A laser scanning confocal fluorescence imaging system and method of a cage coaxial structure

Abstract

The application discloses a laser scanning confocal fluorescence imaging system and method of a cage type coaxial structure, the method utilizes a white light source imaging module to couple white light to a laser incidence module, the white light and laser are irradiated to a sample surface together through a laser light path, excited fluorescence is coupled to an optical property characterization device through a fluorescence light path to realize fluorescence imaging, and the white light reflected back from the sample surface is sequentially imaged through the laser light path and an imaging light path through a CCD camera to obtain topographic imaging, so that fluorescence imaging and topographic imaging are realized simultaneously, and because the laser incidence module, the white light source imaging module and the fluorescence collection module are arranged in the cage type coaxial structure, the structure is simple, the light paths formed by the modules are more stable, therefore, synchronous topographic imaging and fluorescence imaging can be realized stably and efficiently, and different wavelength laser experimental scenes can be quickly adapted.

Description

Technical Field

[0001] This invention patent belongs to the field of optical microscopy imaging technology, specifically relating to a cage-type coaxial structure laser scanning confocal fluorescence imaging system and method. Background Technology

[0002] Laser scanning confocal microscopy breaks away from the field light source and local planar imaging mode of traditional optical microscopes. The laser scanning confocal microscope uses a laser beam as the light source. The laser beam passes through an illumination pinhole, is reflected by a beam splitter to the objective lens, and is focused on the sample. It rapidly scans and images the focal plane of the specimen point by point, line by line, and surface by surface.

[0003] Laser scanning confocal microscopy systems are commonly used for high-precision imaging of fluorescence signals, analysis of single-photon source characteristics, and multi-scale sample characterization. They have become an indispensable professional testing tool in materials science research and biomedicine.

[0004] Traditional laser scanning confocal microscopes are typically complex in structure, have high purchase and maintenance costs, and feature dispersed optical path architecture, numerous optical components, and low positioning accuracy. These drawbacks lead to unstable fluorescence coupling efficiency and weak anti-interference capabilities.

[0005] Patent application CN101013136A discloses a laser-induced fluorescence confocal scanning device and method, belonging to the field of biochip detection technology. Existing devices have low fluorescence collection and utilization efficiency, and focusing control during the scanning process is very difficult. This invention employs a centrally perforated total internal reflection mirror to separate the incident laser and the induced fluorescence. When the laser beam passes through the aperture, energy loss is minimal, and almost all laser energy can irradiate the biochip. Furthermore, the fluorescence collection angle is large, improving both laser incident efficiency and fluorescence collection efficiency. Computer control maintains an approximately confocal relationship between the pinhole of the light source and the pinhole of the detector, requiring only one focusing adjustment before scanning, making focusing control simple and convenient. However, the system disclosed in this patent application cannot be coupled with a morphology imaging system.

[0006] Patent application CN103743718A discloses a confocal micro-Raman spectroscopy (MMR) and laser-induced breakdown spectroscopy (LIBS) combined laser spectrometer. This instrument includes a micro-Raman system, a micro-LIBS system, a high-resolution microscopic imaging system, a confocal microscopic optical path, and a time-resolved spectral receiving system. It can automatically switch between white light microscopic imaging observation mode, autofocus mode, LIBS spectroscopy mode, and Raman spectroscopy mode. A significant feature of this invention is the compact combination of Raman and LIBS using a micro-confocal system, enabling qualitative and quantitative analysis of elemental and molecular structures at the same minute location. Combined with high-resolution imaging, it allows for spatially resolved elemental and structural chemical analysis at the micrometer scale, obtaining complete information such as the spatial distribution of chemical elements, material structure, and physical conditions of the sample. However, the system disclosed in this patent application has poor optical path stability and low multimodal imaging efficiency. Summary of the Invention

[0007] This invention provides a cage-type coaxial structure laser scanning confocal fluorescence imaging system. This system can stably and efficiently achieve simultaneous morphological and fluorescence imaging, and can quickly adapt to experimental scenarios with different wavelengths of laser light. The laser scanning confocal fluorescence imaging system includes a laser incident module, a fluorescence collection module, and a white light source imaging module fixed within a cage-type coaxial structure and located on a supporting body, as well as a microscope objective module and a sample stage located below the supporting body.

[0008] The laser incident module is used to couple the incident laser and white light together to the fluorescence collection module, and also to reflect the white light reflected by the sample back to the white light source imaging module;

[0009] The white light source imaging module is used to couple white light to the laser incident module through a set optical path, so that the white light and the incident laser are in the same optical path. It is also used to reflect the white light reflected by the sample back to the CCD camera, and to image the light through the CCD camera.

[0010] The fluorescence collection module is used to irradiate the sample located on the sample stage with incident laser and white light together through the microscope objective module, and is also used to couple the fluorescence signal generated after sample excitation into the optical property characterization device to realize sample fluorescence imaging.

[0011] Preferably, the laser incident module includes, from top to bottom along the axial direction, a first single-mode fiber, a first lens, a first dichroic mirror, and a first reflecting mirror;

[0012] The first single-mode fiber is used to couple the incident laser into the laser incident module. The coupled incident laser passes through the first lens and then, together with the white light, passes through the first dichroic mirror and the first reflecting mirror in sequence before being coupled into the fluorescence collection module.

[0013] The first dichroic mirror is also used to reflect the white light reflected by the sample back to the white light source imaging module.

