High-precision spectrum type confocal microscopic imaging system

Abstract

The invention discloses a high-precision spectrum type confocal microscopic imaging system, which comprises a light modulation module, a spectrum splitting module, a scanning imaging module, an image detection device and a computer, and is characterized in that a light beam emitted by the light modulation module passes through the scanning imaging module and then enters a sample to excite a fluorescence signal; a fluorescence signal is received by the image detection device after passing through the spectrum splitting module, and the image detection device converts a current signal into a digital signal and transmits the digital signal to the computer to complete imaging; the confocal microscopic imaging device is characterized in that the spectrum splitting module is composed of a first convergent lens, a first pinhole, a first linear long-wave-pass filter, a second convergent lens, a first linear short-wave-pass filter and a third convergent lens which are arranged in sequence. High-precision spectrum confocal imaging is realized by adopting a mode of combining a lens and a linear filter, the problem of spectrum crosstalk in confocal imaging is solved, and the spectral resolution is less than 3.0 nm.

Description

Technical Field

[0001] The present invention relates to a confocal microscopy system, and particularly to a high-precision spectral confocal microscopy 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 sectioning performance, and play an increasingly important role in basic research such as biology and medicine. At present, the demand for studying the interaction mechanisms between cells and organelles has increased sharply. It is necessary to label different organelles with a variety of fluorescent proteins and observe their morphological changes, transport mechanisms, and interactions in real time under a confocal microscope. However, when imaging with multiple fluorescent probes, the fluorescence spectra of different fluorophores are extremely likely to overlap, resulting in fluorescence signal crosstalk, and further distorting the image. Traditional confocal microscopes cannot overcome this problem of spectral crosstalk. Summary of the Invention

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

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

[0005] Compared with the prior art, the advantages of the present invention are 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, solving the problem of spectral crosstalk in confocal imaging, and the spectral resolution is 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 passes through the first dichroic mirror transmissively, and the two-dimensional scanning of the light beam is realized by the two-dimensional scanning device. The light beam is vertically incident on the scanning lens after passing through the second reflector, collimated into a parallel light beam by the tube lens, and 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 the fluorescence signal. The light beam emitted by the light modulation module passes through the first dichroic mirror transmissively, and the two-dimensional scanning of the light beam is realized by the two-dimensional scanning device. The light beam excites 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 along the original path and is collimated into a parallel light, which is reflected by the first dichroic mirror and enters the first converging lens.

[0007] Preferably, 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 a sample, 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 the two-dimensional scanning of the light beam, and then is vertically incident on the scanning lens after being reflected by the second dichroic mirror. After being collimated into a parallel light beam by the tube lens, it is 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, and the scanning lens in sequence along the original path, and then passes through the second dichroic mirror transmissively and enters the first converging lens. Since the fluorescence of biological samples is weak, and the spectral resolution ability at the nanometer level during spectral confocal imaging results in a weak light intensity during imaging, this solution improves the fluorescence detection efficiency in the system and reduces the phototoxicity by designing a non-retroscan optical path, so that the fluorescence signal generated by the sample does not need to pass through the two-dimensional scanning device with a low reflectivity again.

[0008] Preferably, a scanning imaging monitoring module is arranged on the two-dimensional scanning device, 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 mirror, a second beam splitter prism, a fourth lens, and a second camera arranged in sequence. The beam emitted by the LED light source is collimated into a parallel light by the third lens, reaches the second beam splitter prism through the third mirror and is transmitted. The transmitted beam irradiates on the two-dimensional scanning device, and the beam reflected back by the two-dimensional scanning device is reflected by the second beam splitter prism and 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 mirror, a beam shaping device, a first camera, and a first stray light filtering device arranged in sequence. The 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 Gaussian parallel beam is adjusted by the first mirror to make the emitted beam parallel to the optical axis. The beam shaping device changes 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 is incident on the scanning imaging module after passing through the first stray light filtering device, 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 two-dimensional spatial 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 beam is converged at the center of the second pinhole by the first lens and is incident on the scanning imaging module after being collimated by the second lens.

