the circular hole of the optical axis rear retainer 317, forming an initial force balance state and preventing the shaft from shaking freely.

[0089] The rotating shaft pressure plate 324 covers the end of the optical axis rear retainer 317 to prevent dust from entering; one end of the light source module connector 320 is threadedly connected to the light source module rotating shaft 321, and the other end is fixedly connected to the light source collimation and focusing system 1, forming a transmission chain of light source module rotating shaft 321 → light source module connector 320 → light source collimation and focusing system 1, ensuring that the rotational movement of the rotating shaft can be synchronously transmitted to the light source collimation and focusing system 1, realizing the linkage adjustment of the angle, and realizing the synchronous rotation of the light source module rotating shaft 321 and the light source collimation and focusing system 1.

[0090] When it is necessary to adjust the incident angle of the light source collimation and focusing system 1, the control is achieved by tightening or loosening the first limit screw 322, as follows:

[0091] Forward rotation adjustment (e.g., clockwise): If it is necessary to rotate the light source collimating and focusing system 1 around the W axis in a certain direction, such as closer to the microscope optical path, turn the first limiting screw 322 forward, screwing it into the rear retainer 317 of the optical axis. The first limiting screw 322 pushes the second compression spring 323 to further compress, and the spring's restoring force increases. Through the second limiting screw 325, it pushes the light source module rotating shaft 321, causing the rotating shaft to rotate around its own axis in the forward direction (e.g., clockwise). The rotating shaft drives the light source collimating and focusing system 1 to rotate synchronously through the light source module connector 320 until the target angle is reached.

[0092] Reverse rotation adjustment (e.g., counterclockwise): If it is necessary to rotate the light source collimating and focusing system 1 in the opposite direction, such as away from the direction of the microscope optical path, turn the first limiting screw 322 in the opposite direction to unscrew it from the optical axis rear retainer 317. The pressure of the first limiting screw 322 on the compression spring decreases, and the spring's restoring force weakens. The light source module rotating shaft 321 rotates in the opposite direction (e.g., counterclockwise) under the action of its own friction with the inner wall of the circular hole until the spring force and the force on the rotating shaft are rebalanced. The rotating shaft synchronously drives the light source collimating and focusing system 1 to rotate in the opposite direction, thereby realizing angle adjustment.

[0093] After adjustment, the elastic force of the second compression spring 323 continues to act on the light source module shaft 321 through the second limit screw 325, so that the light source module shaft 321 and the inner wall of the circular hole of the optical axis rear retainer 317 are in close contact and the static friction is balanced, thereby stabilizing and fixing the light source collimation and focusing system 1 at the target angle; at the same time, the shaft pressure plate 324 covers the end of the optical axis rear retainer 317 to prevent dust from entering and affecting the rotation accuracy of the shaft, and further ensure the angular stability during long-term use.

[0094] like Figures 9 to 10 As shown, in this embodiment, the system also includes a light source adjustment device, which includes a lens barrel adapter ring 40, an optical fiber positioning component 41, an optical fiber tightening component 42, an optical fiber screwing component 43, an optical fiber output interface 44, a lens barrel 45, and a fixing screw 46.

[0095] The optical fiber output interface 44 passes through the optical fiber tightening member 42 and the optical fiber positioning member 41 in sequence. The end of the optical fiber tightening member 42 is chamfered and three slots are evenly distributed along its central axis.

[0096] The fiber optic tightening member 43 is threadedly connected to the threaded hole on the fiber optic positioning member 41. When the fiber optic tightening member 43 is screwed in, it pushes the fiber optic tightening member 42 to be subjected to force along the chamfered slope, causing the slot of the fiber optic tightening member 42 to shrink and press the fiber optic output interface 44. After the fiber optic output interface 44 is inserted, the fiber optic tightening member 43 is screwed into the fiber optic positioning member 41. The end of the tightening member applies pressure along the chamfered slope of the fiber optic tightening member 42, forcing the three slots of the tightening member to shrink (elastic deformation), thereby tightly wrapping and pressing the fiber optic output interface 44. The fiber optic output interface 44 is rigidly fixed by mechanical force, preventing the fiber from being displaced due to vibration or pulling. At the same time, the central hole of the fiber optic positioning member 41 matches the outer diameter of the fiber optic output interface 44, ensuring that the optical axis of the fiber is strictly aligned with the central axis of the subsequent optical path, reducing the excitation light loss caused by optical axis misalignment.

