Confocal microscope pinhole imaging system

Abstract

The invention relates to a confocal microscope pinhole imaging system, and relates to the field of confocal microscopes, the confocal microscope pinhole imaging system comprises a laser generation module, a focusing lens, a first dichroic spectroscope, a main pinhole, a collimating lens, a scanning module, an objective lens and a receiving imaging module, the main pinhole and the focal plane of the objective lens are arranged in a conjugate mode, the collimating lens is matched with the main pinhole, and the collimating lens is matched with the receiving imaging module. The focusing lens can focus light to the main pinhole, and the collimating lens can collimate the light penetrating through the main pinhole into parallel light; the first dichroic spectroscope, the main pinhole, the collimating lens, the scanning module and the objective lens form a common light path, incident laser passes through the focusing lens and is transmitted to a sample along the common light path, and reflected fluorescence is transmitted to the receiving imaging module along the common light path for imaging. The confocal microscope has the advantages that the number of conjugate devices in the confocal process is reduced, the influence of displacement of the conjugate devices on the imaging resolution of the microscope is improved, and the imaging stability of the confocal microscope is improved. When incident laser passes through the main pinhole, the incident laser is closer to a point light source, and the imaging resolution of the confocal microscope is improved.

Description

Technical Field

[0001] This application relates to the field of confocal microscopy, and in particular to a confocal microscope pinhole imaging system. Background Technology

[0002] Confocal microscopy is a high-end imaging technology that breaks through the resolution limitations of traditional optical microscopes and achieves "optical slicing". Its core principle revolves around "co-positioning of the excitation light focus and the probe light focus" and "pinhole filtering of defocus signals", ultimately achieving high-resolution, high-contrast three-dimensional imaging.

[0003] The existing patent document, CN108414442A, discloses a confocal microscopy system suitable for near-infrared II fluorescence in vivo imaging. In this patent, the laser is reflected by a dichroic mirror and then illuminates the object through a lens and objective lens, generating fluorescence. The fluorescence travels along the light path through the dichroic mirror and passes through a pinhole. A photomultiplier receives the fluorescence passing through the pinhole to form an image, completing the imaging process of the confocal microscope. However, in the prior art, the laser is emitted directly from the end face of a single-mode fiber as a point light source into the subsequent light path. The confocal process requires the point light source, objective lens, and imaging pinhole to be conjugate. If any one of these components deviates from its position, it will affect the resolution of the microscope image, resulting in low imaging stability of the confocal microscope. Summary of the Invention

[0004] In order to improve the problem of low imaging stability caused by the large number of conjugate elements in existing confocal microscopes, this application provides a confocal microscope pinhole imaging system.

[0005] The confocal microscope pinhole imaging system provided in this application adopts the following technical solution: A confocal microscope pinhole imaging system includes a laser generating module, a focusing lens, a first dichroic beam splitter, a main pinhole, a collimating lens, a scanning module, an objective lens, and a receiving imaging module. The main pinhole and the objective lens are arranged conjugately. The collimating lens is adapted to the main pinhole. The focusing lens can focus light onto the main pinhole, and the collimating lens can collimate the light passing through the main pinhole into parallel light. The first dichroic beam splitter, main pinhole, collimating lens, scanning module, and objective lens form a common optical path. The incident laser passes through the focusing lens and is transmitted to the sample along the common optical path. The reflected fluorescence is transmitted to the receiving imaging module along the common optical path for imaging.

[0006] By adopting the above technical solution, in the confocal microscope imaging process, the incident laser is generated by the laser generation module and transmitted to the sample through the focusing lens and the first dichroic beam splitter along the common pipeline to generate reflected fluorescence. The reflected fluorescence is then transmitted along the common optical path to the first dichroic beam splitter and then split to the receiving imaging module, completing the confocal microscope imaging process. During imaging, the incident laser is actively filtered through the main pinhole to form a point light source, and the reflected fluorescence is filtered through the main pinhole and then received by the receiving imaging module. This allows the main pinhole to simultaneously serve as both an illumination pinhole and a detection pinhole, achieving strict conjugation of the pinholes. Therefore, it is only necessary to ensure that the main pinhole and the objective lens are conjugated, reducing the number of conjugated devices in the confocal microscope and improving the imaging stability of the confocal microscope, which is less affected by the displacement of the conjugated devices. When the incident laser passes through the main pinhole, it is closer to a point light source, thereby improving the imaging resolution of the confocal microscope.