[0012] Preferably, 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, which are 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 to make the emitted beam parallel to the optical axis. The beam shaping device changes the Gaussian parallel beam into a flat-top beam. The flat-top beam is incident on the first beam splitter prism after passing through the second stray light filtering device. 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 two-dimensional spatial intensity distribution of the flat-top beam. By combining a white light LED light source, a lens, and a linear filter, the center wavelength and bandwidth of the illumination beam can be adjusted arbitrarily within the visible light range, expanding the selection range of the illumination beam and the product application range and reducing the cost.

[0013] Preferably, the second stray light filtering device is composed of a first lens, a second linear long-pass filter, a fourth converging lens, a second pinhole, a second linear short-pass filter, and a second lens, which are arranged in sequence. The first lens converges the beam on the second linear long-pass filter. The beam emitted from the second linear long-pass filter passes through the fourth converging lens and converges on the center of the second pinhole and is incident on the second linear short-pass filter. The beam emitted from the second linear short-pass filter passes through the second lens and is incident on the first beam splitter prism. Description of the Drawings

[0014] Figure 1 Schematic structural diagram of the backward-scanning high-precision spectral confocal microscopy imaging system according to Embodiment 1 of the present invention; Figure 2 Spectral resolution test chart of the backward-scanning high-precision spectral confocal microscopy imaging system according to Embodiment 1 of the present invention; Figure 3 Schematic structural diagram of the non-backward-scanning high-precision spectral confocal microscopy imaging system according to Embodiment 2 of the present invention; Figure 4 Optical path diagram of the backward-scanning high-precision spectral confocal microscopy imaging system with adjustable center wavelength and bandwidth of the illumination beam according to Embodiment 3 of the present invention; Figure 5 Optical path diagram of the non-backward-scanning high-precision spectral confocal microscopy imaging system with adjustable center wavelength and bandwidth of the illumination beam according to Embodiment 4 of the present invention.

[0015] Description of the Reference Numerals: 1-1, Multi-wavelength laser; 1-2, White light LED light source; 2, Multi-mode optical fiber; 3, Collimating lens; 4, First mirror; 5, Beam shaping device; 6, First beam splitting prism; 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 mirror; 14, Scanning lens; 15, Tube lens; 16, Objective lens; 17, Sample stage; 18, First converging lens; 19, First pinhole; 20, First linear long-pass filter; 21, Second converging lens; 22, First linear short-pass filter; 23, Third converging lens; 24, Image detection device; 25, Computer; 26, LED light source; 27, Third lens; 28, Third mirror; 29, Second beam splitting prism; 30, Fourth lens; 31, Second camera; 32, Second linear long-pass filter; 33, Fourth converging lens; 34, Second linear short-pass filter. Detailed implementation mode

[0016] The present invention will be further described in detail below in conjunction with the embodiments of the accompanying drawings.

[0017] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, 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 part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0018] Embodiment 1: Figure 1 It is a schematic structural diagram of the retrace type high-precision spectral confocal microscopy imaging system provided for the first embodiment. The light modulation module is composed of the following components: multi-wavelength laser 1-1, multi-mode optical fiber 2, collimating lens 3, first mirror 4, beam shaping device 5, first beam splitting prism 6, first camera 7, first lens 8, second pinhole 9, second lens 10; the scanning imaging module is composed of the following components: the first dichroic mirror 11-1 is used to transmit the beam emitted by the multi-wavelength laser 1-1 and reflect the fluorescence signal, two-dimensional scanning device 12, second mirror 13, scanning lens 14, tube lens 15, objective lens 16, sample stage 17; the spectral splitting module is composed of the following components: first converging lens 18, first pinhole 19, first linear long-pass filter 20, second converging lens 21, first linear short-pass filter 22, third converging lens 23; image detection device 24, computer 25, and the scanning imaging monitoring module is composed of the following components: LED light source 26, third lens 27, third mirror 28, second beam splitting prism 29, fourth lens 30 and second camera 31.

