CN120801272B - A cage-type coaxial structure laser scanning confocal fluorescence imaging system and method - Google Patents
A cage-type coaxial structure laser scanning confocal fluorescence imaging system and methodInfo
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- CN120801272B CN120801272B CN202511278149.0A CN202511278149A CN120801272B CN 120801272 B CN120801272 B CN 120801272B CN 202511278149 A CN202511278149 A CN 202511278149A CN 120801272 B CN120801272 B CN 120801272B
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Abstract
本发明公开了一种笼式共轴结构的激光扫描共聚焦荧光成像系统和方法，该方法利用白光源成像模块将白光耦合到激光入射模块，通过激光光路使得白光和激光一起照射样品表面，激发的荧光通过荧光光路耦合到光学性质表征设备中实现荧光成像，经过样品表面反射回的白光依次通过激光光路和成像光路通过CCD相机成像得到形貌成像，从而同时实现了荧光成像和形貌成像，并且由于本发明提供的激光入射模块、白光源成像模块和荧光收集模块均设置在笼式共轴结构内，结构简单，各自形成的光路更为稳定，因此，能够稳定、高效的实现同步的形貌成像和荧光成像，能够快速适配不同波长激光的实验场景。
This invention discloses a cage-type coaxial structure laser scanning confocal fluorescence imaging system and method. The method 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 beam simultaneously illuminate the sample surface. The excited fluorescence is coupled to an optical property characterization device via a fluorescence optical path to achieve fluorescence imaging. The white light reflected back from the sample surface sequentially passes through the laser optical path and the imaging optical path, and is then imaged by a CCD camera to obtain morphological imaging. Thus, fluorescence imaging and morphological imaging are simultaneously achieved. Furthermore, since the laser incident module, white light source imaging module, and fluorescence collection module provided by this invention are all housed within a cage-type 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 wavelengths of laser light.
Description
Technical Field
The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a laser scanning confocal fluorescence imaging system and method of a cage type coaxial structure.
Background
The laser scanning confocal microscope system is separated from a field light source and a local plane imaging mode of a traditional optical microscope, a light path diagram of the laser scanning confocal microscope adopts a laser beam as a light source, the laser beam is reflected to an objective lens through an illumination pinhole and is focused on a sample through a spectroscope, and the sample focal plane is rapidly scanned and imaged point by point, line by line and face by face.
The confocal laser scanning microscopic imaging system is commonly used for high-precision imaging of fluorescent signals, single photon source characteristic analysis and multi-scale sample characterization research. At present, the kit becomes an indispensable professional detection tool in the fields of material science research, biomedicine and the like.
The traditional laser scanning confocal microscope is generally complex in structure, high in purchase and maintenance cost, scattered in light path architecture, more in optical elements and positioning accuracy, and the defects result in unstable fluorescence coupling efficiency and weak anti-interference capability.
The invention patent application with publication number of CN101013136A discloses a laser-induced fluorescence confocal scanning device and a method, and belongs to the technical field of biochip detection. The existing device has low fluorescence collection and utilization efficiency, and the focusing control in the scanning process is very difficult. According to the embodiment of the invention, the middle perforated total reflecting mirror is adopted to split the incident laser and the fluorescence induced and reflected, when the laser beam passes through the aperture, the energy loss is small, almost all laser energy can be irradiated on the biochip, and when the fluorescence is collected, the fluorescence collection angle is large, so that the incidence efficiency of the laser is improved, and the collection efficiency of the fluorescence is improved. The control of the computer is utilized to keep the pinholes of the light source and the pinholes of the detector in approximate confocal relation, focusing is only needed once before scanning, and focusing control is simple and convenient. The system disclosed in this patent application cannot be coupled to a topography imaging system.
The invention patent application publication No. CN103743718A discloses a confocal microscopic Raman (Raman) and Laser Induced Breakdown Spectroscopy (LIBS) combined laser spectrum analyzer. The instrument comprises a microscopic Raman system, a microscopic LIBS system, a high-resolution microscopic imaging system, a confocal microscopic optical path, a spectrum receiving system with a time resolution function and the like, and can automatically switch a white light microscopic imaging observation mode, an automatic focusing mode, a LIBS spectrum working mode, a Raman spectrum working mode and the like. The invention has the remarkable characteristics that the Raman and LIBS are compactly combined by utilizing a microscopic confocal system, qualitative and quantitative analysis of substance elements and molecular structures at the same micro position can be realized, and the chemical analysis of spatially resolved elements and substance structures can be performed on a micrometer scale by combining a high-resolution imaging function, so that complete information such as spatial distribution images of chemical elements, substance structures and physical conditions of a sample can be obtained. However, the confocal microscope disclosed in the patent application of the invention has poor light path stability and low multi-mode imaging efficiency.
Disclosure of Invention
The invention provides a laser scanning confocal fluorescence imaging system with a cage type coaxial structure, which can realize synchronous morphology imaging and fluorescence imaging more stably and efficiently and can rapidly adapt to experimental scenes of lasers with different wavelengths, and comprises a laser incidence module, a fluorescence collection module and a white light source imaging module which are fixed in the cage type coaxial structure and are positioned on a supporting main body, and a microscope objective module and a sample table which are positioned below the supporting main body, wherein:
the laser incidence module is used for coupling incident laser and white light to the fluorescence collection module together and 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 white light to the laser incidence module through a set light path, so that the white light and the incident laser are in a coaxial light path, and the white light reflected by the sample is reflected back to the CCD camera to be imaged through the CCD camera;
the fluorescence collection module is used for irradiating incident laser and white light to a sample on the sample stage through the micro objective module, and is also used for coupling a fluorescence signal generated after the excitation of the sample into the optical property characterization equipment to realize fluorescence imaging of the sample.