[0097] The lens barrel adapter ring 40 is fixedly connected to the fiber optic positioning component 41. The lens barrel adapter ring 40 has a slot that matches the color filter insert plate 21. The color filter insert plate 21 is inserted into the slot. The lens barrel 45 is threadedly connected to the front end of the lens barrel adapter ring 40 for secondary focusing of the excitation light. The color filter insert plate 21 of the color filter switching module 2 is inserted into the lens barrel adapter ring 40 through the slot, so that the color filter 20 is precisely positioned in the optical path between the fiber optic output interface 44 and the lens barrel 45 (i.e., in the optical path between the light source transmission component 11 and the light source collimation and focusing system 1). This ensures that the color filter 20 is aligned with the central axis of the optical path, avoiding the problem of some bands being missed due to the color filter offset. At the same time, the size of the slot matches the color filter insert plate 21, ensuring the stability of the color filter switching and further improving the accuracy of band selection.

[0098] The lens barrel 45 is threadedly connected to the front end of the lens barrel adapter ring 40. It typically integrates an achromatic lens. The excitation light output from the fiber optic output interface 44 enters the lens barrel 45 after being filtered by the color filter 20. The lens inside the lens barrel 45 performs secondary focusing on the diverging excitation light, further compressing the spot size. This provides pre-optimization for the subsequent collimation and focusing system 1 to form a very small spot, which helps reduce the compression pressure on the collimation and focusing system 1 and improves the uniformity and focusing accuracy of the final spot. At the same time, the threaded connection between the lens barrel 45 and the adapter ring allows for fine adjustment of the distance between the lens and the color filter 20, adapting to the divergence angle of different fiber optic output lights and enhancing the system's compatibility with the light source.

[0099] The light source adjustment device is inserted into the circular hole on the light source module connector 320 and can move axially along the circular hole. After adjustment, it is tightened and fixed by the fixing screw 46. When there is a slight axial deviation between the focused spot of the light source collimating and focusing system 1 and the high-magnification objective lens spot, the light source adjustment device is pushed to move axially along the circular hole to fine-tune the overall position of the fiber optic output interface 44, the color filter 20, and the lens barrel 45, so that the optical path of the excitation light is completely coaxial with the optical path of the light source collimating and focusing system 1. After adjustment, the fixing screw 46 is tightened to press the light source adjustment device to ensure stable position. This helps to supplement the adjustment accuracy of the multi-dimensional adjustment bracket 3, solve the problem of slight optical path offset caused by accumulated errors, and ultimately ensure that the tiny spot formed after the excitation light is converged and collimated is completely superimposed on the focused spot of the high-magnification objective lens.

[0100] Furthermore, the present invention also provides a method for using an external fluorescence system applied to a high-magnification stereomicroscope, comprising the following steps:

[0101] S1: System Installation and Fixing:

[0102] The entire external fluorescence system is fixedly connected to the arm support 5 of the high-power stereomicroscope via the adapter plate of the microscope connection part 4, ensuring that the system is stable and secure.

[0103] S2: Connection between light source and optical path:

[0104] One end of the liquid optical fiber of the light source transmission component 11 is connected to the output end of the light source module 10, and the other end is connected to the light source collimation and focusing system 1 through the optical fiber interface to establish a transmission path for the excitation light from the light source to the light source collimation and focusing system 1.

[0105] S3: Excitation band selection:

[0106] According to the fluorescent label type of the sample to be observed, the filter insertion plate 21 of the filter switching module 2 is pushed to move the filter 20 that matches the target output band of the light source module 10 into the optical path between the light source conduction component 11 and the light source collimation and focusing system 1; wherein, the light source module 10 is a four-channel LED light source, and the filter insertion plate 21 is provided with at least four filters 20, and the band of each filter 20 corresponds one-to-one with the four output bands of the four-channel LED light source;

[0107] S4: Multi-dimensional optical path alignment:

[0108] S41: Z-axis direction adjustment:

[0109] The gear rod 303 of the rotating Z-axis adjustment mechanism 30 drives the gear 305 to mesh with the rack on the dovetail groove guide rail 301, driving the dovetail groove slider 302 and the light source collimation and focusing system 1 connected thereto to move linearly along the Z-axis direction, so that the excitation light spot initially falls on the observation area of ​​the sample.

[0110] S42: Y-axis direction adjustment:

[0111] Tighten the adjusting screw 314 of the Y-axis adjustment mechanism 31 to change the distance between the optical axis slider 311 and the optical axis front holder 316, drive the optical axis slider 311 and the light source collimation and focusing system 1 linked with it to move along the Y-axis direction, fine adjust the front and rear position of the light spot on the horizontal plane, so that the light spot gradually approaches the original excitation light spot area of ​​the microscope.