[0019] Adopting the backscanning high-precision spectral confocal microscopy imaging system of the first embodiment, the imaging process is as follows: The multi-wavelength laser 1-1 emits laser light, which is collimated into Gaussian parallel light by the multimode optical fiber 2 and the collimating lens 3. After collimation, the light beam passes through the first mirror 4 to adjust its exit angle, making the light beam parallel to the optical axis. After passing through the beam shaping device 5, the two-dimensional distribution of the light beam changes from Gaussian to flat-top, and the light spot becomes more uniform. The light beam 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, and 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 converged by the first lens 8 to the center of the 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 passes through the first dichroic mirror 11-1 and undergoes two-dimensional scanning by the two-dimensional scanning device 12. It is vertically incident on the scanning lens 14 through the second mirror 13, collimated into a parallel light beam by the tube lens 15, and converged on the sample stage 17 by the objective lens 16. The laser excites the sample to generate a fluorescence signal. The fluorescence signal sequentially passes through the objective lens 16, the tube lens 15, the scanning lens 14, the second mirror 13, and the two-dimensional scanning device 12 and is collimated into a parallel light, and is reflected by the first dichroic mirror 11-1 and enters the spectral splitting module. In the spectral splitting module, the fluorescence signal is converged on the center of the first pinhole 19 by the first converging lens 18. 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 achieve spectral splitting of the fluorescence signal. The fluorescence signal exiting from the first linear short-wave pass filter 22 is converged on the image detection device 24 by the third converging lens 23, and 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 a parallel light beam by the third lens 27, passes through the third mirror 28, and is transmitted at the second beam splitter prism 29. The light beam irradiates on the two-dimensional scanning device 12, and the light beam reflected back by the two-dimensional scanning device 12 is reflected by the second beam splitter prism 29 and imaged on the second camera 31 through the fourth lens 30, and the real-time data is transmitted back to the computer 25 to achieve imaging, and the performance of the two-dimensional scanning device 12 can be monitored in real time, so as to perform scanning control compensation and image distortion calibration at the backend. The high-precision spectral confocal microscopy imaging system of the first embodiment can achieve spectral splitting in the visible light range, such as Figure 2 The transmitted curve of the light beam passing through the linear filter is shown, and the spectral resolution is 2.60 nm.

[0020] Embodiment 2: Figure 3 It is a schematic structural diagram of the non-backscanning high-precision spectral confocal microscopy imaging system provided for the second embodiment. FromFigure 3 As can be seen, the light modulation module, spectral splitting module, scanning imaging monitoring module, image detection device 24, and computer 25 in the second embodiment are the same as those in the first embodiment. The difference lies in that the scanning imaging module consists of the following components: a two-dimensional scanning device 12, a second dichroic mirror 11-2 for reflecting the beam emitted by the multi-wavelength laser 1-1 and transmitting the fluorescence signal, a scanning lens 14, a tube lens 15, an objective lens 16, and a sample stage 17.

[0021] Using the non-backscanning high-precision spectral confocal microscopy imaging system of the second embodiment, the imaging process is as follows: The laser emitted by the multi-wavelength laser 1-1 is collimated into a Gaussian parallel beam through a multi-mode optical fiber 2 and a collimating lens 3. The collimated beam is adjusted by a first mirror 4 to make its exit angle parallel to the optical axis. After passing through a 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 beam is split into two parts by a first beam splitter prism 6. One part of the beam is reflected and imaged on a first camera 7, and the two-dimensional spatial intensity distribution of the illumination beam can be monitored in real time through the first camera 7. The other part of the beam is transmitted and converged on the center of a second pinhole 9 by a first lens 8 to filter out stray light. The beam is collimated again by a second lens 10, and after two-dimensional scanning by the two-dimensional scanning device 12, it is reflected by the second dichroic mirror 11-2 and vertically incident on the scanning lens 14. It is collimated into a parallel beam by the tube lens 15 and converged on the sample stage 17 through 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, and the scanning lens 14 in sequence, and then enters the spectral splitting module after passing through the second dichroic mirror 11-2. In the spectral splitting module, the fluorescence signal is converged on the center of a first pinhole 19 by a first converging lens 18. 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 a 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 splitting of the fluorescence signal. The fluorescence signal exiting from the first linear short-wave pass filter 22 is converged on the image detection device 24 by a third converging lens 23, and the current signal is converted into a digital signal and transmitted to the computer 25 to complete the imaging. In addition, the beam emitted by the LED light source 26 is collimated into a parallel beam through a third lens 27, passes through a third mirror 28, and is transmitted at the second beam splitter prism 29. The beam irradiates on the two-dimensional scanning device 12. The beam reflected back by the two-dimensional scanning device 12 is reflected by the second beam splitter prism 29 and imaged on a second camera 31 through a fourth lens 30, and the real-time data is transmitted back to the computer 25 to achieve imaging, and the performance of the two-dimensional scanning device 12 can be monitored in real time for subsequent scanning control compensation and image distortion calibration.