[0007] Preferably, a first collimating lens is provided between the laser generating module and the focusing lens, and the first collimating lens can collimate the incident laser into parallel light.

[0008] By adopting the above technical solution, a first collimating lens is set between the laser generating module and the focusing lens to collimate the incident laser into parallel light, so that the laser has better directionality and uniformity before entering the focusing lens, which helps to focus the laser more accurately at the main pinhole, thereby further improving the stability and resolution of confocal microscope imaging.

[0009] Preferably, a second collimating lens is provided between the first dichroic beam splitter and the receiving imaging module, and the second collimating lens can collimate the reflected fluorescence into parallel light.

[0010] By adopting the above technical solution, during the process of reflected fluorescence transmission to the receiving imaging module, the second collimating lens collimates the reflected fluorescence into parallel light, which can reduce the scattering and loss of fluorescence light, improve the efficiency and quality of reflected fluorescence transmission, and thus improve the accuracy of the receiving imaging module in receiving fluorescence, making the imaging clearer and more stable.

[0011] Preferably, the receiving imaging module includes a filter wheel and a first fluorescent PMT. The filter wheel is provided with multiple receiving filters with different center wavelengths. The filter wheel can adjust the receiving filters one by one to the optical path of the reflected fluorescence. The first fluorescent PMT can receive the reflected fluorescence transmitted through the receiving filters.

[0012] By adopting the above technical solution, multiple receiving filters with different center wavelengths on the filter wheel can be adjusted one by one to the reflected fluorescence light path, so that the first fluorescence PMT can receive the reflected fluorescence after filtering with different center wavelengths. This significantly enhances the system's adaptability and selectivity to reflected fluorescence of different wavelengths, thereby improving the flexibility and diversity of imaging.

[0013] Preferably, the receiving imaging module further includes a second fluorescent PMT and a first filter. A second dichroic beam splitter is provided in the optical path of the reflected fluorescence between the first fluorescent PMT and the second collimating lens. The second dichroic beam splitter can reflect part of the reflected fluorescence to the first filter, and the second fluorescent PMT can receive the reflected fluorescence passing through the first filter.

[0014] By adopting the above technical solution, a portion of the reflected fluorescence is reflected to the first filter using a second dichroic spectroscope, allowing the second fluorescence PMT to receive the reflected fluorescence passing through the first filter. This enables the separate reception and processing of different parts of the reflected fluorescence, increasing the detection dimensions of the reflected fluorescence, further enriching the imaging information, and improving the accuracy and comprehensiveness of the imaging.

[0015] Preferably, the receiving imaging module further includes a third fluorescent PMT and a second filter. A third dichroic beam splitter is provided in the optical path of reflected fluorescence between the first fluorescent PMT and the second dichroic beam splitter. The third dichroic beam splitter can reflect part of the reflected fluorescence to the second filter. The third fluorescent PMT can receive the reflected fluorescence passing through the second filter. The center wavelength of the second filter is different from the center wavelength of the first filter.

[0016] By adopting the above technical solution, during the confocal microscope imaging process, a third dichroic spectroscope is used to reflect part of the reflected fluorescence to the second filter, and then the third fluorescence PMT receives the reflected fluorescence that passes through the second filter with a center wavelength different from that of the first filter. This enables the separate reception and detection of reflected fluorescence at different wavelengths, enriches the imaging information, and further improves the accuracy and comprehensiveness of the imaging.

[0017] Preferably, the scanning module includes an XY scanning galvanometer and a scanning lens. Light is transmitted sequentially through the XY scanning galvanometer and the scanning lens to the objective lens. The XY scanning galvanometer is used to control the two-dimensional scanning of the laser on the sample.

[0018] By adopting the above technical solution, in the process of confocal microscopy imaging, a scanning module composed of an XY scanning galvanometer and a scanning lens is used to transmit light sequentially through the two to the objective lens. The XY scanning galvanometer controls the laser to perform two-dimensional scanning on the sample, which can expand the scanning range of the laser on the sample, acquire sample information more comprehensively, and thus improve the integrity and accuracy of confocal microscopy imaging.

[0019] Preferably, it also includes a switching mechanism, which has multiple replacement pinholes with different apertures. The main pinhole is located on the switching mechanism. The switching mechanism can switch the positions of the replacement pinholes and the main pinhole, and set the replacement pinholes one by one on the common optical path.

[0020] By adopting the above technical solution, the interchange mechanism can flexibly switch between replacement pinholes of different apertures according to actual needs, so as to adapt to different samples or imaging requirements, thereby enhancing the applicability and flexibility of the confocal microscope pinhole imaging system.