[0112] S43: W-axis angle adjustment:

[0113] The first limiting screw 322 of the W-axis adjustment mechanism 32 drives the rotating shaft 321 of the light source module to rotate through the elastic force change of the second compression spring 323, which in turn drives the light source collimation and focusing system 1 connected to the rotating shaft to rotate around the W-axis until the external excitation light spot completely overlaps with the original excitation light spot of the high-magnification stereomicroscope.

[0114] S5: Brightness Adjustment and Imaging Observation

[0115] Turn on the light source module 10 and adjust the channel power of its corresponding output band to make the excitation light brightness match the sample imaging requirements; switch the objective lens of the high-power stereomicroscope to the 10X objective lens, start the microscope imaging system, and complete the fluorescence imaging observation of the sample.

[0116] S6: Multi-band switching observation:

[0117] If it is necessary to observe samples with different fluorescent labels, repeat steps S3 to S5: push the filter insert 21 to switch to the filter 20 with the wavelength matching the new fluorescent label, and after completing the multi-dimensional optical path alignment and brightness adjustment, start the imaging system for observation.

[0118] Additionally, it should be noted that components not described in detail in this article are existing technologies.

[0119] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An external fluorescence system for use in a high-power stereomicroscope, characterized in that, The application relates to a fluorescence excitation light source system for high-power microscope, which comprises a light source module (10), a light source conducting assembly (11), a light source collimation and light collection system (1), a color filter switching module (2), a multi-dimension adjusting support (3), a microscope connecting part (4) and a light source adjusting device. One end of the light source conducting assembly (11) is connected with the light source module (10), and the other end is in communication with the light source collimation and light collection system (1) to conduct excitation light output by the light source module (10) to the light source collimation and light collection system (1). The light source collimation and light collection system (1) is used for converging excitation light emitted by the light source module (10) into a very small light spot, the size of the very small light spot is matched with the size of a high-power objective lens focusing light spot of a high-power microscope, so that the fluorescence brightness loss caused by the single light path of the high-power objective lens is compensated. The color filter switching module (2) is arranged on the light path between the light source conducting assembly (11) and the light source collimation and light collection system (1) and is used for switching different waveband color filters (20), and each color filter (20) of the color filter switching module (2) corresponds to the output waveband of the light source module (10). The multi-dimension adjusting support (3) is connected with the light source collimation and light collection system (1) and is used for driving the light source collimation and light collection system (1) to realize linear adjustment of at least two axes and angle adjustment of one axis, so that the light spot of the external excitation light and the light spot of the excitation light of the microscope itself module are overlapped through the cooperation of the multi-dimension adjusting support (3) and the axial fine adjustment of the light source adjusting device. The microscope connecting part (4) is used for fixing the whole external fluorescence system on the high-power microscope. The light source adjusting device comprises a lens barrel adapter ring (40), a fiber positioning piece (41), a fiber tightening piece (42), a fiber screwing piece (43), a fiber light outlet interface (44), a lens barrel (45) and a fixing screw (46). The fiber light outlet interface (44) passes through the fiber tightening piece (42) and the fiber positioning piece (41) in sequence, the end of the fiber tightening piece (42) is provided with a chamfer, and three grooves are uniformly arranged along the axial direction of the fiber tightening piece (42). The fiber screwing piece (43) is threadedly connected with the threaded hole of the fiber positioning piece (41), when the fiber screwing piece (43) is screwed in, the fiber tightening piece (42) is forced to move along the chamfer, so that the grooves of the fiber tightening piece (42) are contracted to press the fiber light outlet interface (44). The lens barrel adapter ring (40) is fixedly connected with the fiber positioning piece (41), the lens barrel adapter ring (40) is provided with an insertion groove matched with a color filter insertion board (21) of the color filter switching module (2), the color filter insertion board (21) is inserted into the insertion groove, the lens barrel (45) is threadedly connected with the front end of the lens barrel adapter ring (40) and is used for secondarily converging excitation light. The fixing screw (46) is used for fixing the light source adjusting device after movement adjustment, and the movement adjustment makes the light path of the excitation light coaxial with the light path of the light source collimation and light collection system (1).

2. The external fluorescence system for high magnification microscopy of claim 1, wherein, The light source transmission assembly (11) is a liquid optical fiber, one end of the liquid optical fiber is connected with the output end of the light source module (10), and the other end is connected with the light source collimation and light collection system (1) through an optical fiber interface.