[0022] Compared with the solution of the second embodiment, the solution of the second embodiment improves the fluorescence detection efficiency of the entire system, reduces the phototoxicity of the illumination optical path, and can further expand the application range of the product.

[0023] Embodiment Three: Figure 4 The following is a schematic structural diagram of a push-scanning high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on a white light LED provided in this third embodiment. As can be seen Figure 4 from it, the scanning imaging module, spectral splitting module, scanning imaging monitoring module, image detection device 24, and computer 25 in the third embodiment are the same as those in the first embodiment. The difference lies in 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-pass filter 32, fourth converging lens 33, second pinhole 9, second linear short-pass filter 34, second lens 10, first beam splitter prism 6, and first camera 7.

[0024] When using the push-scanning high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on a white light LED in this third embodiment, the imaging process is as follows: The illumination beam emitted by the white light LED light source 1-2 is collimated into a Gaussian-type parallel light by the multimode optical fiber 2 and the collimating lens 3. After collimation, the beam passes through the first reflector 4 to adjust its exit 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 type to flat-top type, and the light spot is more uniform. The beam passes through the first lens 8 and converges on the second linear long-pass filter 32, passes through the fourth converging lens 33 and converges on the center of the second pinhole 9 and enters the second linear short-pass filter 34. The second linear short-pass filter 34 is placed behind the second pinhole 9. The beam emitted by the second linear short-pass filter 34 is collimated into a parallel beam by the second lens 10. The second linear long-pass filter 32 and the second linear short-pass filter 34 are precisely moved by a motor with closed-loop control. Arbitrary adjustment of the central wavelength and bandwidth of the illumination beam is achieved through the two-way precise movement of the two linear filters. The 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 beam is reflected and imaged on the first camera 7. The spatial two-dimensional intensity distribution of the illumination beam can be monitored in real time through the first camera 7. The other part of the beam is transmitted and then incident on the first dichroic mirror 11-1. The beam is transmitted through the first dichroic mirror 11-1, and two-dimensional scanning of the beam is achieved through the two-dimensional scanning device 12. It is vertically incident on the scanning lens 14 through the second reflector 13 and is collimated into a parallel beam by the tube lens 15 and converges on the sample stage 17 through the objective lens 16. The white light excites the sample to generate a fluorescence signal. The fluorescence signal sequentially 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 and is collimated into a parallel light, and is reflected by the first dichroic mirror 11-1 and enters the spectral splitting module. In the spectral splitting module, the fluorescence signal converges on the center of the first pinhole 19 through the first converging lens 18. The first pinhole 19 can effectively filter out stray light. The first linear long-pass filter 20 is placed behind the first pinhole 19. The fluorescence signal converges on the first linear short-pass filter 22 through the first linear long-pass filter 20 and the second converging lens 21. The combination of the linear long-pass filter and the short-pass filter can achieve spectral splitting of the fluorescence signal. The fluorescence signal emitted from the first linear short-pass filter 22 converges on the image detection device 24 through the third converging lens 23. The current signal is converted into a digital signal and transmitted to the computer 25 to complete imaging. In addition, the beam emitted by the LED light source 26 is collimated into a parallel light by the third lens 27, passes through the third reflector 28, and is transmitted at the second beam splitter prism 29. The beam irradiates on the two-dimensional scanning device 12. The beam reflected back by the two-dimensional scanning device 12 is reflected by the second beam splitter prism 29 and imaged on the second camera 31 through the fourth lens 30, and the real-time data is transmitted back to the computer 25 to achieve imaging, and the performance of the two-dimensional scanning device 12 can be monitored in real time for subsequent scanning control compensation and image distortion calibration.

[0025] The solution of the third embodiment can excite the strongest peak intensity for different fluorescent markers compared with the first embodiment, improve the signal-to-noise ratio of the system, reduce the phototoxicity of the illumination optical path, and further expand the application range of the product.