[0014] Preferably, the white light source imaging module includes a second lens, a second reflector, a second dichroic mirror, a third reflector, and a third lens;

[0015] The illumination optical path is constructed by a white light source, a second lens, a second reflector, a second dichroic mirror, and a third reflector. The white light generated by the white light source is coupled to the laser incident module through the illumination optical path, so that the white light and the incident laser are in the same optical path.

[0016] An imaging optical path is constructed using the third reflecting mirror, the second dichroic mirror, the third lens, and the CCD camera. The white light reflected from the sample is reflected back to the CCD camera through the imaging optical path, and then imaged by the CCD camera.

[0017] Preferably, the fluorescence collection module includes a third dichroic mirror, a filter, a fourth lens, and a second single-mode optical fiber;

[0018] The third dichroic mirror is used to reflect the incident laser and white light together to the microscope objective module, and also to reflect the white light passing through the sample back to the laser incident module.

[0019] The third dichroic mirror, filter, fourth lens, and second single-mode fiber form a fluorescence optical path, through which the fluorescence signal generated after sample excitation is coupled into the optical property characterization device to achieve sample fluorescence imaging.

[0020] The third dichroic mirror and filter are also used to filter out stray light and residual laser light.

[0021] Preferably, the optical property characterization device includes a single-photon detector, a data acquisition card, and a PC.

[0022] The single-photon detector is connected to a second single-mode fiber, and the single-photon detector is used to count photons in the received fluorescence signal to obtain an electrical pulse signal.

[0023] The data acquisition card is connected to the single-photon detector. The data acquisition card is used to convert electrical pulse signals into digital signals and transmit the digital signals to the PC. The PC is used to realize fluorescence imaging and real-time monitoring of fluorescence intensity.

[0024] Preferably, the cage-type coaxial structure includes a first cage-type coaxial module, a second cage-type coaxial module, and a third cage-type coaxial module. The first cage-type coaxial module is connected to the second cage-type coaxial module and the third cage-type coaxial module, respectively. The axes of the first cage-type coaxial module, the second cage-type coaxial module, and the third cage-type coaxial module are parallel to each other and are each located independently on the supporting body.

[0025] The laser incident module is fixed inside the first cage-type coaxial module, the fluorescence collection module is fixed inside the third cage-type coaxial module, and the white light source imaging module is fixed inside the second cage-type coaxial module.

[0026] Preferably, the first cage-type coaxial module and the second cage-type coaxial module each independently include an XY two-dimensional translation adjustment frame, a Z-axis translation mounting base, a first two-dimensional optical adjustment frame, a first cage-type connecting support rod, and two first cage-type cubes arranged vertically.

[0027] In the first cage-type coaxial module or the second cage-type coaxial module, the XY two-dimensional translation adjustment frame, the Z-axis translation mounting base, the first two-dimensional optical adjustment frame and the first cage-type cube are fixedly connected from top to bottom by the first cage-type connecting support rod;

[0028] The XY two-dimensional translation adjustment frame and the first two-dimensional optical adjustment frame are used to adjust the incident direction and spatial position of the incident laser, and also to optimize the coupling efficiency of the fluorescence signal.

[0029] The Z-axis translation mount is used to change the beam waist position and size of the incident laser;

[0030] The first cage-type coaxial module has a first reflector and a first dichroic mirror respectively installed in the two first cage-type cubes. The second cage-type coaxial module has a third dichroic mirror installed in the first cage-type cube. The first cage-type cube with the third dichroic mirror is connected to the first cage-type cube with the first dichroic mirror, so that the incident laser and white light are coupled together to the fluorescence collection module. It can also reflect the white light reflected by the sample back to the white light source imaging module. The first reflector and the first dichroic mirror are optical elements of the laser incident module, and the third dichroic mirror is an optical element of the fluorescence collection module.

[0031] The third cage-type coaxial module includes a first cage-type coaxial sub-module, a second cage-type coaxial sub-module, and a second cage-type cube. The first cage-type coaxial sub-module and the second cage-type coaxial sub-module each independently include a second two-dimensional optical adjustment frame, a second cage-type connecting support rod, and a third cage-type cube.

[0032] In the first cage-type coaxial submodule or the second cage-type coaxial submodule, the second two-dimensional optical adjustment frame and the third cage-type cube, which are set from top to bottom, are fixedly connected by the second cage-type connecting support rod.

[0033] The first cage-type coaxial submodule and the second cage-type coaxial submodule are respectively provided with a second reflector and a second dichroic mirror in the third cage-type cube. The second cage-type cube is provided with a third reflector. The third cage-type cube, the second cage-type cube and the first cage-type cube provided with a first dichroic mirror are interconnected, thereby coupling the white light generated by the white light source to the laser incident module. The second reflector, the second dichroic mirror and the third reflector are optical elements of the white light source imaging module.

[0034] Preferably, the microscope objective module includes a microscope objective suspension arm and a microscope objective converter;

[0035] One end of the microscope objective suspension arm is connected to the cage-type coaxial structure, and the other end is connected to the microscope objective converter. The microscope objective suspension arm is used to fix the microscope objective converter and to make the microscope objective converter coaxial with the fluorescence collection module, so that the incident laser and white light are incident on the microscope objective converter together.

[0036] The microscope objective converter includes multiple microscopes with different magnifications. By adjusting the field of view and fluorescence collection efficiency of white light source imaging through microscopes with different magnifications, multi-scale sample characterization can be achieved.