Preferably, the laser incidence module sequentially comprises a first single-mode optical fiber, a first lens, a first dichroic mirror and a first reflecting mirror from top to bottom along the axial direction;
the first single-mode optical fiber is used for coupling incident laser into the laser incidence module, and the coupled incident laser passes through the first lens and then is coupled with the white light into the fluorescence collection module through the first dichroic mirror and the first reflecting mirror in sequence;
the first dichroic mirror is also used to reflect white light reflected by the sample back to the white light source imaging module.
Preferably, the white light source imaging module includes a second lens, a second mirror, a second dichroic mirror, a third mirror, and a third lens;
An illumination light path is constructed through a white light source, a second lens, a second reflecting mirror, a second dichroic mirror and a third reflecting mirror, and white light generated by the white light source is coupled to the laser incidence module through the illumination light path, so that the white light and incident laser are in a coaxial light path;
And constructing an imaging light path through the third reflecting mirror, the second dichroic mirror, the third lens and the CCD camera, reflecting the white light reflected by the sample back to the CCD camera through the imaging light path, and imaging through the CCD camera.
Preferably, the fluorescence collection module comprises a third dichroic mirror, an optical filter, a fourth lens and a second single mode optical fiber;
The third dichroic mirror is used for reflecting incident laser and white light to the micro objective lens module together and is also used for reflecting the white light passing through the sample back to the laser incidence module;
the third dichroic mirror, the optical filter, the fourth lens and the second single-mode optical fiber construct a fluorescence light path, and a fluorescence signal generated after the excitation of the sample is coupled into optical property characterization equipment through the fluorescence light path to realize fluorescence imaging of the sample;
The third dichroic mirror and the filter are also used for filtering out stray light and remaining laser light.
Preferably, 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 for carrying out photon counting on the received fluorescent signal to obtain an electric pulse signal;
The data acquisition card is connected with the single photon detector and is used for converting an electric pulse signal into a digital signal and transmitting the digital signal to the PC end, and fluorescent imaging and real-time monitoring of fluorescent intensity are realized through the PC end.
Preferably, the cage type coaxial structure comprises a first cage type coaxial module, a second cage type coaxial module and a third cage type coaxial module, wherein the first cage type coaxial module is respectively connected with the second cage type coaxial module and the third cage type coaxial module, and the axes of the first cage type coaxial module, the second cage type coaxial module and the third cage type coaxial module are mutually parallel and are respectively and independently positioned on the supporting main body;
the laser incidence module is fixed in the first cage type coaxial module, the fluorescence collection module is fixed in the third cage type coaxial module, and the white light source imaging module is fixed in the second cage type coaxial module.
Preferably, the first cage type coaxial module and the second cage type coaxial module independently comprise an XY two-dimensional translation adjusting frame, a Z-axis translation mounting seat, a first two-dimensional optical adjusting frame, a first cage type connecting support rod and two first cage type cubes which are arranged up and down;
In the first cage type coaxial module or the second cage type coaxial module, the XY two-dimensional translation adjusting frame, the Z-axis translation mounting seat, the first two-dimensional optical adjusting frame and the first cage type cube are fixedly connected from top to bottom through a first cage type connecting supporting rod;
the XY two-dimensional translation adjusting frame and the first two-dimensional optical adjusting frame are used for adjusting the incidence direction and the space position of the incident laser and optimizing and adjusting the coupling efficiency of the fluorescent signals;
the Z-axis translation mounting seat is used for changing the beam waist position and the beam waist size of incident laser;
A first reflecting mirror and a first dichroic mirror are respectively arranged in two first cage cubes of the first cage coaxial module, a third dichroic mirror is arranged in the first cage cube of the second cage coaxial module, the first cage cube provided with the third dichroic mirror is communicated with the first cage cube provided with the first dichroic mirror, so that incident laser and white light are coupled to the fluorescence collection module together, white light reflected by a sample can be reflected back to the white light source imaging module, the first reflecting mirror and the first dichroic mirror are optical elements of the laser incidence module, and the third dichroic mirror is an optical element of the fluorescence collection module;
the third cage type coaxial module comprises a first cage type coaxial sub-module, a second cage type coaxial sub-module and a second cage type cube, and the first cage type coaxial sub-module and the second cage type coaxial sub-module independently comprise a second two-dimensional optical adjusting frame, a second cage type connecting support rod and a third cage type cube respectively;
in the first cage type coaxial sub-module or the second cage type coaxial sub-module, a second two-dimensional optical adjusting frame and a third cage type cube which are arranged from top to bottom are fixedly connected through a second cage type connecting supporting rod;
The first cage type coaxial sub-module and the second cage type coaxial sub-module are internally provided with a second reflecting mirror and a second dichroic mirror respectively, the second cage type cube is internally provided with a third reflecting mirror, and the third cage type cube, the second cage type cube and the first cage type cube provided with the first dichroic mirror are mutually communicated, so that white light generated by the white light source is coupled to the laser incidence module, and the second reflecting mirror, the second dichroic mirror and the third reflecting mirror are optical elements of the white light source imaging module.