[0021] Preferably, a condenser lens is provided in the optical path between the second collimating lens and the first dichroic beam splitter, and a displacement mechanism is externally connected to the second collimating lens, the displacement mechanism being able to adjust the distance between the second collimating lens and the condenser lens.

[0022] By adopting the above technical solution, a condenser lens is set in the optical path of the reflected fluorescence, and the distance between the second collimating lens and the condenser lens is adjusted by a displacement mechanism. With the change of the aperture of the replacement pinhole, the focusing state of the reflected fluorescence can be flexibly adjusted to adapt to different imaging requirements, thereby further improving the imaging quality and the adaptability of the system.

[0023] Preferably, the maximum distance between the second collimating lens and the condenser lens is less than the focal length of the condenser lens.

[0024] By adopting the above technical solution, limiting the maximum distance between the second collimating lens and the condenser lens to be less than the focal length of the condenser lens, the reflected fluorescence can be kept in a suitable focused state in the optical path, avoiding excessive divergence or convergence of light, thereby optimizing the fluorescence quality received by the receiving imaging module and further improving the clarity and accuracy of confocal microscope imaging.

[0025] In summary, this application includes at least one of the following beneficial technical effects: 1. The main pinhole serves as both the illumination and detection pinhole, achieving strict conjugation. Only the main pinhole and the objective lens need to be conjugated, reducing the number of conjugated components and avoiding the impact on imaging resolution due to the misalignment of multiple conjugated components, thus improving the imaging stability of the confocal microscope. 2. The incident laser light is actively filtered through the main pinhole to form light that is closer to a point source, thus improving the imaging resolution of the confocal microscope; 3. By setting receiving filters with different center wavelengths, the PMT can receive reflected fluorescence after filtering with different center wavelengths, which significantly enhances the system's adaptability and selectivity to reflected fluorescence of different wavelengths. By setting three PMTs and filters with different center wavelengths, the detection dimensions of reflected fluorescence are increased, further enriching the imaging information and improving the accuracy and comprehensiveness of imaging. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of a confocal microscope pinhole imaging system according to Embodiment 1 of this application.

[0027] Figure 2 This is a schematic diagram of a confocal microscope pinhole imaging system according to Embodiment 2 of this application.

[0028] Explanation of reference numerals in the attached drawings: 11. Laser generating module; 12. First collimating lens; 13. Focusing lens; 21. First dichroic beam splitter; 22. Main pinhole; 23. Collimating lens; 24. Scanning module; 241. XY scanning galvanometer; 242. Scanning lens; 25. Objective lens; 31. Second collimating lens; 32. Receiving imaging module; 321. Filter wheel; 322. First fluorescence PMT; 323. Second fluorescence PMT; 324. First filter; 325. Third fluorescence PMT; 326. Second filter; 327. Second dichroic beam splitter; 328. Third dichroic beam splitter; 329. Receiving filter; 41. Changing mechanism; 411. Pinhole wheel; 42. Replacement pinhole; 43. Condensing lens; 44. Displacement mechanism. Detailed Implementation

[0029] The following will be combined with the appendix Figures 1-2 The technical solutions in the embodiments of the present invention are further described in detail below. The described embodiments are only possible technical implementations of the present invention, but are not limited thereto. Those skilled in the art can certainly combine the embodiments of the present invention to obtain other embodiments without creative effort, which are also within the protection scope of the present invention.

[0030] This application mainly uses the main pinhole 22 to realize the illumination and detection functions, improve the imaging stability, and achieve the effect of reducing the number of conjugate devices and improving imaging resolution and stability. The following is a further detailed description of this application.

[0031] Example 1 Reference Figure 1 A confocal microscope pinhole imaging system includes a laser generating module 11, a first collimating lens 12, a focusing lens 13, a first dichroic beam splitter 21, a main pinhole 22, a collimating lens 23, a scanning module 24, an objective lens 25, a second collimating lens 31, and a receiving imaging module 32.

[0032] The laser generating module 11 is a semiconductor laser generator. The laser's emission port is connected to a single-mode fiber. The laser is first injected into the single-mode fiber through a fiber coupler and then directly emitted from the end face of the single-mode fiber, serving as a point source in the subsequent optical path. The main pinhole 22 is conjugate to the focal plane of the objective lens 25. The incident laser is transmitted to the first dichroic beam splitter 21 after passing through the first collimating lens 12 and focusing lens 13. The first dichroic beam splitter 21 transmits the laser and reflects fluorescence. The common optical path formed by the first dichroic beam splitter 21, main pinhole 22, collimating lens 23, scanning module 24, and objective lens 25 is shared by the laser and fluorescence. The incident laser, passing through the first dichroic beam splitter 21, is transmitted along this common optical path to the sample slice. The sample generates reflected fluorescence, which is transmitted along the common optical path to the first dichroic beam splitter 21 and reflected by it to the second collimating lens 31. The receiving imaging module 32 receives and images the reflected fluorescence.