[0026] Embodiment 4: Figure 5 It is a schematic structural diagram of a non-descanning high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on a white LED provided in this embodiment. As can be seen Figure 5 from it, the light modulation module in Embodiment 4 is the same as that in Embodiment 3, and the scanning imaging module, spectral splitting module, scanning imaging monitoring module, image detection device 24, and computer 25 are the same as those in Embodiment 2.

[0027] Using the non-descanning high-precision spectral confocal microscopy imaging system with adjustable illumination beam center and bandwidth based on a white LED in this Embodiment 4, the imaging process is as follows: The illumination beam emitted by the white light LED light source 1-2 is collimated into a Gaussian-type parallel light by the multimode optical fiber 2 and the collimating lens 3. After collimation, the beam passes through the first reflector 4 to adjust its exit 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 type to flat-top type, and the light spot becomes more uniform. The beam passes through the first lens 8 and converges on the second linear long-pass filter 32, and then converges on the center of the second pinhole 9 through the fourth converging lens 33 and enters the second linear short-pass filter 34. The second linear short-pass filter 34 is placed behind the second pinhole 9. The second linear long-pass filter 32 and the second linear short-pass filter 34 are precisely moved by a motor with closed-loop control. Arbitrary adjustment of the central wavelength and bandwidth of the illumination beam is achieved through the two-way precise movement of the two linear filters. The beam emitted from the second linear short-pass filter 34 is collimated into a parallel beam by the second lens 10 and enters the first beam splitter prism 6. The beam is split into two parts by the first beam splitter prism 6. One part of the beam is reflected and imaged on the first camera 7, and the two-dimensional spatial intensity distribution of the illumination beam can be monitored in real time through the first camera 7. The other part of the beam is transmitted and then undergoes two-dimensional scanning by the two-dimensional scanning device 12. After being reflected by the second dichroic mirror 11-2, it is vertically incident on the scanning lens 14, collimated into a parallel beam by the tube lens 15, and converges 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 turn along the original path, and enters the spectral splitting module after passing through the second dichroic mirror 11-2. In the spectral splitting module, the fluorescence signal converges on the center of the first pinhole 19 through the first converging lens 18. The first pinhole 19 can effectively filter out stray light. The first linear long-pass filter 20 is placed behind the first pinhole 19. The fluorescence signal converges on the first linear short-pass filter 22 through the first linear long-pass filter 20 and the second converging lens 21. The combination of the linear long-pass filter and the short-pass filter can achieve spectral splitting of the fluorescence signal. The fluorescence signal emitted from the first linear short-pass filter 22 converges on the image detection device 24 through the third converging lens 23. The current signal is converted into a digital signal and transmitted to the computer 25 to complete imaging. In addition, the beam emitted by the LED light source 26 is collimated into a parallel light by the third lens 27, passes through the third reflector 28, and is transmitted at the second beam splitter prism 29. The beam irradiates on the two-dimensional scanning device 12. The beam reflected back by the two-dimensional scanning device 12 is reflected by the second beam splitter prism 29 and imaged on the second camera 31 through the fourth lens 30, and the real-time data is transmitted back to the computer 25 to achieve imaging, and the performance of the two-dimensional scanning device 12 can be monitored in real time for subsequent scanning control compensation and image distortion calibration.

[0028] Compared with the solution of Embodiment 3, the solution of Embodiment 4 improves the signal-to-noise ratio of the system, reduces the phototoxicity of the illumination optical path, and can further expand the application range of the product.

[0029] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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 spectral splitting module, a scanning imaging module, an image detection device and a computer. The beam emitted by the light modulation module is incident on a sample through the scanning imaging module to excite a fluorescence signal. The fluorescence signal is received by the image detection device after passing through the spectral splitting module. The image detection device converts the current signal into a digital signal and then transmits it to the computer to complete imaging. It is characterized in that, The described spectral splitting module consists of a first converging lens, a first pinhole, a first linear long-pass filter, a second converging lens, a first linear short-pass filter, and a third converging lens arranged in sequence. The first converging lens converges the fluorescence signal to the center of the first pinhole and enters the first linear long-pass filter. The fluorescence signal emerging from the first linear long-pass filter is converged by the second converging lens onto the first linear short-pass filter. The fluorescence signal emerging from the first linear short-pass filter is converged by the third converging lens onto the image detection device.