Preferably, the microscope objective module comprises a microscope objective suspension arm and a microscope objective changer;
one end of the microscope objective suspension arm is connected with the cage type coaxial structure, the other end of the microscope objective suspension arm 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 incident laser and white light are incident to the microscope objective converter together;
The microscope objective converter comprises a plurality of microscopes with different multiplying powers, and the field of view size and fluorescence collection efficiency of the white light source imaging are adjusted through the microscopes with different multiplying powers, so that the multi-scale sample characterization is realized.
Preferably, the sample platform comprises a wide-range electric triaxial displacement platform, a high-precision piezoelectric ceramic nano electric triaxial displacement platform and a sample placing flat plate;
the sample placing flat plate is positioned on the high-precision piezoelectric ceramic nano electric triaxial displacement table and is used for placing a sample;
The high-precision piezoelectric ceramic nano electric triaxial displacement table is positioned on a wide-range electric triaxial displacement table and is used for realizing nano-scale scanning;
The wide-range electric triaxial displacement table is used for realizing wafer-level range scanning.
In another aspect, the present invention further provides a laser scanning confocal fluorescent imaging method of a cage-type coaxial structure, and a laser scanning confocal fluorescent imaging system adopting the cage-type coaxial structure includes:
The method comprises the steps that incident laser generated by a laser irradiates a sample on a sample table through a laser incidence module, a fluorescence collection module and a microscope objective module, and a fluorescence signal generated after the sample is excited is coupled into optical property characterization equipment through the fluorescence collection module to realize fluorescence imaging of the sample;
The white light generated by the white light source is coupled to the laser incidence module through the white light source imaging module, sequentially irradiates the sample on the sample table through the laser incidence module, the fluorescence collection module and the microscope objective module, sequentially reflects the white light reflected by the sample 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 images through the CCD camera.
Compared with the prior art, the invention has the beneficial effects that:
The invention utilizes the white light source imaging module to couple the white light to the laser incidence module, the white light and the laser light irradiate the surface of the sample together through the laser light path, the excited fluorescence is coupled to the optical property characterization equipment through the fluorescence light path to realize fluorescence imaging, the white light reflected back from the surface of the sample sequentially passes through the laser light path and the imaging light path to be imaged through the CCD camera to obtain morphology imaging, thereby realizing fluorescence imaging and morphology imaging simultaneously.
Drawings
FIG. 1 is a schematic diagram of a laser scanning confocal fluorescence imaging system with a cage-type coaxial structure according to an embodiment of the present invention;
Fig. 2 is a schematic diagram of an optical path of a laser scanning confocal fluorescence imaging system with a cage-type coaxial structure according to an embodiment of the present invention.
1-Cage coaxial structure, 11-first cage coaxial module, 12-second cage coaxial module, 13-third cage coaxial module, 131-first cage coaxial sub-module, 132 second cage coaxial sub-module, 14-optical mounting plate, a-XY two-dimensional translation adjusting frame, b-Z axis translation mounting seat, c-first two-dimensional optical adjusting frame, d-first cage connecting support bar, e-first cage cube, f-second cage cube, g-second two-dimensional optical adjusting frame, h-second cage connecting support bar, i-third cage cube, 2-support body, 3-laser incidence module, 31-first single mode fiber, 32-first lens, 33-first two-dimensional color mirror, 34-first reflecting mirror, 35-laser light path, 36-first optical fiber adapter, 4-fluorescence collecting module, 41-third dichroic mirror, 42-optical filter, 43-fourth lens, 44-second single mode optical fiber, 45-fluorescence light path, 46-second optical fiber 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 light path, 57-imaging light path, 6-micro objective lens module, 61-micro objective lens suspension arm, 62-micro objective lens converter, 621-micro objective lens, 7-sample stage, 71-wide range electric triaxial displacement stage, 72-high precision piezoelectric ceramic nanometer electric triaxial displacement stage, 73-sample placing plate, 8-white light source and 9-CCD camera.
Detailed Description
In the prior art, although the confocal coupling scheme exists, the problems of poor light path stability and low multi-mode imaging efficiency of the confocal microscope exist, the specific embodiment of the invention separates an illumination light path and an imaging light path of the white light source imaging module, white light can be coupled into a light path of laser through the illumination light path, the light path of stable laser irradiates the surface of a sample, the fluorescent signal generated by excitation and the white light passing through the sample are skillfully separated by utilizing a third dichroic mirror of the fluorescent collecting module, the fluorescent signal realizes fluorescent imaging through the fluorescent light path, the white light passing through the sample realizes morphology imaging through the light path and the imaging light path of the laser, so that fluorescent imaging and morphology imaging are simultaneously obtained, and the specific embodiment of the invention skillfully designs the light path trend under the cage type coaxial structure, so that stable and efficient synchronous morphology imaging and fluorescent imaging can be realized by utilizing the stability of the cage type coaxial structure, and experimental scenes of lasers with different wavelengths can be quickly adapted.