[0037] Preferably, the sample stage includes a wide-range electric triaxial displacement stage, a high-precision piezoelectric ceramic nano-electric triaxial displacement stage, and a sample placement plate;

[0038] The sample placement plate is located on a high-precision piezoelectric ceramic nano-electric triaxial displacement stage, and the sample placement plate is used to place the sample.

[0039] The high-precision piezoelectric ceramic nano-electric triaxial displacement stage is located on a wide-range electric triaxial displacement stage, and the high-precision piezoelectric ceramic nano-electric triaxial displacement stage is used to realize nanoscale scanning.

[0040] The wide-range electric three-axis displacement stage is used to achieve wafer-level range scanning.

[0041] On another front, the present invention also provides a laser scanning confocal fluorescence imaging method with a cage-like coaxial structure, comprising:

[0042] The incident laser generated by the laser is irradiated onto the sample on the sample stage through the laser incident module, the fluorescence collection module and the microscope objective module. The fluorescence signal generated after the sample is excited is coupled into the optical property characterization device through the fluorescence collection module to realize the fluorescence imaging of the sample.

[0043] The white light generated by the white light source is coupled to the laser incident module through the white light source imaging module, and then sequentially illuminates the sample on the sample stage through the laser incident module, the fluorescence collection module, and the microscope objective module. The white light reflected from the sample then sequentially passes through the microscope objective module, the fluorescence collection module, the laser incident module, and is reflected back to the CCD camera through the white light source imaging module, and is then imaged by the CCD camera.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] This invention utilizes a white light source imaging module to couple white light to a laser incident module. Through the laser optical path, the white light and laser light illuminate the sample surface together. The excited fluorescence is coupled to an optical property characterization device through a fluorescence optical path to achieve fluorescence imaging. The white light reflected back from the sample surface passes sequentially through the laser optical path and the imaging optical path to obtain morphological imaging through a CCD camera. Thus, fluorescence imaging and morphological imaging are achieved simultaneously. Furthermore, since the laser incident module, white light source imaging module, and fluorescence collection module provided by this invention are all set in a cage-like coaxial structure, the structure is simple, and the optical paths formed by each module are more stable. Therefore, it can stably and efficiently achieve synchronous morphological imaging and fluorescence imaging, and can quickly adapt to experimental scenarios with different wavelength lasers. Attached Figure Description

[0046] Figure 1 A schematic diagram of a cage-type coaxial structure laser scanning confocal fluorescence imaging system provided in a specific embodiment of the present invention;

[0047] Figure 2 This is a schematic diagram of the optical path of a cage-type coaxial structure laser scanning confocal fluorescence imaging system provided in a specific embodiment of the present invention.

[0048] 1-Cage-type coaxial structure, 11-First cage-type coaxial module, 12-Second cage-type coaxial module, 13-Third cage-type coaxial module, 131-First cage-type coaxial sub-module, 132-Second cage-type coaxial sub-module, 14-Optical mounting plate, a-XY two-dimensional translation adjustment frame, b-Z-axis translation mounting seat, c-First two-dimensional optical adjustment frame, d-First cage-type connecting support rod, e-First cage-type cube, f-Second cage-type cube, g-Second two-dimensional optical adjustment frame, h-Second cage-type connecting support rod, i-Third cage-type cube, 2-Support body, 3-Laser incident module, 31-First single-mode fiber, 32-First lens, 33-First dichroic mirror, 34-First reflector, 35-Laser optical path, 36- 4-Fluorescence collection module, 41-Third dichroic mirror, 42-Filter, 43-Fourth lens, 44-Second single-mode fiber, 45-Fluorescence optical path, 46-Second fiber optic adapter, 5-White light source imaging module, 51-Second lens, 52-Second reflecting mirror, 53-Second dichroic mirror, 54-Third reflecting mirror, 55-Third lens, 56-Illumination optical path, 57-Imaging optical path, 6-Microscope objective module, 61-Microscope objective suspension arm, 62-Microscope objective converter, 621-Microscope objective, 7-Sample stage, 71-Wide range motorized triaxial stage, 72-High precision piezoelectric ceramic nano-motorized triaxial stage, 73-Sample placement plate, 8-White light source, 9-CCD camera. Detailed Implementation

[0049] While existing technologies offer confocal coupling solutions, they suffer from poor optical path stability and low multimodal imaging efficiency in confocal microscopes. This invention, in its specific embodiment, separates the illumination and imaging optical paths of the white light source imaging module. The white light is coupled into the laser's optical path via the illumination path, and the sample surface is illuminated by the stable laser path. The third dichroic mirror of the fluorescence collection module cleverly separates the excitation-generated fluorescence signal from the white light passing through the sample. This fluorescence signal is used for fluorescence imaging via the fluorescence optical path, while the white light passing through the sample is used for morphology imaging via the laser and imaging optical paths. Thus, both fluorescence and morphology imaging are obtained simultaneously. Furthermore, this invention cleverly designs the optical path routing within a cage-like coaxial structure, leveraging the stability of the cage-like coaxial structure to achieve stable and efficient simultaneous morphology and fluorescence imaging, enabling rapid adaptation to experimental scenarios with different wavelengths of laser light.