3. The external fluorescence system for high magnification microscopy of claim 2, wherein, The color filter switching module (2) further comprises at least four color filters (20), and the color filter plugboard (21) is provided with mounting grooves corresponding to the color filters (20); the color filters (20) are correspondingly embedded in the mounting grooves. The color filter plugboard (21) can move back and forth along a preset horizontal direction to switch different color filters (20) to the light path between the light source transmission assembly (11) and the light source collimation and light collection system (1), and the wave bands of the color filters (20) correspond to the four output wave bands of the light source module (10) one by one.

4. The external fluorescence system for high power light microscopy according to claim 3, wherein, The multi-dimensional adjustment support (3) comprises a Z-axis adjustment mechanism (30), a Y-axis adjustment mechanism (31) and a W-axis adjustment mechanism (32). The Z-axis adjustment mechanism (30) is connected with the light source collimation and light collection system (1) and is used to drive the light source collimation and light collection system (1) to move linearly along the Z-axis direction; one end of the Y-axis adjustment mechanism (31) is fixedly connected with the Z-axis adjustment mechanism (30), and the other end is used to cooperate with a fixed structure of a high-power stereomicroscope and is used to drive the Z-axis adjustment mechanism (30) and the light source collimation and light collection system (1) to move linearly along the Y-axis direction; the W-axis adjustment mechanism (32) is connected between the Y-axis adjustment mechanism (31) and the light source collimation and light collection system (1) and is used to drive the light source collimation and light collection system (1) to rotate around a preset W-axis, and the rotation angle range is 0°-90°.

5. The external fluorescence system for high power light microscopy of claim 4, wherein, The Z-axis adjustment mechanism (30) comprises a dovetail groove guide rail (301), a dovetail groove sliding block (302), a gear rod (303), a locking screw (304) and a gear (305). The dovetail groove guide rail (301) and the dovetail groove sliding block (302) are connected through dovetail groove sliding cooperation, the dovetail groove sliding block (302) can move linearly along the length direction of the dovetail groove guide rail (301), the gear rod (303) horizontally penetrates a circular groove provided on the dovetail groove sliding block (302), the gear (305) is fixedly sleeved on the end of the gear rod (303), the upper end surface of the dovetail groove guide rail (301) is provided with a rack, the rack is engaged with the gear (305), when the gear rod (303) rotates around its axis, the gear (305) is driven to rotate, and then the dovetail groove sliding block (302) is driven to move along the dovetail groove guide rail (301); A dovetail groove notch is provided on the side surface of the dovetail groove sliding block (302), the locking screw (304) horizontally penetrates the side wall of the dovetail groove sliding block (302) and tightly presses the dovetail groove notch, so that the dovetail groove notch is elastically deformed to press the dovetail groove guide rail (301), and the relative fixation of the dovetail groove sliding block (302) and the dovetail groove guide rail (301) is realized.

6. The external fluorescence system for high power light microscopy of claim 5, wherein, The Y-axis adjusting mechanism (31) comprises an optical axis guide rail (310), an optical axis slider (311), a rotating hinge (312), a Z-axis connecting piece (313), an adjusting screw (314), a first compression spring (315), an optical axis front holder (316), an optical axis rear holder (317) and a positioning screw (318); The optical axis guide rail (310) is provided with two groups, and the two groups of optical axis guide rails (310) are arranged in parallel and pass through the round holes formed in the optical axis slider (311), and the two ends of the optical axis guide rail (310) are connected with the optical axis front holder (316) and the optical axis rear holder (317) respectively; The first compression spring (315) is arranged in the groove hole formed in the optical axis slider (311), the positioning screw (318) passes through the first compression spring (315) and is screwed with the optical axis front holder (316) in a threaded manner, and the first compression spring (315) is in a slightly compressed state, and the restoring force of the first compression spring (315) pushes the optical axis slider (311) to always abut against the optical axis front holder (316); The adjusting screw (314) is screwed into the threaded hole formed in the optical axis front holder (316), the distance between the optical axis slider (311) and the optical axis front holder (316) is changed by adjusting the screwing depth of the adjusting screw (314), the Z-axis connecting piece (313) is fixedly connected with the Z-axis adjusting mechanism (30), one side of the rotating hinge (312) is fixed with the Z-axis connecting piece (313), and the other side is fixed with the optical axis slider (311).