The embodiment of the invention provides a laser scanning confocal fluorescence imaging system with a cage type coaxial structure, which integrates a white light source imaging module 5 in the cage type coaxial structure and is positioned behind a fluorescence collecting module 4 and a laser incidence module 3, white light is coupled into a light path of the laser incidence module 3 from the rear through a reflecting mirror, the system synchronously completes appearance imaging and fluorescence imaging of a sample, realizes appearance imaging and fluorescence imaging synchronous analysis, and the specific structure is shown in fig. 1 and 2, and comprises:
The laser scanning confocal fluorescence imaging system provided by the embodiment of the invention comprises a laser incidence module 3, a fluorescence collection module 4 and a white light source imaging module 5 which are fixed in a cage type coaxial structure 1 and positioned on a supporting main body 2, and a microscope objective module 6 and a sample table 7 which are positioned below the supporting main body 2.
The laser incidence module 3 provided in the embodiment of the present invention is used for coupling the incident laser and the white light together to the fluorescence collection module 4, and is also used for reflecting the white light reflected by the sample back to the white light source imaging module 5.
In a specific embodiment, the laser incidence module 3 provided in the embodiment of the present invention includes, in order from top to bottom along an axial direction, a first single-mode optical fiber 31, a first lens 32, a first dichroic mirror 33, and a first reflecting mirror 34;
the first single mode fiber 31 provided in the embodiment of the present invention is used for coupling the incident laser into the laser incidence module 3, and the incident laser after being coupled into the first lens 32 and the white light after being coupled into the first dichroic mirror 33 and the first reflecting mirror 34 are coupled into the fluorescence collection module 4.
Specifically, the first mirror 34 provided in this embodiment is 45 ° and is mounted within the cage.
The first dichroic mirror 33 provided in the 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.
In one embodiment, the fluorescence collection module 4 provided by the embodiment of the present invention is used for illuminating the incident laser light and the white light together to the sample on the sample stage 7 through the micro objective lens module 6, and is further used for coupling the fluorescence signal generated after the excitation of the sample into the optical property characterization device to realize fluorescence imaging of the sample.
In a specific embodiment, the fluorescence collection module 4 provided in this embodiment includes a third dichroic mirror 41, an optical filter 42, a fourth lens 43, and a second single-mode optical fiber 44;
The third dichroic mirror 41 provided in this embodiment is used for reflecting incident laser light together with white light to the microscope objective module 6, and also for reflecting the white light passing through the sample back to the laser light incidence module 3.
The third dichroic mirror 41, the optical filter 42, the fourth lens 43 and the second single-mode optical fiber 44 provided in the present embodiment construct a fluorescence light path 45, and a fluorescence signal generated after excitation of the sample is coupled into the optical property characterization device through the fluorescence light path 45 to realize fluorescence imaging of the sample.
The third dichroic mirror 41 and the filter 42 provided in the present embodiment also serve to filter out stray light and remaining laser light.
Specifically, the optical filter 42 provided in this embodiment is implemented to further filter the laser signal, enhance the signal-to-noise ratio of the fluorescent signal, and is located on the upper side of the third dichroic mirror 41.
Specifically, the optical property characterization device provided by the embodiment includes 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 for carrying out photon counting on the received fluorescent signal to obtain an electric pulse signal;
The data acquisition card is connected with the single photon detector and is used for converting an electric pulse signal into a digital signal and transmitting the digital signal to the PC end, and fluorescent imaging and real-time monitoring of fluorescent intensity are realized through the PC end.
In a specific embodiment, the laser incidence module 3 and the fluorescence collection module 4 provided in this embodiment are installed in parallel along the front row, the horizontal heights are consistent and can be adjusted independently, the incident laser is coupled into the laser incidence module 3 through the first single mode fiber 31, is transmitted downwards through the first lens 32 and the first dichroic mirror 33 in sequence, is reflected leftwards through the first reflecting mirror 34 and is reflected downwards through the third dichroic mirror 41, and then is injected into the microscope objective converter 62, and is focused on the surface of a sample through the microscope objective 621, the fluorescence signal radiated by the sample to be measured is collected and converted into near-parallel light through the same microscope objective 621, and is then transmitted upwards through the third dichroic mirror 41 to the fluorescence collection module 4, and finally is coupled into a pulse signal through the second single mode fiber 44, and is received by the PC and is converted into an electrical signal.
The white light source imaging module 5 provided by the embodiment of the invention is used for coupling white light to the laser incidence module 3 through a set light path, so that the white light and the incident laser are in a coaxial light path, and the white light reflected by the sample is also reflected back to the CCD camera 9 to be imaged through the CCD camera 9.
In a specific embodiment, the white light source imaging module 5 provided in this embodiment includes a second lens 51, a second mirror 52, a second dichroic mirror 53, a third mirror 54, and a third lens 55.
The present embodiment constructs an illumination light path through the white light source 8, the second lens 51, the second reflecting mirror 52, the second dichroic mirror 53 and the third reflecting mirror 54, and the white light generated by the white light source 8 is coupled to the laser incidence module 3 through the illumination light path 56, so that the white light and the incident laser are in a coaxial light path, and then focused on the sample surface on the sample stage by the microscope objective.
The present embodiment also constructs an imaging optical path 57 through the third reflecting mirror 54, the second dichroic mirror 53, the third lens 55, and the CCD camera 9, and reflects the white light reflected by the sample back to the CCD camera through the imaging optical path 57, and images through the CCD camera. The embodiment of the invention separates the imaging light path from the illumination light path, can realize flexible regulation and control of the light path, and can improve the stability.
