A high-precision spectral confocal microscopy system

Abstract

The present invention discloses a high-precision spectral confocal microscopy imaging system, comprising a light modulation module, a spectrum splitting module, a scanning imaging module, an image detection device and a computer. The light beam emitted by the light modulation module passes through the scanning imaging module and is incident on a sample to excite a fluorescence signal. The fluorescence signal passes through the spectrum splitting module and is received by the image detection device. The image detection device converts the current signal into a digital signal and transmits it to the computer to complete imaging. The system is characterized in that the spectrum splitting module consists of a first converging lens, a first pinhole, a first linear long-wave pass filter, a second converging lens, a first linear short-wave pass filter and a third converging lens arranged in sequence. The system has the advantage of realizing high-precision spectral confocal imaging by combining lenses and linear filters on the basis of the traditional confocal microscopy imaging principle, solving the problem of spectral crosstalk in confocal imaging, and achieving a spectral resolution of less than 3.0 nm.

Description

Technical Field

[0001] The present invention relates to a confocal microscopic imaging system, in particular to a high-precision spectral confocal microscopic imaging system. Background Art

[0002] In recent years, confocal microscopes have become one of the most widely used fluorescence microscopes due to their high signal-to-noise ratio and good layer-cutting performance. They play an increasingly important role, especially in basic research in biology and medicine. At present, the demand for studying the interaction mechanisms between cells and organelles has increased dramatically. It is necessary to use a variety of fluorescent proteins to label different organelles and observe their morphological changes, transmission mechanisms, and interactions in real time under a confocal microscope. However, when using multiple fluorescent probes to label imaging, the fluorescence spectra of different fluorophores are very easy to overlap, resulting in fluorescence signal crosstalk, which in turn distorts the image. Traditional confocal microscopes cannot overcome the problem of spectral crosstalk. Summary of the Invention

[0003] The object of the present invention is to provide a high-precision spectral confocal microscopy system that can solve the spectral crosstalk problem and achieve nanometer-level spectral resolution.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: a high-precision spectral confocal microscopy imaging system, including a light modulation module, a spectrum splitting module, a scanning imaging module, an image detection device and a computer, wherein the light beam emitted by the light modulation module passes through the scanning imaging module and is incident on the sample to excite a fluorescence signal, and the fluorescence signal is received by the image detection device after passing through the spectrum splitting module. The image detection device converts the current signal into a digital signal and transmits it to the computer to complete imaging, and the spectrum splitting module is composed of a first converging lens, a first pinhole, a first linear long-wave pass filter, a second converging lens, a first linear short-wave pass filter and a third converging lens arranged in sequence, the first converging lens converges the fluorescence signal at the center of the first pinhole and is incident on the first linear long-wave pass filter, the fluorescence signal emitted from the first linear long-wave pass filter passes through the second converging lens and is converged on the first linear short-wave pass filter, and the fluorescence signal emitted from the first linear short-wave pass filter passes through the third converging lens and converges on the image detection device.

[0005] Compared with the existing technology, the advantage of the present invention is that on the basis of the traditional confocal microscopy principle, a combination of lenses and linear filters is used to achieve high-precision spectral confocal imaging, which solves the problem of spectral crosstalk in confocal imaging and achieves a spectral resolution of less than 3.0 nm.

[0006] Preferably, the scanning imaging module is composed of a first dichroic mirror, a two-dimensional scanning device, a second reflector, a scanning lens, a tube lens, an objective lens and a sample stage for placing a sample, which are arranged in sequence. The light beam emitted by the light modulation module is transmitted through the first dichroic mirror, and is two-dimensionally scanned by the two-dimensional scanning device. The light beam is vertically incident on the scanning lens through the second reflector, and is collimated into a parallel light beam by the tube lens. The light beam is converged on the sample stage through the objective lens. The first dichroic mirror is used to transmit the light beam emitted by the light modulation module and reflect a fluorescence signal. The light beam emitted by the light modulation module is transmitted through the first dichroic mirror, and is two-dimensionally scanned by the two-dimensional scanning device. The light beam excites the sample on the sample stage to generate a fluorescence signal. The fluorescence signal is collimated into parallel light through the objective lens, the tube lens, the scanning lens, the second reflector and the two-dimensional scanning device in sequence. The light beam is reflected by the first dichroic mirror and enters the first converging lens.