[0033] During the confocal microscope imaging process, the incident laser is generated by the laser generating module 11 and transmitted through the focusing lens 13 and the first collimating lens 12 to the first dichroic beam splitter 21. The light then passes through the first dichroic beam splitter 21 and is transmitted along the common optical path to the sample, generating reflected fluorescence. The reflected fluorescence is transmitted along the common optical path to the first dichroic beam splitter 21, and then splits and passes through the second collimating lens 31 to the receiving imaging module 32, completing the confocal microscope imaging process. During the imaging process, the incident laser is actively filtered by the main pinhole 22 to form a point light source, and the reflected fluorescence is filtered by the main pinhole 22 and received by the receiving imaging module 32, so that the main pinhole 22 simultaneously serves as both an illumination pinhole and a detection pinhole, achieving strict conjugation of the pinholes. Therefore, it is only necessary to ensure that the main pinhole 22 is conjugated with the objective lens 25, reducing the number of conjugated devices in the confocal microscope and improving the resolution of the microscope imaging. This is less affected by the displacement of the conjugated devices and improves the imaging stability of the confocal microscope. When the incident laser passes through the main pinhole 22, it becomes closer to a point light source, thereby improving the imaging resolution of the confocal microscope.

[0034] The scanning module 24 in this embodiment includes an XY scanning galvanometer 241 and a scanning lens 242. The XY scanning galvanometer 241 comprises two scanning galvanometers with mutually perpendicular rotation axes. The incident laser is reflected onto the scanning galvanometer, and through two reflections, the incident laser is transmitted downwards. After passing through the XY scanning galvanometer 241, the incident laser beam is expanded by the scanning lens 242. The expanded parallel light is focused into a point by the objective lens 25 to excite the probe, forming the focal plane of the objective lens 25 on the sample. By controlling the laser to perform two-dimensional scanning on the sample using the XY scanning galvanometer 241, the scanning range of the laser on the sample can be expanded, and sample information can be acquired more comprehensively, thereby improving the integrity and accuracy of confocal microscopy imaging.

[0035] The receiving imaging module 32 in this embodiment includes a filter wheel 321, a first fluorescent PMT 322, a second fluorescent PMT 323, a first filter 324, a third fluorescent PMT 325, and a second filter 326. A second dichroic beam splitter 327 and a third dichroic beam splitter 328 are provided in the optical path of the reflected fluorescence after passing through the second collimating lens 31. The incident fluorescence is transmitted to the filter wheel 321 through the second and third dichroic beam splitters 327 and 328. In this embodiment, the filter wheel 321 is a six-aperture filter wheel 321, with six apertures. Four of these apertures contain receiving filters 329, each with a different center wavelength: 405 nm, 488 nm, 561 nm, and 640 nm, respectively, enabling the reception of fluorescence of different wavelengths.

[0036] The first fluorescence PMT 322 is positioned behind the filter wheel 321. Fluorescence passing through the receiving filter 329 is received by the first fluorescence PMT 322, which converts the optical signal into an electrical signal and applies a certain magnification to display the image information of the sample on the focal plane of the objective lens 25. Multiple receiving filters 329 with different center wavelengths on the filter wheel 321 can be individually adjusted to the reflected fluorescence path, enabling the first fluorescence PMT 322 to receive reflected fluorescence filtered at different center wavelengths, significantly enhancing the system's adaptability and selectivity to reflected fluorescence of different wavelengths.

[0037] The second fluorescence PMT323, the first filter 324, and the second dichroic beam splitter 327 are arranged opposite each other. The second dichroic beam splitter 327 separates multicolor fluorescence and reflects one color of fluorescence to the first filter 324. The center wavelength of the first filter 324 is not opposite to the center wavelength of the four receiving filters 329 on the filter wheel 321. The first filter 324 filters the fluorescence of the target wavelength. The second fluorescence PMT323 receives the fluorescence filtered by the first filter 324 and performs imaging operation.