Specifically, the CCD camera 9 provided in this embodiment is used for imaging the surface morphology of the sample, and is used for observing and positioning the structural unit and the research area of the surface of the sample, and is located at the rear of the laser incidence module 3 and connected through the cage-type coaxial structure optomechanical assembly.
The microscope objective module 6 provided in the embodiment of the present invention includes a microscope objective suspension arm 61 and a microscope objective converter 62, where the microscope objective suspension arm 61 provided in the embodiment of the present invention is connected to a cage-type coaxial structure at the lower side of the cover of the support body 2, and 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 light path through the cage-type coaxial structure.
In one embodiment, the cage-type coaxial structure provided in the embodiment of the present invention further includes an optical mounting plate 14, wherein the optical mounting plate 14 is fixed on the support body 2 through a threaded connection, the cage-type coaxial structure is fixedly connected with the support body 2 through the optical mounting plate, and the micro objective lens converter 62 is fixed on the optical mounting plate through a precision fixture.
The microscope objective changer 62 according to the embodiment of the present invention is provided with a plurality of microscope objectives 621 having different magnifications for adjusting the size of the field of view and the fluorescence collection efficiency of the white light source imaging.
The sample stage 7 provided by the embodiment of the invention comprises a wide-range electric triaxial displacement stage 71, a high-precision piezoelectric ceramic nano electric triaxial displacement stage 72 and a sample placing flat plate 73.
The sample placing flat plate 73 provided by the embodiment of the invention is positioned on the high-precision piezoelectric ceramic nano electric triaxial displacement table 72, and the sample placing flat plate 73 is used for placing a sample.
The high-precision piezoelectric ceramic nano electric triaxial displacement table 72 provided by the embodiment of the invention is positioned on the wide-range electric triaxial displacement table 71, and the high-precision piezoelectric ceramic nano electric triaxial displacement table 72 is used for adopting piezoelectric ceramic driving and capacitance or resistance displacement sensing technology to meet the precision scanning and positioning of nano-scale structures such as silicon carbide color centers and the like. The multi-scale characterization compatibility is realized, and the wafer level large-scale scanning and the nano level accurate positioning and scanning are realized.
The wide-range electric triaxial displacement table 71 provided by the embodiment of the invention is used for realizing the rapid global positioning of a wafer-level sample based on a direct-drive servo motor and closed-loop feedback.
The embodiment of the invention realizes the large-scale three-dimensional movement of the sample by adjusting the wide-range electric triaxial displacement table 71 at the PC end, moves the sample to a required characterization surface, and then controls the high-precision piezoelectric ceramic nano electric triaxial displacement table 72 to focus the laser beam on the sample to be measured. In order to realize multi-scale characterization compatibility, a proper microscope objective can be selected according to a wafer-level large-range scanning and nano-level accurate positioning and scanning two-mode rotation microscope objective converter.
The specific embodiment of the invention realizes the compatibility of wafer-level large-scale scanning and nanoscale accurate positioning and multi-scale characterization of scanning by means of the combination of the wide-range electric triaxial displacement table and the high-precision piezoelectric ceramic nano electric triaxial displacement table.
The wide-range electric triaxial displacement table 71 and the high-precision piezoelectric ceramic nano electric triaxial displacement table 72 are coordinated, the wide-range electric triaxial displacement table 71 is responsible for large-range global positioning, the high-precision piezoelectric ceramic nano electric triaxial displacement table 72 performs precise scanning, the wafer-level-nano-level multi-scale characterization capability is highlighted, the multi-scale analysis requirement of semiconductor materials such as silicon carbide is met, and the technical advantage is enhanced.
In one embodiment, the cage type coaxial structure 1 provided by the embodiment of the invention comprises a first cage type coaxial module 11, a second cage type coaxial module 12 and a third cage type coaxial module 13, wherein the first cage type coaxial module 11 is respectively connected with the second cage type coaxial module 12 and the third cage type coaxial module 13, and 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 mutually parallel and are respectively and independently positioned on the supporting main body 2. The laser light path 35, the fluorescence light path 45, the imaging light path 57 and the illumination light path 56 provided by the embodiment of the invention are perpendicular to the supporting body, that is, the optical elements of the laser incidence module 3, the fluorescence collection module 4 and the white light source imaging module 5 provided by the embodiment of the invention are parallel to the supporting body 2, so that the optical elements cannot cause optical deviation due to the influence of gravity, and therefore, the stability is good.
The laser incidence module 3 provided by the embodiment of the invention is fixed in the first cage type coaxial module 11, wherein a first single-mode fiber 31 is arranged above an XY two-dimensional translation adjusting frame a through a first fiber adapter 36, an aspheric first lens 32 is fixed above a Z-axis translation mounting seat b, and the XY two-dimensional translation adjusting frame a, the Z-axis translation mounting seat b, a first two-dimensional optical adjusting frame c and a cage type cube are connected and fixed by virtue of a cage type connecting support rod d. The incidence direction and the space position of the incident laser can be accurately adjusted through the XY two-dimensional translation adjusting frame a and the first two-dimensional optical adjusting frame c, the Z-axis translation mounting seat b is adjusted, and the position and the size of the laser beam waist are changed according to different requirements. The cage-type cube for supporting the laser incidence module 3 fixed on the optical mounting plate 14 comprises two first cage-type cubes e spliced up and down, wherein a first dichroic mirror 33 with 45 degrees backward is arranged in the upper cube, a first reflecting mirror 34 with 45 degrees leftward is arranged in the lower cube, the two mirrors keep the optical paths coaxial, the optical mounting plate 14 is horizontally arranged above the supporting body 2, and the two mirrors are rigidly connected by bolts and nuts, so that the structural stability is ensured.