[0007] Preferably, the scanning imaging module comprises a two-dimensional scanning device, a second dichroic mirror, a scanning lens, a tube lens, an objective lens, and a sample stage for placing a sample, arranged in sequence. The second dichroic mirror is used to reflect the light beam emitted by the light modulation module and transmit the fluorescence signal. The light beam emitted by the light modulation module is incident on the two-dimensional scanning device to achieve two-dimensional scanning of the light beam. It is then reflected by the second dichroic mirror and perpendicularly incident on the scanning lens. After being collimated into a parallel beam by the tube lens, it is converged by the objective lens onto the sample stage, thereby exciting the sample on the sample stage to produce a fluorescence signal. The fluorescence signal passes through the objective lens, the tube lens, and the scanning lens in the original path, and then transmits through the second dichroic mirror and enters the first converging lens. Due to the weak fluorescence of biological samples and the nanometer-level spectral resolution of spectral confocal imaging, the light intensity during imaging is relatively weak. This solution, by designing a non-de-scanning optical path, eliminates the need for the fluorescence signal generated by the sample to pass through the two-dimensional scanning device with lower reflectivity, thereby improving the fluorescence detection efficiency within the system and reducing phototoxicity.

[0008] Preferably, the two-dimensional scanning device is provided with a scanning imaging monitoring module, and the scanning imaging monitoring module is used to monitor the performance of the two-dimensional scanning device in real time.

[0009] Preferably, the scanning imaging monitoring module is composed of an LED light source, a third lens, a third reflector, a second dichroic prism, a fourth lens and a second camera arranged in sequence. The light beam emitted by the LED light source is collimated into parallel light by the third lens, passes through the third reflector to reach the second dichroic prism and is transmitted. The transmitted light beam is irradiated on the two-dimensional scanning device. The light beam reflected back by the two-dimensional scanning device is reflected by the second dichroic prism and is imaged on the second camera through the fourth lens. The second camera transmits real-time data back to the computer to monitor the performance of the two-dimensional scanning device in real time.

[0010] Preferably, the light modulation module is composed of a multi-wavelength laser, a multi-mode optical fiber, a collimating lens, a first reflector, a beam shaping device, a first camera, and a first stray light filtering device arranged in sequence. The light beam emitted by the multi-wavelength laser is collimated into a Gaussian parallel beam by the multi-mode optical fiber and the collimating lens. The output angle of the Gaussian parallel beam is adjusted by the first reflector so that the output beam is parallel to the optical axis. The beam shaping device converts the Gaussian parallel beam into a flat-top beam. After the flat-top beam passes through the first beam splitter prism, the transmitted flat-top beam passes through the first stray light filtering device and enters the scanning imaging module. The reflected flat-top beam is imaged on the first camera. The first camera transmits real-time data back to the computer for real-time monitoring of the spatial two-dimensional intensity distribution of the flat-top beam.

[0011] Preferably, the first stray light filtering device is composed of a first lens, a second pinhole and a second lens. The transmitted light beam is converged at the center of the second pinhole through the first lens, and is collimated by the second lens before entering the scanning imaging module.

[0012] Preferably, the light modulation module comprises a white light LED light source, a multimode optical fiber, a collimating lens, a first reflector, a beam shaping device, a second stray light filtering device, a first beam splitter prism, and a first camera, arranged in sequence. The illumination beam emitted by the white light LED light source is collimated into a Gaussian parallel beam by the multimode optical fiber and the collimating lens. The collimated parallel beam is adjusted by the first reflector at an output angle parallel to the optical axis. The beam shaping device converts the Gaussian parallel beam into a flat-top beam. The flat-top beam passes through the second stray light filtering device and is incident on the first beam splitter prism. The transmitted flat-top beam is incident on the scanning imaging module and the reflected flat-top beam is imaged on the first camera. The first camera transmits real-time data back to the computer for real-time monitoring of the spatial two-dimensional intensity distribution of the flat-top beam. The combination of the white light LED light source, the lens, and the linear filter allows for arbitrary adjustment of the central wavelength and bandwidth of the illumination beam within the visible light range, expanding the range of illumination beam options and product applications while reducing costs.