[0038] The third fluorescence PMT 325, the second filter 326, and the third dichroic beam splitter 328 are positioned opposite each other. The third dichroic beam splitter 328 separates multicolor fluorescence and reflects one color of fluorescence to the second filter 326. The center wavelength of the second filter 326 is not opposite to the center wavelengths of the four receiving filters 329 and the first filter 324 on the filter wheel 321. The second filter 326 filters fluorescence of the target wavelength. The third fluorescence PMT 325 receives the fluorescence filtered by the second filter 326 and performs imaging operations.

[0039] By using a second dichroic beam splitter 327 to reflect a portion of the reflected fluorescence onto a first filter 324, a second fluorescence PMT 323 receives the reflected fluorescence passing through the first filter 324, and a third dichroic beam splitter 328 to reflect a portion of the reflected fluorescence onto a second filter 326, a third fluorescence PMT 325 receives the reflected fluorescence passing through the second filter 326. This allows for the separate reception and processing of different portions of the reflected fluorescence, increasing the detection dimensions of the reflected fluorescence, further enriching the imaging information, and improving the accuracy and comprehensiveness of the imaging.

[0040] The implementation principle of Example 1 is as follows: During the confocal microscope imaging process, the incident laser generated by the laser generating module 11 is first collimated into parallel light by the first collimating lens 12, and then focused by the focusing lens 13 onto the main pinhole 22 to form a point light source. The light from the point light source is reflected by the first dichroic beam splitter 21 into the common optical path, and then illuminates the sample through the scanning module 24 and the objective lens 25, generating reflected fluorescence. The reflected fluorescence returns along the common optical path, passes through the objective lens 25, the scanning module 24, and the collimating lens 23 again, and after reaching the first dichroic beam splitter 21, it is transmitted to the receiving imaging module 32. In this process, the main pinhole 22 simultaneously serves as both an illumination pinhole and a detection pinhole, achieving strict conjugation of the pinholes. It is only necessary to ensure that the main pinhole 22 is conjugated with the objective lens 25, reducing the number of conjugated devices and reducing the impact of conjugated device position deviation on imaging resolution, thereby improving the imaging stability of the confocal microscope. At the same time, the incident laser is closer to a point light source when passing through the main pinhole 22, improving the imaging resolution. The receiving imaging module 32 can detect and image reflected fluorescence of different wavelengths by means of beam splitting and filtering, thereby improving the imaging efficiency and information acquisition capability of the system.

[0041] Example 2 Reference Figure 2 The difference between this embodiment and Embodiment 1 is that the confocal microscope imaging system in this embodiment also includes a switching mechanism 41, which is a pinhole wheel 411. The main pinhole 22 is set on the pinhole wheel 411. In addition, the pinhole wheel 411 is also provided with multiple replacement pinholes 42 of different aperture sizes. In order to balance spatial resolution and signal strength / signal-to-noise ratio, the aperture size of the pinhole needs to be adjusted. By rotating the pinhole wheel 411, the positions of the replacement pinholes 42 of the corresponding aperture size and the main pinhole 22 are switched, thereby realizing the adjustment of pinholes of different aperture sizes to adapt to different samples or imaging requirements, and enhancing the applicability and flexibility of the confocal microscope pinhole imaging system.

[0042] To improve the concentration of the reflected fluorescence beam, a condenser lens 43 is provided in the optical path between the second collimating lens 31 and the first dichroic beam splitter 21. The condenser lens 43 is a convex lens. The fluorescence beam reflected by the first dichroic beam splitter 21 is focused by the condenser lens 43 and then collimated into parallel light by the second collimating lens 31. The second collimating lens 31 is mounted on the displacement mechanism 44. In this embodiment, the displacement mechanism 44 is a lead screw and nut mechanism. The displacement mechanism 44 drives the second collimating lens 31 to move closer to or further away from the condenser lens 43, adjusting the distance between the second collimating lens 31 and the condenser lens 43. The maximum distance between the second collimating lens 31 and the condenser lens 43 is less than the focal length of the condenser lens 43. As the aperture of the pinhole is adjusted, the diameter of the corresponding fluorescence beam also changes. The distance between the second collimating lens 31 and the condenser lens 43 is adjusted by the displacement mechanism 44, so that the second collimating lens 31 contacts the fluorescence cone surface after focusing by the condenser lens 43 at different positions, so that the second collimating lens 31 collimates into fluorescence beams of different diameters, in order to meet the needs of different imaging resolutions, further improve the imaging quality and the adaptability of the system.