The fluorescence collection module 4 provided by the embodiment of the invention is fixed in the second cage type coaxial module 12, a second single-mode fiber 44 is arranged on an XY two-dimensional translation adjusting frame a through a second optical fiber adapter 46, an aspheric fourth lens 43 is fixed above a Z-axis translation mounting seat b, and the XY two-dimensional translation adjusting frame a, the Z-axis translation mounting seat b, a first two-dimensional optical adjusting frame c and a first cage type cube are connected and fixed through a first cage type connecting support rod d. Through XY two-dimensional translation regulating frame a, first two-dimensional optics regulating frame c, Z axle translation mount pad b are adjusted in coordination, optimize fluorescent signal's coupling efficiency and improve fluorescent signal's collection efficiency. The cage-type cubes used for supporting the fluorescent collection module and fixed on the optical mounting plate 14 comprise two first cage-type cubes e spliced up and down, wherein the upper cubes are empty, a third dichroic mirror 41 which is arranged right at an angle of 45 degrees is arranged in the lower cubes, fluorescent signals radiated by a wiped sample are transmitted upwards, a light filter 42 is arranged between the first two-dimensional optical adjusting frame c and the Z-axis translation mounting seat b, and a long-pass light filter, a band-pass light filter or a short-pass light filter is flexibly selected according to fluorescent signals of the sample to be tested excited by incident lasers with different wavelengths.
The white light source imaging module 5 provided by the embodiment of the invention is fixed in the third cage type coaxial module 13, the white light source imaging module 5 is installed at the rear side of the laser incidence module 3 in parallel, and the optical path is coaxial through the third reflector 54 in the second cage type cube f. The illumination light path 56 is composed of a white light source 8, a second two-dimensional optical adjusting frame g, a second lens 51, a second reflecting mirror 52 and a cage type supporting component, illumination white light is reflected leftwards by the second reflecting mirror 52, the second dichroic mirror 53 is transmitted leftwards, the third reflecting mirror 54 is reflected forwards to the first dichroic mirror 33 and is coaxial with the light path of incident laser light, uniform illumination is provided for imaging the appearance of a sample, the imaging light path 57 is composed of the CCD camera 9, the second two-dimensional optical adjusting frame g, the third lens 55, the second dichroic mirror 53, the third reflecting mirror 54 and the cage type supporting component, and imaging light reflected back by the illumination white light is driven into the CCD camera through the backward reflection of the third reflecting mirror 54, the upward reflection of the second dichroic mirror 53, and the imaging of the appearance of the sample is completed.
In a specific embodiment, the first cage-type coaxial module 11 and the second cage-type coaxial module 12 provided in this embodiment include, respectively, an XY two-dimensional translation adjusting frame a, a Z-axis translation mounting seat b, a first two-dimensional optical adjusting frame c, a first cage-type connection supporting rod d, and two first cage-type cubes e disposed up and down.
In the first cage-type coaxial module 11 or the second cage-type coaxial module 12, the XY two-dimensional translational adjustment frame a, the Z-axis translational 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 a first cage-type connection support rod d, which is fixed to the support body 2 by an optical mounting plate 14.
The XY two-dimensional translation adjusting frame a and the first two-dimensional optical adjusting frame c provided by the embodiment of the invention are used for adjusting the incidence direction and the space position of incident laser, and are also used for optimizing and adjusting the coupling efficiency of fluorescent signals, so that the coupling efficiency of the fluorescent signals and single-mode fibers can be optimized efficiently.
The Z-axis translation mounting seat b provided by the embodiment of the invention is used for changing the position and the size of the tube bundle waist of the incident laser.
The two first cage cubes e of the first cage coaxial module 11 provided by the embodiment of the present invention are respectively provided with a first reflecting mirror 34 and a first dichroic mirror 33, the first cage cube e of the second cage coaxial module 12 is internally provided with a third dichroic mirror 41, and the first cage cube e provided with the third dichroic mirror 41 is communicated with the first cage cube e provided with the first dichroic mirror 33, so that the incident laser light and the white light are coupled to the fluorescence collection module 4 together, and the white light reflected by the sample can be reflected back to the white light source imaging module 5.
The third cage type coaxial module 13 provided by the embodiment of the invention comprises a first cage type coaxial sub-module 131, a second cage type coaxial sub-module 132 and a second cage type cube f, wherein the first cage type coaxial sub-module 131 and the second cage type coaxial sub-module 132 independently comprise a second two-dimensional optical adjusting frame g, a second cage type connecting support rod h and a third cage type cube i respectively.
In the specific embodiment of the invention, in the first cage type coaxial submodule 131 or the second cage type coaxial submodule 132, a second two-dimensional optical adjusting frame g and a third cage type cube i which are arranged from top to bottom are fixedly connected through a second cage type connecting supporting rod h.