[0050] The specific embodiment of the present invention provides a cage-type coaxial structure laser scanning confocal fluorescence imaging system. This system integrates a white light source imaging module 5 within the cage-type coaxial structure, located behind the fluorescence collection module 4 and the laser incident module 3. White light is coupled into the optical path of the laser incident module 3 from the rear via a reflector. This system simultaneously completes sample morphology imaging and fluorescence imaging, achieving simultaneous analysis of morphology and fluorescence imaging. The specific structure is as follows: Figure 1 , Figure 2 As shown, it includes:

[0051] The laser scanning confocal fluorescence imaging system provided in a specific embodiment of the present invention includes a laser incident module 3, a fluorescence collection module 4, and a white light source imaging module 5 fixed in a cage-like coaxial structure 1 and located on a support body 2, as well as a microscope objective module 6 and a sample stage 7 located below the support body 2.

[0052] The laser incident module 3 provided in the specific embodiment of the present invention is used to couple the incident laser and white light together to the fluorescence collection module 4, and is also used to reflect the white light reflected by the sample back to the white light source imaging module 5.

[0053] In one specific embodiment, the laser incident module 3 provided by the present invention includes, from top to bottom along the axial direction, a first single-mode fiber 31, a first lens 32, a first dichroic mirror 33, and a first reflector 34;

[0054] The first single-mode fiber 31 provided in the specific embodiment of the present invention is used to couple incident laser into the laser incident module 3. The coupled incident laser passes through the first lens 32 and then couples with the coupled white light through the first dichroic mirror 33 and the first reflecting mirror 34 into the fluorescence collection module 4.

[0055] Specifically, in this embodiment, the first reflector 34 is at a 45° angle and is installed inside a cage-like cube.

[0056] The first dichroic mirror 33 provided in the specific embodiment of the present invention is also used to reflect the white light reflected by the sample back to the white light source imaging module 5.

[0057] In one specific embodiment, the fluorescence collection module 4 provided in this embodiment of the invention is used to irradiate the sample located on the sample stage 7 with incident laser and white light together through the microscope objective module 6, and is also used to couple the fluorescence signal generated after sample excitation into the optical property characterization device to realize sample fluorescence imaging.

[0058] In one specific embodiment, the fluorescence collection module 4 provided in this embodiment includes a third dichroic mirror 41, a filter 42, a fourth lens 43, and a second single-mode optical fiber 44;

[0059] The third dichroic mirror 41 provided in this embodiment is used to reflect the incident laser and white light together to the microscope objective module 6, and also to reflect the white light passing through the sample back to the laser incident module 3.

[0060] The third dichroic mirror 41, filter 42, fourth lens 43, and second single-mode fiber 44 provided in this embodiment form a fluorescence optical path 45. The fluorescence signal generated after sample excitation is coupled into the optical property characterization device through the fluorescence optical path 45 to realize sample fluorescence imaging.

[0061] The third dichroic mirror 41 and filter 42 provided in this embodiment are also used to filter out stray light and residual laser light.

[0062] Specifically, the filter 42 provided in this embodiment further filters the laser signal and enhances the signal-to-noise ratio of the fluorescence signal, and is located on the upper side of the third dichroic mirror 41.

[0063] Specifically, the optical property characterization device provided in this embodiment includes a single-photon detector, a data acquisition card, and a PC.

[0064] The single-photon detector is connected to a second single-mode fiber, and the single-photon detector is used to count photons in the received fluorescence signal to obtain an electrical pulse signal.

[0065] The data acquisition card is connected to the single-photon detector. The data acquisition card is used to convert electrical pulse signals into digital signals and transmit the digital signals to the PC. The PC is used to realize fluorescence imaging and real-time monitoring of fluorescence intensity.

[0066] In one specific embodiment, the laser incident module 3 and the fluorescence collection module 4 provided in this embodiment are installed parallel to each other along the front row, with the same horizontal height and adjustable independently. The incident laser is coupled into the laser incident module 3 through the first single-mode fiber 31, and is transmitted downward through the first lens 32 and the first dichroic mirror 33 in sequence. It is reflected to the left by the first reflecting mirror 34, and then reflected downward through the third dichroic mirror 41 before entering the microscope objective converter 62. It is then focused onto the sample surface by the microscope objective 621. The fluorescence signal emitted by the sample to be tested is collected by the same microscope objective 621 and converted into near-parallel light. It is then transmitted upward through the third dichroic mirror 41 to the fluorescence collection module 4. Finally, it is coupled to the single-photon detector through the second single-mode fiber 44 and converted into a pulse signal. The pulse signal is received by the PC and converted into an electrical signal.

[0067] The white light source imaging module 5 provided in the specific embodiment of the present invention is used to couple white light to the laser incident module 3 through a set optical path, so that the white light and the incident laser are in the same optical path. It is also used to reflect the white light reflected by the sample back to the CCD camera 9, and to image the light through the CCD camera 9.

[0068] In one specific embodiment, the white light source imaging module 5 provided in this embodiment includes a second lens 51, a second reflector 52, a second dichroic mirror 53, a third reflector 54, and a third lens 55.

[0069] In this embodiment, an illumination optical path is constructed using a white light source 8, a second lens 51, a second reflector 52, a second dichroic mirror 53, and a third reflector 54. The white light generated by the white light source 8 is coupled to the laser incident module 3 through the illumination optical path 56, so that the white light and the incident laser are in the same optical path. Then, the white light is focused onto the sample surface on the sample stage by the microscope objective.