7. The external fluorescence system for high magnification microscopy of claim 6, wherein, The W-axis adjusting mechanism (32) comprises a light source module connecting piece (320), a light source module rotating shaft (321), a first limiting screw (322), a second compression spring (323), a rotating shaft pressing plate (324) and a second limiting screw (325); The light source module rotating shaft (321) passes through the round hole formed in the optical axis rear holder (317) in a horizontal manner, and the second limiting screw (325) is screwed into the light source module rotating shaft (321) from the right side through hole of the optical axis rear holder (317) and abuts against the light source module rotating shaft (321); The second compression spring (323) is arranged in the optical axis rear holder (317) and abuts against the first limiting screw (322) and the second limiting screw (325) at two ends, the second compression spring (323) is in a compressed state, the first limiting screw (322) is screwed into the through hole threaded hole formed in the optical axis rear holder (317), and the rotating angle of the light source module rotating shaft (321) is adjusted by loosening or tightening the first limiting screw (322); The rotating shaft pressing plate (324) covers the end of the optical axis rear holder (317), one end of the light source module connecting piece (320) is screwed with the light source module rotating shaft (321) in a threaded manner, the other end is connected with the light source collimation and light condensation system (1), and synchronous rotation of the light source module rotating shaft (321) and the light source collimation and light condensation system (1) is realized.

8. The external fluorescence system for high magnification microscopy of claim 7, wherein, The light source adjusting device is installed in the round hole of the light source module connector (320) and can move along the axial direction of the round hole, and is fixed by the fixing screw (46) after adjustment.

9. The external fluorescence system for high magnification microscopy of claim 8, wherein, The microscope connecting part (4) is an adapter plate, one side of the adapter plate is fixedly connected with the multi-dimensional adjusting support (3), and the other side is used for being fixedly connected with a vertical arm support (5) of a high-power visual microscope, so that the whole external fluorescence system and the microscope are fixed.

10. A method of using an external fluorescence system for a high-power optical microscope according to claim 9, characterized in that, The method comprises the following steps: S1: system installation and fixation: Through the adapter plate of the microscope connecting part (4), the whole external fluorescence system is fixedly connected with the vertical arm support (5) of the high-power visual microscope, so that the system is stable and free from loosening; S2: light source and light path connection: One end of the liquid optical fiber of the light source transmission assembly (11) is connected with the output end of the light source module (10), and the other end is conducted through the optical fiber interface and the light source collimation and light collection system (1), so as to establish a transmission path of excitation light from the light source to the light source collimation and light collection system (1); S3: excitation wave band selection: According to the fluorescence label type of the sample to be observed, the color filter plug-in board (21) of the color filter switching module (2) is pushed, and the color filter (20) matched with the target output wave band of the light source module (10) is moved into the light path between the light source transmission assembly (11) and the light source collimation and light collection system (1); wherein the light source module (10) is a four-channel LED light source, and at least four color filters (20) are arranged on the color filter plug-in board (21), and the wave bands of the color filters (20) correspond to the four output wave bands of the four-channel LED light source one by one; S4: multi-dimensional light path alignment: S41: Z-axis direction adjustment: The gear rod (303) of the Z-axis adjusting mechanism (30) is rotated, the gear (305) is engaged with the rack on the dovetail groove guide rail (301), the dovetail groove slider (302) and the light source collimation and light collection system (1) connected therewith are driven to move linearly along the Z-axis direction, and the excitation light spot is preliminarily located in the observation area of the sample; S42: Y-axis direction adjustment: The adjusting screw (314) of the Y-axis adjusting mechanism (31) is screwed, the distance between the optical axis slider (311) and the optical axis front holder (316) is changed, the optical axis slider (311) and the light source collimation and light collection system (1) connected therewith are driven to move along the Y-axis direction, the front and back positions of the light spot on the horizontal plane are finely adjusted, and the light spot gradually approaches the light spot area of the original excitation light of the microscope; S43: W-axis angle adjustment: The first limiting screw (322) of the W-axis adjusting mechanism (32) is loosened and tightened, the light source module rotating shaft (321) is driven to rotate through the elastic force change of the second compression spring (323), the light source collimation and light collection system (1) connected with the rotating shaft is driven to rotate around the W-axis, and the external excitation light spot is completely overlapped with the original excitation light spot of the high-power visual microscope; S5: brightness adjustment and imaging observation: Open the light source module (10), adjust the channel power of its corresponding output wave band, and make the excitation light brightness adapt to the sample imaging requirement; switch the objective lens of the high-power visual microscope to the 10X objective lens, start the microscope imaging system, and complete the fluorescence imaging observation of the sample; S6: Multi-wave band switching observation: If different fluorescently labeled samples need to be observed, repeat steps S3 to S5, push the color filter insertion plate (21) to switch to the color filter (20) matched with the new fluorescent label, re-complete the multi-dimensional light path alignment and brightness adjustment, and then start the imaging system for observation.

Images