[0013] Preferably, the second stray light filtering device is composed of a first lens, a second linear long-wave pass filter, a fourth converging lens, a second pinhole, a second linear short-wave pass filter and a second lens arranged in sequence. The first lens converges the light beam on the second linear long-wave pass filter, the light beam emitted from the second linear long-wave pass filter is converged at the center of the second pinhole through the fourth converging lens and is incident on the second linear short-wave pass filter, and the light beam emitted from the second linear short-wave pass filter is incident on the first dichroic prism through the second lens. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the structure of the backscan high-precision spectral confocal microscopy imaging system according to the first embodiment of the present invention;

[0015] Figure 2 This is a spectral resolution test chart of the backscan high-precision spectral confocal microscopy imaging system according to the first embodiment of the present invention;

[0016] Figure 3 This is a schematic structural diagram of a non-de-scanning high-precision spectral confocal microscopy imaging system according to a second embodiment of the present invention;

[0017] Figure 4 This is an optical path diagram of a backscan high-precision spectral confocal microscopy imaging system with adjustable illumination beam center wavelength and bandwidth according to a third embodiment of the present invention;

[0018] Figure 5 This is an optical path diagram of a non-de-scanning high-precision spectral confocal microscopic imaging system with adjustable central wavelength and bandwidth of the illumination beam according to the fourth embodiment of the present invention.

[0019] Description of reference numerals:

[0020] 1-1, multi-wavelength laser; 1-2, white light LED light source; 2, multimode optical fiber; 3, collimating lens; 4, first reflector; 5, beam shaping device; 6, first beam splitter; 7, first camera; 8, first lens; 9, second pinhole; 10, second lens; 11-1, first dichroic mirror; 11-2, second dichroic mirror; 12, two-dimensional scanning device; 13, second reflector; 14, scanning lens; 15, tube lens; 16, objective lens; 17, sample stage; 18, first Converging lens; 19. First pinhole; 20. First linear long-wave pass filter; 21. Second converging lens; 22. First linear short-wave pass filter; 23. Third converging lens; 24. Image detection device; 25. Computer; 26. LED light source; 27. Third lens; 28. Third reflector; 29. ​​Second spectroscopic prism; 30. Fourth lens; 31. Second camera; 32. Second linear long-wave pass filter; 33. Fourth converging lens; 34. Second linear short-wave pass filter. DETAILED DESCRIPTION

[0021] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0023] Example 1: Figure 1This is a schematic diagram of the structure of the backscan high-precision spectral confocal microscopy imaging system provided in the first embodiment of the present invention. The light modulation module is composed of the following components: a multi-wavelength laser 1-1, a multimode optical fiber 2, a collimating lens 3, a first reflector 4, a beam shaping device 5, a first beam splitter prism 6, a first camera 7, a first lens 8, a second pinhole 9, and a second lens 10; the scanning imaging module is composed of the following components: a first dichroic mirror 11-1 for transmitting the light beam emitted by the multi-wavelength laser 1-1 and reflecting the fluorescence signal, a two-dimensional scanning device 12, a second Reflector 13, scanning lens 14, tube lens 15, objective lens 16, sample stage 17; spectrum splitting module consists of the following components: first converging lens 18, first pinhole 19, first linear long-wave pass filter 20, second converging lens 21, first linear short-wave pass filter 22, third converging lens 23; image detection device 24, computer 25, scanning imaging monitoring module consists of the following components: LED light source 26, third lens 27, third reflector 28, second dichroic prism 29, fourth lens 30 and second camera 31.