2. The high-precision spectral confocal microscopy imaging system according to claim 1, wherein The described scanning imaging module consists of a first dichroic mirror, a two-dimensional scanning device, a second mirror, a scanning lens, a tube lens, an objective lens, and a sample stage for placing the sample, arranged in sequence. The first dichroic mirror is used to transmit the beam emitted by the light modulation module and reflect the fluorescence signal. The beam emitted by the light modulation module passes through the first dichroic mirror, and two-dimensional scanning of the beam is achieved by the two-dimensional scanning device. The beam is vertically incident on the scanning lens through the second mirror, collimated into a parallel beam by the tube lens, converged by the objective lens onto the sample stage, and excites the sample on the sample stage to generate a fluorescence signal. The fluorescence signal sequentially passes back through the objective lens, the tube lens, the scanning lens, the second mirror, and the two-dimensional scanning device and is collimated into a parallel light, which is reflected by the first dichroic mirror and enters the first converging lens.

3. The high-precision spectral confocal microscopy imaging system according to claim 1, wherein The described scanning imaging module consists 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 the sample, arranged in sequence. The second dichroic mirror is used to reflect the beam emitted by the light modulation module and transmit the fluorescence signal. The beam emitted by the light modulation module is incident on the two-dimensional scanning device to achieve two-dimensional scanning of the beam, and then is vertically incident on the scanning lens after being reflected by the second dichroic mirror. After being collimated into a parallel beam by the tube lens, it is converged by the objective lens onto the sample stage, and excites the sample on the sample stage to generate a fluorescence signal. The fluorescence signal sequentially passes back through the objective lens, the tube lens, and the scanning lens, and then passes through the second dichroic mirror and enters the first converging lens.

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

5. The high-precision spectral confocal microscopy imaging system according to claim 4, characterized in that The described scanning imaging monitoring module is composed of an LED light source, a third lens, a third mirror, a second beam splitter prism, a fourth lens, and a second camera arranged in sequence. The beam emitted by the LED light source is collimated into a parallel beam by the third lens, reaches the second beam splitter prism after passing through the third mirror, is transmitted at the second beam splitter prism, the transmitted beam irradiates on the two-dimensional scanning device, the beam reflected back by the two-dimensional scanning device is reflected by the second beam splitter prism, passes through the fourth lens and is imaged on the second camera, and 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. The high-precision spectral confocal microscopy imaging system according to claim 1, characterized in that The described light modulation module is composed of a multi-wavelength laser, a multi-mode optical fiber, a collimating lens, a first mirror, a beam shaping device, a first beam splitter prism, a first camera, and a first stray light filtering device. The 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 Gaussian parallel beam is adjusted by the first mirror to make the emitted beam parallel to the optical axis. The beam shaping device changes 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 enters the scanning imaging module after passing through the first stray light filtering device, 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 two-dimensional spatial intensity distribution of the flat-top beam.

7. The high-precision spectral confocal microscopy imaging system according to claim 6, characterized in that, The described first stray light filtering device is composed of a first lens, a second pinhole, and a second lens. The transmitted beam converges 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. A high-precision spectral confocal microscopy imaging system according to claim 1, wherein The described light modulation module is composed of a white light LED light source, a multi-mode optical fiber, a collimating lens, a first mirror, 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 multi-mode optical fiber and the collimating lens. The collimated parallel beam is adjusted by the first mirror to make the emitted beam parallel to the optical axis. The beam shaping device changes the Gaussian parallel beam into a flat-top beam. After the flat-top beam passes through the second stray light filtering device, it enters the first beam splitter prism. The transmitted flat-top beam enters 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 two-dimensional spatial intensity distribution of the flat-top beam.

9. The high-precision spectral confocal microscopy imaging system according to claim 8, wherein, The second stray light filtering device described is composed of a first lens, a second linear long-pass filter, a fourth converging lens, a second pinhole, a second linear short-pass filter, and a second lens arranged in sequence. The first lens converges the light beam on the second linear long-pass filter. The light beam emerging from the second linear long-pass filter passes through the fourth converging lens and converges on the center of the second pinhole and then enters the second linear short-pass filter. The light beam emerging from the second linear short-pass filter passes through the second lens and enters the first beam splitter prism.

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