[0043] The implementation principle of Example 2 is as follows: a condenser lens 43 is set in the optical path of the reflected fluorescence, and the distance between the second collimating lens 31 and the condenser lens 43 is adjusted by the displacement mechanism 44. With the change of the aperture of the replacement pinhole 42, the focusing state of the reflected fluorescence can be flexibly adjusted to adapt to different imaging requirements, thereby further improving the imaging quality and the adaptability of the system.

[0044] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A confocal microscope pinhole imaging system, characterized in that: The system includes a laser generating module (11), a focusing lens (13), a first dichroic beam splitter (21), a main pinhole (22), a collimating lens (23), a scanning module (24), an objective lens (25), and a receiving imaging module (32). The main pinhole (22) and the objective lens (25) are set conjugately. The collimating lens (23) is adapted to the main pinhole (22). The focusing lens (13) can focus light onto the main pinhole (22). The collimating lens (23) can collimate the light passing through the main pinhole (22) into parallel light. The first dichroic beam splitter (21), main pinhole (22), collimating lens (23), scanning module (24), and objective lens (25) form a common optical path. The incident laser passes through the focusing lens (13) and is transmitted to the sample along the common optical path. The reflected fluorescence is transmitted to the receiving imaging module (32) along the common optical path for imaging.

2. The confocal microscope pinhole imaging system according to claim 1, characterized in that: A first collimating lens (12) is provided between the laser generating module (11) and the focusing lens (13), and the first collimating lens (12) can collimate the incident laser into parallel light.

3. The confocal microscope pinhole imaging system according to claim 1, characterized in that: A second collimating lens (31) is provided between the first dichroic beam splitter (21) and the receiving imaging module (32). The second collimating lens (31) can collimate the reflected fluorescence into parallel light.

4. The confocal microscope pinhole imaging system according to claim 3, characterized in that: The receiving imaging module (32) includes a filter wheel (321) and a first fluorescence PMT (322). The filter wheel (321) is provided with a plurality of receiving filters (329) with different center wavelengths. The filter wheel (321) can adjust the receiving filters (329) one by one to the optical path of the reflected fluorescence. The first fluorescence PMT (322) can receive the reflected fluorescence transmitted through the receiving filters (329).

5. The confocal microscope pinhole imaging system according to claim 4, characterized in that: The receiving imaging module (32) further includes a second fluorescent PMT (323) and a first filter (324). A second dichroic beam splitter (327) is provided in the optical path of the reflected fluorescence between the first fluorescent PMT (322) and the second collimating lens (31). The second dichroic beam splitter (327) can reflect part of the reflected fluorescence to the first filter (324). The second fluorescent PMT (323) can receive the reflected fluorescence passing through the first filter (324).

6. The confocal microscope pinhole imaging system according to claim 5, characterized in that: The receiving imaging module (32) further includes a third fluorescent PMT (325) and a second filter (326). A third dichroic beam splitter (328) is provided in the optical path of reflected fluorescence between the first fluorescent PMT (322) and the second dichroic beam splitter (327). The third dichroic beam splitter (328) can reflect part of the reflected fluorescence to the second filter (326). The third fluorescent PMT (325) can receive the reflected fluorescence passing through the second filter (326). The center wavelength of the second filter (326) is different from the center wavelength of the first filter (324).

7. The confocal microscope pinhole imaging system according to claim 1, characterized in that: The scanning module (24) includes an XY scanning galvanometer (241) and a scanning lens (242). Light is transmitted sequentially through the XY scanning galvanometer (241) and the scanning lens (242) to the objective lens (25). The XY scanning galvanometer (241) is used to control the two-dimensional scanning of the laser on the sample.

8. The confocal microscope pinhole imaging system according to claim 1, characterized in that: It also includes a switching mechanism (41), which has multiple replacement pinholes (42) with different apertures. The main pinhole (22) is located on the switching mechanism (41). The switching mechanism (41) can switch the positions of the replacement pinhole (42) and the main pinhole (22), and set the replacement pinholes (42) one by one on the common optical path.

9. The confocal microscope pinhole imaging system according to claim 8, characterized in that: A condenser lens (43) is provided in the optical path between the second collimating lens (31) and the first dichroic beam splitter (21). The second collimating lens (23) is externally connected to a displacement mechanism (44), which can adjust the distance between the second collimating lens (31) and the condenser lens (43).

10. The confocal microscope pinhole imaging system according to claim 9, characterized in that: The maximum distance between the second collimating lens (31) and the condenser lens (43) is less than the focal length of the condenser lens (43).

Images