[0024] The imaging process of the backscan high-precision spectral confocal microscopy imaging system of the first embodiment is as follows:

[0025] Laser light emitted by multi-wavelength laser 1-1 is collimated into Gaussian parallel light through multimode fiber 2 and collimating lens 3. The collimated beam passes through first reflector 4 to adjust its exit angle, aligning it with the optical axis. After passing through beam shaping device 5, the two-dimensional distribution of the beam changes from Gaussian to flat-top, resulting in a more uniform light spot. The beam passes through first beam-splitting prism 6 and is split into two parts. One part is reflected and imaged by first camera 7, which allows real-time monitoring of the spatial two-dimensional intensity distribution of the illumination beam. The other part is transmitted through first lens 8 and converged at the center of second pinhole 9 to filter out stray light. The light beam is collimated again by the second lens 10 and incident on the first dichroic mirror 11-1. The light beam transmits through the first dichroic mirror 11-1, passes through the two-dimensional scanning device 12 to realize two-dimensional scanning of the light beam, passes through the second reflector 13 and vertically enters the scanning lens 14, is collimated into a parallel light beam by the tube lens 15, and is converged on the sample stage 17 by the objective lens 16. The laser excites the sample to generate a fluorescence signal. The fluorescence signal passes through the objective lens 16, the tube lens 15, the scanning lens 14, the second reflector 13, and the two-dimensional scanning device 12 in the original path, is collimated into parallel light, and is reflected by the first dichroic mirror 11-1 and enters the spectrum splitting module. In the spectrum splitting module, The fluorescence signal passes through the first converging lens 18 and converges at the center of the first pinhole 19. The first pinhole 19 can effectively filter out stray light. A first linear long-wave pass filter 20 is placed behind the first pinhole 19. The fluorescence signal passes through the first linear long-wave pass filter 20 and the second converging lens 21 and converges on the first linear short-wave pass filter 22. The combination of the linear long-wave pass filter and the short-wave pass filter can realize the spectral splitting of the fluorescence signal. The fluorescence signal emitted from the first linear short-wave pass filter 22 passes through the third converging lens 23 and converges on the image detection device 24. The current signal is converted into a digital signal and transmitted to the computer 25 to complete the imaging. In addition, the light beam emitted by the LED light source 26 is collimated into parallel light by the third lens 27, passes through the third reflector 28, and is transmitted through the second dichroic prism 29. The light beam is irradiated on the two-dimensional scanning device 12. The light beam reflected back by the two-dimensional scanning device 12 is reflected by the second dichroic prism 29, passes through the fourth lens 30, and is imaged on the second camera 31. The real-time data is transmitted back to the computer 25 to realize imaging, which can realize real-time monitoring of the performance of the two-dimensional scanning device 12 so that the back-end can perform scanning control compensation and image distortion calibration. The high-precision spectral confocal microscopy imaging system of this embodiment can realize spectral separation in the visible light range, such as Figure 2 Shown is the transmission curve of the beam through the linear filter with a spectral resolution of 2.60 nm.

[0026] Example 2: Figure 3 This is a schematic diagram of the structure of the non-de-scanning high-precision spectral confocal microscopy imaging system provided in the second embodiment. Figure 3As can be seen, the light modulation module, spectrum splitting module, scanning imaging monitoring module, image detection device 24 and computer 25 in Example 2 are the same as those in Example 1, except that the scanning imaging module is composed of the following components: a two-dimensional scanning device 12, a second dichroic mirror 11-2 for reflecting the light beam emitted by the multi-wavelength laser 1-1 and transmitting the fluorescent signal, a scanning lens 14, a tube lens 15, an objective lens 16, and a sample stage 17.

[0027] The non-de-scanning high-precision spectral confocal microscopy imaging system of the second embodiment is used, and the imaging process is as follows:

[0028] Laser light emitted by multi-wavelength laser 1-1 is collimated into Gaussian parallel light through multimode fiber 2 and collimating lens 3. The collimated beam passes through first reflector 4 to adjust its exit angle, aligning it with the optical axis. After passing through beam shaping device 5, the two-dimensional distribution of the beam changes from Gaussian to flat-top, resulting in a more uniform light spot. The beam passes through first beam-splitting prism 6 and is split into two parts. One part is reflected and imaged by first camera 7, which allows real-time monitoring of the spatial two-dimensional intensity distribution of the illumination beam. The other part is transmitted through first lens 8 and converged at the center of second pinhole 9 to filter out stray light. The light beam is collimated again by the second lens 10, and after being scanned in two dimensions by the two-dimensional scanning device 12, it is reflected by the second dichroic mirror 11-2 and vertically enters the scanning lens 14. It is collimated into a parallel light beam by the tube lens 15, and converged on the sample stage 17 through the objective lens 16. The laser excites the sample to produce a fluorescence signal. The fluorescence signal passes through the objective lens 16, the tube lens 15, and the scanning lens 14 in the original path, and is transmitted through the second dichroic mirror 11-2 and enters the spectrum splitting module. In the spectrum splitting module, the fluorescence signal passes through the first converging lens 18 and converges into the first pinhole 19. The first pinhole 19 can effectively filter out stray light. A first linear long-wave pass filter 20 is placed behind the first pinhole 19. The fluorescence signal passes through the first linear long-wave pass filter 20 and the second converging lens 21, and is converged onto the first linear short-wave pass filter 22. The combination of the linear long-wave pass filter and the short-wave pass filter can achieve spectral separation of the fluorescence signal. The fluorescence signal emitted from the first linear short-wave pass filter 22 passes through the third converging lens 23 and is converged onto the image detection device 24. The current signal is converted into a digital signal and transmitted to the computer 25 to complete the imaging. In addition, the light beam emitted by the LED light source 26 is collimated into parallel light by the third lens 27, passes through the third reflector 28, and is transmitted through the second beam splitter prism 29. The light beam is irradiated onto the two-dimensional scanning device 12. The light beam reflected back from the two-dimensional scanning device 12 is reflected by the second beam splitter prism 29, passes through the fourth lens 30, and is imaged on the second camera 31. The real-time data is transmitted back to the computer 25 to complete the imaging. This allows for real-time monitoring of the performance of the two-dimensional scanning device 12, facilitating back-end scanning control compensation and image distortion calibration.

[0029] Compared with the first embodiment, the solution of the second embodiment improves the fluorescence detection efficiency of the entire system, reduces the phototoxicity of the illumination light path, and can further expand the application range of the product.

[0030] Example 3: Figure 4 This is a schematic diagram of the structure of the backscan high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on white light LEDs provided in the third embodiment. Figure 4 As can be seen, the scanning imaging module, spectrum splitting module, scanning imaging monitoring module, image detection device 24 and computer 25 in Example 3 are the same as those in Example 1, except that the light modulation module is composed of the following components: white light LED light source 1-2, multimode optical fiber 2, collimating lens 3, first reflector 4, beam shaping device 5, first lens 8, second linear long-wave pass filter 32, fourth converging lens 33, second pinhole 9, second linear short-wave pass filter 34, second lens 10, first beam splitter prism 6, and first camera 7.

[0031] The imaging process of the backscan high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on white light LEDs of the third embodiment is as follows:

[0032] The illumination beam emitted by the white light LED light source 1-2 is collimated into Gaussian parallel light through the multimode optical fiber 2 and the collimating lens 3. The collimated beam passes through the first reflector 4 to adjust its output angle so that the beam is parallel to the optical axis. After passing through the beam shaping device 5, the two-dimensional distribution of the beam changes from Gaussian to flat top, and the light spot is more uniform. The light beam passes through the first lens 8 and converges on the second linear long-wave pass filter 32, passes through the fourth converging lens 33 and converges at the center of the second pinhole 9 to be incident on the second linear short-wave pass filter 34. The second linear short-wave pass filter 34 is placed behind the second pinhole 9, and the light beam emitted by the second linear short-wave pass filter 34 is collimated into a parallel light beam by the second lens 10. The second linear long-wave pass filter 32 and the second linear short-wave pass filter 34 are precisely moved by a closed-loop controlled motor, and the central wavelength and bandwidth of the illumination light beam can be arbitrarily adjusted through the bidirectional precision movement of the two linear filters. The light beam is incident on the first beam splitter prism 6 and is divided into two parts by the first beam splitter prism 6. One part of the light beam is reflected and imaged on the first camera 7. The spatial two-dimensional intensity distribution of the illumination light beam can be monitored in real time through the first camera 7. The other part of the light beam is incident on the first dichroic mirror 11-1 after transmission. The light beam is transmitted through the first dichroic mirror 11-1 and passes through the two-dimensional scanning device 12 to realize two-dimensional scanning of the light beam, and passes through the second reflector 13 for vertical incident scanning. The scanning lens 14 is collimated into a parallel light beam by the tube lens 15, and converged on the sample stage 17 through the objective lens 16. The white light excites the sample to produce a fluorescence signal. The fluorescence signal passes through the objective lens 16, the tube lens 15, the scanning lens 14, the second reflector 13, and the two-dimensional scanning device 12 in the original path to be collimated into parallel light, and is reflected by the first dichroic mirror 11-1 and enters the spectrum splitting module. In the spectrum splitting module, the fluorescence signal passes through the first converging lens 18 and converges on the center of the first pinhole 19. The first pinhole 19 can effectively filter out stray light. A first linear long-wave pass filter 20 is placed behind the first pinhole 19. The fluorescence signal passes through the first linear long-wave pass filter 20 and the second converging lens 21 and converges on the first linear short-wave pass filter 22. The combination of the linear long-wave pass filter and the short-wave pass filter can realize spectral splitting of the fluorescence signal. The fluorescence signal emitted from the first linear short-wave pass filter 22 passes through the third converging lens 23 and converges on the image detection device 24. The current signal is converted into a digital signal and transmitted to the computer 25 to complete imaging. In addition, the light beam emitted by the LED light source 26 is collimated into parallel light by the third lens 27, passes through the third reflector 28, and is transmitted at the second dichroic prism 29. The light beam is irradiated on the two-dimensional scanning device 12. The light beam reflected back by the two-dimensional scanning device 12 is reflected by the second dichroic prism 29, passes through the fourth lens 30 and is imaged on the second camera 31. The real-time data is transmitted back to the computer 25 to realize imaging, which can realize real-time monitoring of the performance of the two-dimensional scanning device 12 so that the back-end can perform scanning control compensation and image distortion calibration.

[0033] Compared with the first embodiment, the solution of the third embodiment can excite different fluorescent markers to their strongest peak intensity, improve the signal-to-noise ratio of the system, reduce the phototoxicity of the illumination light path, and further expand the application range of the product.

[0034] Example 4: Figure 5 The schematic diagram of the structure of the non-de-scanning high-precision spectral confocal microscopy imaging system based on the white light LED illumination beam center and adjustable bandwidth provided in this embodiment is as follows: Figure 5 It can be seen that the light modulation module in the fourth embodiment is the same as that in the third embodiment, and the scanning imaging module, spectrum splitting module, scanning imaging monitoring module, image detection device 24 and computer 25 are the same as those in the second embodiment.

[0035] The non-de-scanning high-precision spectral confocal microscopic imaging system with adjustable illumination beam center and bandwidth based on white light LED of the fourth embodiment is used, and the imaging process is as follows:

[0036] The illumination beam emitted by white light LED source 1-2 is collimated into Gaussian parallel light through multimode fiber 2 and collimating lens 3. The collimated beam passes through first reflector 4 to adjust its exit angle, making the beam parallel to the optical axis. After passing through beam shaping device 5, the two-dimensional distribution of the beam changes from Gaussian to flat-top, making the spot more uniform. The beam passes through first lens 8 and converges onto second linear long-wavepass filter 32. Then, through fourth converging lens 33, it converges onto the center of second pinhole 9 and is incident on second linear short-wavepass filter 34. Second linear short-wavepass filter 34 is placed behind second pinhole 9. The second linear long-wavepass filter 32 and second linear short-wavepass filter 34 are precisely moved by a closed-loop controlled motor. The precise bidirectional movement of the two linear filters allows for arbitrary adjustment of the central wavelength and bandwidth of the illumination beam. The light beam emitted by the second linear short-wave pass filter 34 is collimated into a parallel light beam by the second lens 10 and enters the first beam splitter prism 6. It is divided into two parts by the first beam splitter prism 6. One part of the light beam is reflected and imaged on the first camera 7. The spatial two-dimensional intensity distribution of the illumination light beam can be monitored in real time through the first camera 7. The other part of the light beam is transmitted and passes through the two-dimensional scanning device 12 to realize two-dimensional scanning of the light beam. After that, it is reflected by the second dichroic mirror 11-2 and vertically enters the scanning lens 14. It is collimated into a parallel light beam by the tube lens 15 and converged on the sample stage 17 through the objective lens 16. The white light excites the sample to generate a fluorescence signal. The fluorescence signal passes through the objective lens 16, the tube lens 15, and the scanning lens 14 in the original path, and is transmitted through the second dichroic mirror After the mirror 11-2, it enters the spectrum splitting module. In the spectrum splitting module, the fluorescence signal passes through the first converging lens 18 and converges on the center of the first pinhole 19. The first pinhole 19 can effectively filter out stray light. A first linear long-wave pass filter 20 is placed behind the first pinhole 19. The fluorescence signal passes through the first linear long-wave pass filter 20 and the second converging lens 21 and converges on the first linear short-wave pass filter 22. The combination of the linear long-wave pass filter and the short-wave pass filter can realize the spectrum splitting of the fluorescence signal. The fluorescence signal emitted from the first linear short-wave pass filter 22 passes through the third converging lens 23 and converges on the image detection device 24. The current signal is converted into a digital signal and transmitted to the computer 25 to complete the imaging. In addition, the light beam emitted by the LED light source 26 is collimated into parallel light by the third lens 27, passes through the third reflector 28, and is transmitted at the second dichroic prism 29. The light beam is irradiated on the two-dimensional scanning device 12. The light beam reflected back by the two-dimensional scanning device 12 is reflected by the second dichroic prism 29, and is imaged on the second camera 31 through the fourth lens 30. The real-time data is transmitted back to the computer 25 to realize imaging, which can realize real-time monitoring of the performance of the two-dimensional scanning device 12 so that the back-end can perform scanning control compensation and image distortion calibration.

[0037] Compared with the solution of the third embodiment, the solution of the fourth embodiment improves the signal-to-noise ratio of the system, reduces the phototoxicity of the illumination light path, and can further expand the application range of the product.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A high-precision spectral confocal microscopy imaging system, comprising a light modulation module, a spectrum splitting module, a scanning imaging module, an image detection device, and a computer. The light beam emitted by the light modulation module passes through the scanning imaging module and is incident on a sample to excite a fluorescence signal. The fluorescence signal passes through the spectrum splitting module and is received by the image detection device. The image detection device converts the current signal into a digital signal and transmits it to the computer to complete imaging. The system is characterized in that: The spectrum splitting module is composed of a first converging lens, a first pinhole, a first linear long-wave pass filter, a second converging lens, a first linear short-wave pass filter and a third converging lens arranged in sequence. The first converging lens converges the fluorescence signal at the center of the first pinhole and incidents it on the first linear long-wave pass filter. The fluorescence signal emitted from the first linear long-wave pass filter is converged on the first linear short-wave pass filter through the second converging lens. The fluorescence signal emitted from the first linear short-wave pass filter is converged on the image detection device through the third converging lens.

2. A high-precision spectral confocal microscopy imaging system according to claim 1, characterized in that: The scanning imaging module is composed of a first dichroic mirror, a two-dimensional scanning device, a second reflector, a scanning lens, a tube lens, an objective lens and a sample stage for placing a sample, which are arranged in sequence. The first dichroic mirror is used to transmit the light beam emitted by the light modulation module and reflect the fluorescence signal. The light beam emitted by the light modulation module is transmitted through the first dichroic mirror, and the two-dimensional scanning device realizes two-dimensional scanning of the light beam. The light beam passes through the second reflector and is vertically incident on the scanning lens. It is collimated into a parallel light beam by the tube lens and converged on the sample stage through the objective lens, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal passes through the objective lens, the tube lens, the scanning lens, the second reflector and the two-dimensional scanning device in sequence to be collimated into parallel light, and is reflected by the first dichroic mirror and enters the first converging lens.