[0070] This embodiment also constructs an imaging optical path 57 using the third reflecting mirror 54, the second dichroic mirror 53, the third lens 55, and the CCD camera 9. This imaging optical path 57 reflects the white light reflected from the sample back to the CCD camera, which then forms the image. This specific embodiment of the invention separates the imaging optical path from the illumination optical path, enabling flexible control of the optical path and improving stability.

[0071] Specifically, the CCD camera 9 provided in this embodiment enables imaging of the sample surface morphology, used to observe and locate the structural units and study areas of the sample surface; it is located behind the laser incident module 3 and connected through a cage-type coaxial structured optomechanical assembly.

[0072] The microscope objective module 6 provided in a specific embodiment of the present invention includes a microscope objective suspension arm 61 and a microscope objective converter 62. The microscope objective suspension arm 61 provided in the embodiment of the present invention is located on the lower side of the cover of the support body 2 and is connected to a cage-type coaxial structure. The microscope objective converter 62 is connected to the microscope objective suspension arm 61, and the microscope objective converter 62 is coaxial with the fluorescence optical path through the cage-type coaxial structure.

[0073] In one specific embodiment, the cage-type coaxial structure provided by the present invention further includes an optical mounting plate 14, which is fixed to the support body 2 by a threaded connection. The cage-type coaxial structure is fixedly connected to the support body 2 by the optical mounting plate, and the microscope objective converter 62 is fixed to the optical mounting plate by a precision clamp.

[0074] The microscope objective converter 62 provided in the specific embodiment of the present invention is equipped with multiple microscope objectives 621 with different magnifications, which are used to adjust the field of view size and fluorescence collection efficiency of white light source imaging.

[0075] The sample stage 7 provided in the specific embodiment of the present invention includes a wide-range electric triaxial displacement stage 71, a high-precision piezoelectric ceramic nano-electric triaxial displacement stage 72, and a sample placement plate 73.

[0076] The sample placement plate 73 provided in the specific embodiment of the present invention is located on a high-precision piezoelectric ceramic nano-electric triaxial displacement stage 72, and the sample placement plate 73 is used to place the sample.

[0077] The high-precision piezoelectric ceramic nano-electric triaxial displacement stage 72 provided in this specific embodiment of the invention is located on a wide-range electric triaxial displacement stage 71. The high-precision piezoelectric ceramic nano-electric triaxial displacement stage 72 is used to employ piezoelectric ceramic drive and capacitive or resistive displacement sensing technology to meet the requirements of precise scanning and positioning of nanoscale structures such as silicon carbide color centers. It achieves multi-scale characterization compatibility: wafer-level large-range scanning and nanoscale precise positioning and scanning.

[0078] The wide-range electric three-axis displacement stage 71 provided in the specific embodiment of the present invention is used to realize rapid global positioning of wafer-level samples based on direct-drive servo motors and closed-loop feedback.

[0079] In a specific embodiment of this invention, a wide-range electric triaxial stage 71 is adjusted via a PC to achieve large-scale three-dimensional movement of the sample, moving it to the desired characterization surface. Then, a high-precision piezoelectric ceramic nano-electric triaxial stage 72 is controlled to focus the laser beam onto the sample. To achieve multi-scale characterization compatibility, the appropriate microscope objective can be selected by rotating the microscope objective converter according to both wafer-level large-scale scanning and nanoscale precise positioning and scanning modes.

[0080] In specific embodiments of the present invention, a combination of a wide-range electric triaxial displacement stage and a high-precision piezoelectric ceramic nano-electric triaxial displacement stage is used to achieve multi-scale characterization that is compatible with both wafer-level large-scale scanning and nano-level precise positioning and scanning.

[0081] In a specific embodiment of the present invention, a wide-range electric triaxial stage 71 and a high-precision piezoelectric ceramic nano-electric triaxial stage 72 are coordinated. The wide-range electric triaxial stage 71 is responsible for large-scale global positioning, while the high-precision piezoelectric ceramic nano-electric triaxial stage 72 performs precision scanning, highlighting the multi-scale characterization capability from wafer level to nanoscale, meeting the multi-scale analysis needs of semiconductor materials such as silicon carbide, and strengthening the technical advantages.

[0082] In one specific embodiment, the cage-type coaxial structure 1 provided by this invention includes a first cage-type coaxial module 11, a second cage-type coaxial module 12, and a third cage-type coaxial module 13. The first cage-type coaxial module 11 is connected to the second cage-type coaxial module 12 and the third cage-type coaxial module 13, respectively. The axes of the first cage-type coaxial module 11, the second cage-type coaxial module 12, and the third cage-type coaxial module 13 are parallel to each other and are each located independently on the support body 2. The laser optical path 35, the fluorescence optical path 45, the imaging optical path 57, and the illumination optical path 56 provided by this invention are all perpendicular to the support body. That is, the optical elements of the laser incident module 3, the fluorescence collection module 4, and the white light source imaging module 5 provided by this invention are all parallel to the support body 2, so that the optical elements will not have optical deviations due to the influence of gravity, thus having good stability.