The third cage cubes i of the first cage coaxial submodule 131 and the second cage coaxial submodule 132 provided by the embodiment of the invention are respectively provided with a second reflecting mirror 52 and a second dichroic mirror 53, the second cage cube f is internally provided with a third reflecting mirror 54, and the two third cage cubes i, the second cage cube f and the first cage cube e provided with the first dichroic mirror are mutually communicated, so that white light generated by the white light source is coupled to the laser incidence module 3.
The specific embodiment of the invention ensures the coaxial transmission of multiple light paths by means of the precise angle configuration (such as 45 DEG reflection/transmission) of the cage cube, the reflecting mirror and the dichroic mirror, reduces the optical path difference and the energy loss and improves the system integration level and the signal acquisition efficiency.
The cage-type coaxial-structure laser scanning confocal fluorescence imaging system provided by the embodiment of the invention has the advantages of high integration level, easiness in installation and debugging, strong anti-interference capability, long maintenance period and the like. The device can ensure stable and accurate light path through a cage type coaxial light path framework and a precise positioning pin fixing core component, realize efficient collection and analysis of fluorescent signals by utilizing a single-mode optical fiber, a large-value microscope objective, a single-photon detector and the like, realize multi-scale characterization compatibility of wafer-level large-scale scanning and nano-scale precise positioning and scanning by means of combination of a wide-range electric triaxial displacement table and a high-precision piezoelectric ceramic nano-electric triaxial displacement table, and synchronously complete sample morphology imaging and fluorescent signal acquisition by an integrated white light source imaging module, and realize synchronous analysis of fluorescent imaging-morphology imaging and data recording and storage.
On the other hand, the invention also provides a laser scanning confocal fluorescence imaging method of the cage type coaxial structure, and the laser scanning confocal fluorescence imaging system adopting the cage type coaxial structure comprises the following steps:
The method comprises the steps that incident laser generated by a laser irradiates a sample on a sample table through a laser incidence module, a fluorescence collection module and a microscope objective module, and a fluorescence signal generated after the sample is excited is coupled into optical property characterization equipment through the fluorescence collection module to realize fluorescence imaging of the sample;
The white light generated by the white light source is coupled to the laser incidence module through the white light source imaging module, sequentially irradiates the sample on the sample table through the laser incidence module, the fluorescence collection module and the microscope objective module, sequentially reflects the white light reflected by the sample 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 images through the CCD camera.
Claims (9)
1. The utility model provides a coaxial structure's of cage laser scanning confocal fluorescence imaging system which characterized in that, laser scanning confocal fluorescence imaging system is including fixing in the coaxial structure of cage, and is located laser incidence module, fluorescence collection module and the white light source imaging module on the support main part, and is located microscope objective module and the sample platform of support main part below, wherein:
the laser incidence module is used for coupling incident laser and white light to the fluorescence collection module together and 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 white light to the laser incidence module through a set light path, so that the white light and the incident laser are in a coaxial light path, and is also used for reflecting the white light reflected by the sample back to the CCD camera to form an image through the CCD camera;
The fluorescence collection module is used for irradiating incident laser and white light to a sample positioned on the sample stage through the micro objective module, and is also used for coupling a fluorescence signal generated after the excitation of the sample into the optical property characterization equipment to realize fluorescence imaging of the sample;
the cage type coaxial structure comprises a first cage type coaxial module, a second cage type coaxial module and a third cage type coaxial module, wherein the first cage type coaxial module is respectively connected with the second cage type coaxial module and the third cage type coaxial module, and the axes of the first cage type coaxial module, the second cage type coaxial module and the third cage type coaxial module are mutually parallel and are respectively and independently positioned on the supporting main body;
The laser incidence module is fixed in the first cage type coaxial module, the fluorescence collection module is fixed in the second cage type coaxial module, and the white light source imaging module is fixed in the third cage type coaxial module.
2. The cage-type coaxial-structured laser scanning confocal fluorescence imaging system of claim 1, wherein the laser incidence module comprises a first single-mode fiber, a first lens, a first dichroic mirror, and a first reflecting mirror in order from top to bottom along an axial direction;
the first single-mode optical fiber is used for coupling incident laser into the laser incidence module, and the coupled incident laser passes through the first lens and then is coupled with the white light into the fluorescence collection module through the first dichroic mirror and the first reflecting mirror in sequence;
the first dichroic mirror is also used to reflect white light reflected by the sample back to the white light source imaging module.
3. The cage-type coaxially structured laser scanning confocal fluorescence imaging system of claim 1, wherein said white light source imaging module comprises a second lens, a second mirror, a second dichroic mirror, a third mirror, and a third lens;
An illumination light path is constructed through a white light source, a second lens, a second reflecting mirror, a second dichroic mirror and a third reflecting mirror, and white light generated by the white light source is coupled to the laser incidence module through the illumination light path, so that the white light and incident laser are in a coaxial light path;
And constructing an imaging light path through the third reflecting mirror, the second dichroic mirror, the third lens and the CCD camera, reflecting the white light reflected by the sample back to the CCD camera through the imaging light path, and imaging through the CCD camera.