3. A high-precision spectral confocal microscopy imaging system according to claim 1, characterized in that: The scanning imaging module is composed of a two-dimensional scanning device, a second dichroic mirror, a scanning lens, a tube lens, an objective lens and a sample stage for placing samples, which are arranged in sequence. The second dichroic mirror is used to reflect the light beam emitted by the light modulation module and transmit the fluorescence signal. The light beam emitted by the light modulation module is incident on the two-dimensional scanning device to realize two-dimensional scanning of the light beam, and then reflected by the second dichroic mirror and vertically incident on the scanning lens. After being collimated into a parallel light beam by the tube lens, it is converged on the sample stage by the objective lens, exciting the sample on the sample stage to generate a fluorescence signal. The fluorescence signal passes through the objective lens, the tube lens and the scanning lens in sequence along the original path, and is transmitted through the second dichroic mirror to enter the first converging lens.

4. A high-precision spectral confocal microscopy imaging system according to claim 2 or 3, characterized in that: The two-dimensional scanning device is provided with a scanning imaging monitoring module, and the scanning imaging monitoring module is used to monitor the performance of the two-dimensional scanning device in real time.

5. A high-precision spectral confocal microscopy imaging system according to claim 4, characterized in that: The scanning imaging monitoring module is composed of an LED light source, a third lens, a third reflector, a second dichroic prism, a fourth lens and a second camera arranged in sequence. The light beam emitted by the LED light source is collimated into parallel light by the third lens, passes through the third reflector to reach the second dichroic prism and is transmitted. The transmitted light beam is irradiated on the two-dimensional scanning device. The light beam reflected back by the two-dimensional scanning device is reflected by the second dichroic prism and is imaged on the second camera through the fourth lens. The second camera transmits real-time data back to the computer to monitor the performance of the two-dimensional scanning device in real time.

6. A high-precision spectral confocal microscopy imaging system according to claim 1, characterized in that: The light modulation module is composed of a multi-wavelength laser, a multi-mode optical fiber, a collimating lens, a first reflector, a beam shaping device, a first beam splitter prism, a first camera, and a first stray light filtering device. The light beam emitted by the multi-wavelength laser is collimated into a Gaussian parallel beam by the multi-mode optical fiber and the collimating lens. The output angle of the Gaussian parallel beam is adjusted by the first reflector so that the output beam is parallel to the optical axis. The beam shaping device converts the Gaussian parallel beam into a flat-top beam. After the flat-top beam passes through the first beam splitter prism, the transmitted flat-top beam passes through the first stray light filtering device and enters the scanning imaging module. The reflected flat-top beam is imaged on the first camera. The first camera transmits real-time data back to the computer for real-time monitoring of the spatial two-dimensional intensity distribution of the flat-top beam.

7. A high-precision spectral confocal microscopy imaging system according to claim 6, characterized in that: The first stray light filtering device is composed of a first lens, a second pinhole and a second lens. The transmitted light beam is converged at the center of the second pinhole through the first lens, and is collimated by the second lens before entering the scanning imaging module.

8. The high-precision spectral confocal microscopy imaging system according to claim 1, wherein: The light modulation module is composed of a white light LED light source, a multimode optical fiber, a collimating lens, a first reflector, a beam shaping device, a second stray light filtering device, a first beam splitter prism and a first camera. The illumination beam emitted by the white light LED light source is collimated into a Gaussian parallel beam by the multimode optical fiber and the collimating lens. The collimated parallel beam is adjusted by the first reflector at an output angle so that the output beam is parallel to the optical axis. The beam shaping device converts the Gaussian parallel beam into a flat-top beam. After passing through the second stray light filtering device, the flat-top beam is incident on the first beam splitter prism. The transmitted flat-top beam is incident on the scanning imaging module. The reflected flat-top beam is imaged on the first camera. The first camera transmits real-time data back to the computer for real-time monitoring of the spatial two-dimensional intensity distribution of the flat-top beam.

9. A high-precision spectral confocal microscopy imaging system according to claim 8, characterized in that: The second stray light filtering device is composed of a first lens, a second linear long-wave pass filter, a fourth converging lens, a second pinhole, a second linear short-wave pass filter and a second lens arranged in sequence. The first lens converges the light beam on the second linear long-wave pass filter. The light beam emitted from the second linear long-wave pass filter is converged at the center of the second pinhole through the fourth converging lens and incident on the second linear short-wave pass filter. The light beam emitted from the second linear short-wave pass filter is incident on the first dichroic prism through the second lens.

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