[0083] In a specific embodiment of the present invention, the laser incident module 3 is fixed inside the first cage-type coaxial module 11. The first single-mode optical fiber 31 is mounted above the XY two-dimensional translation adjustment frame a via the first optical fiber adapter 36, and the aspherical first lens 32 is fixed above the Z-axis translation mounting base b. The XY two-dimensional translation adjustment frame a, the Z-axis translation mounting base b, the first two-dimensional optical adjustment frame c, and the cage-type cube are connected and fixed via a cage-type connecting support rod d. The incident direction and spatial position of the incident laser can be precisely adjusted via the XY two-dimensional translation adjustment frame a and the first two-dimensional optical adjustment frame c; the position and size of the laser beam waist can be changed according to different requirements by adjusting the Z-axis translation mounting base b. The cage-like cube used to support the laser incident module 3 fixed to the optical mounting plate 14 includes two first cage-like cubes e spliced ​​together vertically. The upper cube is equipped with a first dichroic mirror 33 facing backward at 45°, and the lower cube is equipped with a first reflector 34 facing left at 45°. The two maintain the optical path coaxial. The optical mounting plate 14 is laid flat on top of the support body 2 and is rigidly connected by bolts and nuts to ensure structural stability.

[0084] In a specific embodiment of the present invention, the fluorescence collection module 4 is fixed inside the second cage-type coaxial module 12. The second single-mode optical fiber 44 is mounted on the XY two-dimensional translation adjustment frame a via the second optical fiber adapter 46. The aspherical fourth lens 43 is fixed above the Z-axis translation mounting base b. The XY two-dimensional translation adjustment frame a, the Z-axis translation mounting base b, the first two-dimensional optical adjustment frame c, and the first cage-type cube are connected and fixed via the first cage-type connecting support rod d. Through the coordinated adjustment of the XY two-dimensional translation adjustment frame a, the first two-dimensional optical adjustment frame c, and the Z-axis translation mounting base b, the coupling efficiency of the fluorescence signal is optimized and the acquisition efficiency of the fluorescence signal is improved. The cage-like cube used to support the fluorescence collection module fixed on the optical mounting plate 14 includes two first cage-like cubes e spliced ​​together vertically. The upper cube is empty, and a third dichroic mirror 41 arranged at 45° to the right is installed in the lower cube to transmit the fluorescence signal radiated by the sample to be wiped upward. A filter 42 is installed between the first two-dimensional optical adjustment frame c and the Z-axis translation mounting seat b. According to the fluorescence signal of the sample to be tested excited by the incident laser of different wavelengths, a long-pass filter, a band-pass filter or a short-pass filter can be flexibly selected.

[0085] In a specific embodiment of the present invention, the white light source imaging module 5 is fixed inside the third cage-type coaxial module 13. The white light source imaging module 5 is installed parallel to the rear side of the laser incident module 3, and the optical path is coaxial through the third reflecting mirror 54 inside the second cage-type cube f. The illumination optical path 56 consists of a white light source 8, a second two-dimensional optical adjustment frame g, a second lens 51, a second reflecting mirror 52, and a cage-type support assembly. The illumination white light is reflected to the left by the second reflecting mirror 52, transmitted to the left by the second dichroic mirror 53, and reflected forward by the third reflecting mirror 54 onto the first dichroic mirror 33, coaxial with the optical path of the incident laser, providing uniform illumination for sample morphology imaging. The imaging optical path 57 consists of a CCD camera 9, a second two-dimensional optical adjustment frame g, a third lens 55, a second dichroic mirror 53, a third reflecting mirror 54, and a cage-type support assembly. The imaging light reflected back by the third reflecting mirror 54 and upward by the second dichroic mirror 53 is then injected into the CCD camera to complete the sample morphology imaging.

[0086] In one specific embodiment, the first cage-type coaxial module 11 and the second cage-type coaxial module 12 provided in this embodiment each independently include an XY two-dimensional translation adjustment frame a, a Z-axis translation mounting seat b, a first two-dimensional optical adjustment frame c, a first cage-type connecting support rod d, and two first cage-type cubes e arranged vertically.

[0087] In the first cage-type coaxial module 11 or the second cage-type coaxial module 12, the XY two-dimensional translation adjustment frame a, the Z-axis translation mounting seat b, the first two-dimensional optical adjustment frame c and the first cage-type cube e are fixedly connected from top to bottom by the first cage-type connecting support rod d. The first cage-type connecting support rod d is fixed to the support body 2 by the optical mounting plate 14.

[0088] The XY two-dimensional translation adjustment frame a and the first two-dimensional optical adjustment frame c provided in the specific embodiments of the present invention are used to adjust the incident direction and spatial position of the incident laser, and are also used to optimize the coupling efficiency of the fluorescence signal, which can efficiently optimize the coupling efficiency between the fluorescence signal and the single-mode optical fiber.

[0089] The Z-axis translation mounting base b provided in the specific embodiment of the present invention is used to change the position and size of the beam waist of the incident laser.

[0090] In a specific embodiment of the present invention, the first cage-type coaxial module 11 has a first reflector 34 and a first dichroic mirror 33 respectively in its two first cage-type cubes e. The second cage-type coaxial module 12 has a third dichroic mirror 41 in its first cage-type cube e. The first cage-type cube e with the third dichroic mirror 41 is connected to the first cage-type cube e with the first dichroic mirror 33, so that the incident laser and white light are coupled together to the fluorescence collection module 4, and the white light reflected by the sample can also be reflected back to the white light source imaging module 5.