4. The cage-type coaxial-structured laser scanning confocal fluorescence imaging system of claim 1, wherein the fluorescence collection module comprises a third dichroic mirror, an optical filter, a fourth lens, and a second single-mode optical fiber;
The third dichroic mirror is used for reflecting incident laser and white light to the micro objective lens module together and is also used for reflecting the white light passing through the sample back to the laser incidence module;
the third dichroic mirror, the optical filter, the fourth lens and the second single-mode optical fiber construct a fluorescence light path, and a fluorescence signal generated after the excitation of the sample is coupled into optical property characterization equipment through the fluorescence light path to realize fluorescence imaging of the sample;
The third dichroic mirror and the filter are also used for filtering out stray light and remaining laser light.
5. The coaxial-structure-based 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 for carrying out photon counting on the received fluorescent signal to obtain an electric pulse signal;
The data acquisition card is connected with the single photon detector and is used for converting an electric pulse signal into a digital signal and transmitting the digital signal to the PC end, and fluorescent imaging and real-time monitoring of fluorescent intensity are realized through the PC end.
6. The coaxial-structure-based confocal laser fluorescence imaging system of claim 1, wherein the first and second coaxial-cage modules comprise an XY two-dimensional translational adjustment frame, a Z-axis translational mount, a first two-dimensional optical adjustment frame, a first cage-type connection support bar, and two first cage-type cubes arranged up and down, respectively;
In the first cage type coaxial module or the second cage type coaxial module, the XY two-dimensional translation adjusting frame, the Z-axis translation mounting seat, the first two-dimensional optical adjusting frame and the first cage type cube are fixedly connected from top to bottom through a first cage type connecting supporting rod;
the XY two-dimensional translation adjusting frame and the first two-dimensional optical adjusting frame are used for adjusting the incidence direction and the space position of the incident laser and optimizing and adjusting the coupling efficiency of the fluorescent signals;
the Z-axis translation mounting seat is used for changing the beam waist position and the beam waist size of incident laser;
A first reflecting mirror and a first dichroic mirror are respectively arranged in two first cage cubes of the first cage coaxial module, a third dichroic mirror is arranged in the first cage cube of the second cage coaxial module, the first cage cube provided with the third dichroic mirror is communicated with the first cage cube provided with the first dichroic mirror, so that incident laser and white light are coupled to the fluorescence collection module together, white light reflected by a sample can be reflected back to the white light source imaging module, the first reflecting mirror and the first dichroic mirror are optical elements of the laser incidence module, and the third dichroic mirror is an optical element of the fluorescence collection module;
the third cage type coaxial module comprises a first cage type coaxial sub-module, a second cage type coaxial sub-module and a second cage type cube, and the first cage type coaxial sub-module and the second cage type coaxial sub-module independently comprise a second two-dimensional optical adjusting frame, a second cage type connecting support rod and a third cage type cube respectively;
in the first cage type coaxial sub-module or the second cage type coaxial sub-module, a second two-dimensional optical adjusting frame and a third cage type cube which are arranged from top to bottom are fixedly connected through a second cage type connecting supporting rod;
The first cage type coaxial sub-module and the second cage type coaxial sub-module are internally provided with a second reflecting mirror and a second dichroic mirror respectively, the second cage type cube is internally provided with a third reflecting mirror, and the two third cage type cubes, the second cage type cube and the first cage type cube provided with the first dichroic mirror are mutually communicated, so that white light generated by the white light source is coupled to the laser incidence module, and the second reflecting mirror, the second dichroic mirror and the third reflecting mirror are optical elements of the white light source imaging module.
7. The coaxial-structure-based laser scanning confocal fluorescence imaging system of claim 1, wherein the microobjective module comprises a microobjective suspension arm and a microobjective transducer;
one end of the microscope objective suspension arm is connected with the cage type coaxial structure, the other end of the microscope objective suspension arm 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 incident laser and white light are incident to the microscope objective converter together;
The microscope objective converter comprises a plurality of microscopes with different multiplying powers, and the field of view size and fluorescence collection efficiency of the white light source imaging are adjusted through the microscopes with different multiplying powers, so that the multi-scale sample characterization is realized.
8. The laser scanning confocal fluorescence imaging system of a cage-type coaxial structure according to claim 1, wherein the sample stage comprises a wide-range electric triaxial displacement stage, a high-precision piezoelectric ceramic nano electric triaxial displacement stage and a sample placing flat plate;
the sample placing flat plate is positioned on the high-precision piezoelectric ceramic nano electric triaxial displacement table and is used for placing a sample;
The high-precision piezoelectric ceramic nano electric triaxial displacement table is positioned on a wide-range electric triaxial displacement table and is used for realizing nano-scale scanning;
The wide-range electric triaxial displacement table is used for realizing wafer-level range scanning.
9. A method for laser scanning confocal fluorescence imaging of a cage-type coaxial structure, characterized in that the system for laser scanning confocal fluorescence imaging of a cage-type coaxial structure according to any one of claims 1 to 8 is employed, comprising:
The method comprises the steps that incident laser generated by a laser irradiates a sample on a sample table through a laser incidence module, a fluorescence collection module and a microscope objective module, and a fluorescence signal generated after the sample is excited is coupled into optical property characterization equipment through the fluorescence collection module to realize fluorescence imaging of the sample;
The white light generated by the white light source is coupled to the laser incidence module through the white light source imaging module, sequentially irradiates the sample on the sample table through the laser incidence module, the fluorescence collection module and the microscope objective module, sequentially reflects the white light reflected by the sample 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 images through the CCD camera.
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