[0091] The third cage-type coaxial module 13 provided in the specific embodiment of the present invention includes a first cage-type coaxial submodule 131, a second cage-type coaxial submodule 132, and a second cage-type cube f. The first cage-type coaxial submodule 131 and the second cage-type coaxial submodule 132 each include a second two-dimensional optical adjustment frame g, a second cage-type connecting support rod h, and a third cage-type cube i.

[0092] In a specific embodiment of the present invention, in the first cage-type coaxial submodule 131 or the second cage-type coaxial submodule 132, the second two-dimensional optical adjustment frame g and the third cage-type cube i, which are arranged from top to bottom, are fixedly connected by the second cage-type connecting support rod h.

[0093] In a specific embodiment of the present invention, the third cage cube i of the first cage coaxial submodule 131 and the second cage coaxial submodule 132 are respectively provided with a second reflector 52 and a second dichroic mirror 53, and the second cage cube f is provided with a third reflector 54. The two third cage cubes i, the second cage cube f and the first cage cube e provided with the first dichroic mirror are interconnected, thereby coupling the white light generated by the white light source to the laser incident module 3.

[0094] In specific embodiments of the present invention, the precise angular configuration of the cage-like cube, reflector, and dichroic mirror (such as 45° reflection / transmission) ensures coaxial transmission of multiple optical paths, reduces optical path difference and energy loss, and improves system integration and signal acquisition efficiency.

[0095] The cage-type coaxial structure laser scanning confocal fluorescence imaging system provided in this invention has advantages such as high integration, ease of installation and debugging, strong anti-interference ability, and long maintenance cycle. The cage-type coaxial optical path architecture and precision positioning pins secure the core components, ensuring optical path stability and accuracy. Single-mode optical fibers, large numerical microscope objectives, and single-photon detectors enable efficient collection and analysis of fluorescence signals. A combination of a wide-range motorized triaxial stage and a high-precision piezoelectric ceramic nano-motorized triaxial stage achieves multi-scale characterization compatibility, including wafer-level large-range scanning and nanoscale precise positioning and scanning. Simultaneously, a white light source imaging module is integrated to synchronously complete sample morphology imaging and fluorescence signal acquisition, enabling simultaneous fluorescence imaging-morphology imaging analysis and data recording and storage.

[0096] On the other hand, the present invention also provides a laser scanning confocal fluorescence imaging method with a cage-like coaxial structure, wherein the laser scanning confocal fluorescence imaging system employing the aforementioned cage-like coaxial structure includes:

[0097] The incident laser generated by the laser is irradiated onto the sample on the sample stage through the laser incident module, the fluorescence collection module and the microscope objective module. The fluorescence signal generated after the sample is excited is coupled into the optical property characterization device through the fluorescence collection module to realize the fluorescence imaging of the sample.

[0098] The white light generated by the white light source is coupled to the laser incident module through the white light source imaging module, and then sequentially illuminates the sample on the sample stage through the laser incident module, the fluorescence collection module, and the microscope objective module. The white light reflected from the sample then sequentially passes through the microscope objective module, the fluorescence collection module, the laser incident module, and is reflected back to the CCD camera through the white light source imaging module, and is then imaged by the CCD camera.

Claims

1. A laser scanning confocal fluorescence imaging system of a cage coaxial structure, characterized in that, The laser scanning confocal fluorescence imaging system comprises a laser incidence module, a fluorescence collection module and a white light source imaging module fixed in a cage coaxial structure and located on a support body, and a microscope objective module and a sample stage located below the support body, wherein: The laser incidence module is used for coupling the incident laser and the white light together into the fluorescence collection module, and is also used for reflecting the white light reflected by the sample back to the white light source imaging module; The white light source imaging module is used for coupling the white light to the laser incidence module through a set optical path, so that the white light is in the coaxial optical path with the incident laser, and is also used for reflecting the white light reflected by the sample back to the CCD camera for imaging by the CCD camera; The fluorescence collection module is used for irradiating the incident laser and the white light together to the sample located on the sample stage through the microscope objective module, and is also used for coupling the fluorescence signal generated after the sample is excited into an optical property characterization device to realize sample fluorescence imaging; The cage coaxial structure comprises a first cage coaxial module, a second cage coaxial module and a third cage coaxial module, the first cage coaxial module is connected with the second cage coaxial module and the third cage coaxial module respectively, the axes of the first cage coaxial module, the second cage coaxial module and the third cage coaxial module are parallel to each other and are located on the support body respectively; The laser incidence module is fixed in the first cage coaxial module, the fluorescence collection module is fixed in the second cage coaxial module, and the white light source imaging module is fixed in the third cage coaxial module.

2. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The laser incidence module comprises a first single-mode optical fiber, a first lens, a first dichroic mirror and a first mirror in sequence from top to bottom along the axial direction; The first single-mode optical fiber is used for coupling the incident laser into the laser incidence module, and the coupled incident laser passes through the first lens and then the first dichroic mirror and the first mirror in sequence with the white light to be coupled into the fluorescence collection module; The first dichroic mirror is also used for reflecting the white light reflected by the sample back to the white light source imaging module.

3. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The white light source imaging module comprises a second lens, a second mirror, a second dichroic mirror, a third mirror and a third lens; The illumination light path is constructed through the white light source, the second lens, the second mirror, the second dichroic mirror and the third mirror, and the white light generated by the white light source is coupled into the laser incidence module through the illumination light path, so that the white light is in the coaxial optical path with the incident laser; The imaging light path is constructed through the third mirror, the second dichroic mirror, the third lens and the CCD camera, and the white light reflected by the sample is reflected back to the CCD camera through the imaging light path for imaging by the CCD camera.

4. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The fluorescence collection module comprises a third dichroic mirror, a filter, a fourth lens and a second single-mode optical fiber; The third dichroic mirror is used for reflecting the incident laser and the white light together to the microscope objective module, and is also used for reflecting the white light passing through the sample back to the laser incidence module; The third dichroic mirror, the filter, the fourth lens and the second single-mode optical fiber construct a fluorescence light path, through which the fluorescence signal generated after excitation of the sample is coupled into the optical property characterization device to realize sample fluorescence imaging; The third dichroic mirror and the filter are also used to filter out stray light and residual laser light.

5. The laser scanning confocal fluorescence imaging system of claim 4, wherein, The optical property characterization device comprises a single-photon detector, a data acquisition card and a PC end; The single-photon detector is connected with the second single-mode optical fiber, and is used to perform photon counting on the received fluorescence signal to obtain an electric pulse signal; The data acquisition card is connected with the single-photon detector, and is used to convert the electric pulse signal into a digital signal and transmit the digital signal to the PC end, through which fluorescence imaging and real-time monitoring of fluorescence intensity are realized.

6. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The first cage coaxial module and the second cage coaxial module each independently comprise an XY two-dimensional translation adjustment frame, a Z-axis translation mounting seat, a first two-dimensional optical adjustment frame, a first cage connecting support rod and two first cage cubes arranged in an up-down manner; In the first cage coaxial module or the second cage coaxial module, the XY two-dimensional translation adjustment frame, the Z-axis translation mounting seat, the first two-dimensional optical adjustment frame and the first cage cubes are fixedly connected from top to bottom through the first cage connecting support rod; The XY two-dimensional translation adjustment frame and the first two-dimensional optical adjustment frame are used to adjust the incident direction and spatial position of incident laser light and also to optimize the coupling efficiency of the fluorescence signal; The Z-axis translation mounting seat is used to change the beam waist position and size of the incident laser light; The first cage coaxial module comprises a first cage coaxial sub-module and a second cage coaxial sub-module and a second cage cube, and the first cage coaxial sub-module and the second cage coaxial sub-module each independently comprise a second two-dimensional optical adjustment frame, a second cage connecting support rod and a third cage cube; In the first cage coaxial sub-module or the second cage coaxial sub-module, the second two-dimensional optical adjustment frame and the third cage cube arranged in an up-down manner are fixedly connected through the second cage connecting support rod; The second two-dimensional optical adjustment frame and the third cage cube are used to adjust the incident direction and spatial position of incident laser light and also to optimize the coupling efficiency of the fluorescence signal. The third cage-shaped cube of the first cage-shaped coaxial sub-module and the second cage-shaped coaxial sub-module is respectively provided with a second mirror and a second dichroic mirror, the third cage-shaped cube is provided with a third mirror, and the two third cage-shaped cubes, the second cage-shaped cube and the first cage-shaped cube provided with the first dichroic mirror are in communication with each other, so as to couple the white light generated by the white light source to the laser incidence module, and the second mirror, the second dichroic mirror and the third mirror are optical elements of the white light source imaging module.

7. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The microscope objective module comprises a microscope objective suspension arm and a microscope objective converter; One end of the microscope objective suspension arm is connected with the cage-shaped coaxial structure, and the other end is connected with the microscope objective converter, the microscope objective suspension arm is used for fixing the microscope objective converter, and the microscope objective converter is coaxial with the fluorescence collection module, so that the incident laser and the white light are incident to the microscope objective converter together; The microscope objective converter comprises a plurality of microscopes with different magnifications, the field of view size of the white light source imaging and the fluorescence collection efficiency are adjusted by the microscopes with different magnifications, and multi-scale sample characterization is realized.

8. The laser scanning confocal fluorescence imaging system of claim 1, wherein, The sample stage comprises a wide-range electric three-axis displacement stage, a high-precision piezoelectric ceramic nanometer electric three-axis displacement stage and a sample placing plate. The sample placing plate is located on the high-precision piezoelectric ceramic nanometer electric three-axis displacement stage, and is used for placing a sample. The high-precision piezoelectric ceramic nanometer electric three-axis displacement stage is located on the wide-range electric three-axis displacement stage, and is used for realizing nanometer scale scanning. The wide-range electric three-axis displacement stage is used for realizing wafer-level range scanning.

9. A method of laser scanning confocal fluorescence imaging of a caged coaxial structure, characterized by, The laser scanning confocal fluorescence imaging system with the cage-shaped coaxial structure according to any one of claims 1-8 comprises: The incident laser generated by the laser is irradiated to the sample on the sample stage through the laser incidence module, the fluorescence collection module and the microscope objective module, the fluorescence signal generated after the sample is excited is coupled into the optical property characterization equipment through the fluorescence collection module to realize sample fluorescence imaging; The white light generated by the white light source is coupled to the laser incidence module through the white light source imaging module, and is sequentially irradiated to the sample on the sample stage through the laser incidence module, the fluorescence collection module and the microscope objective module, the white light reflected by the sample is then sequentially reflected back to the CCD camera through the microscope objective module, the fluorescence collection module, the laser incidence module and the white light through the white light source imaging module, and imaging is realized through the CCD camera.

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