CN109828364B - Vacuum intracavity confocal microscopic imaging system and method based on cage structure - Google Patents

Abstract

The invention discloses a vacuum intracavity confocal microscopic imaging system and method based on a cage structure, wherein the system comprises the following steps: the device comprises a vacuum cavity, a vacuum cavity sealing cover, a quartz window sheet, a three-dimensional electric displacement table, a galvanometer system, a microscope objective, a fluorescent microscope tube, a cage-type structure optical-mechanical component, a laser, a single-mode optical fiber, an optical fiber beam splitter, a dichroic mirror, an optical filter, an imaging CCD (charge coupled device), a single photon detector, a pulse counter, a coincidence instrument, a computer and the like. The three-dimensional electric displacement table and the microscope objective are arranged in the vacuum cavity, the vibrating mirror system and the microscope system main body are arranged outside the vacuum cavity, and the microscope system main body adopts a stable cage structure and is assembled by a cage structure optical-mechanical assembly which is easy to install. The invention has the advantages of high integration level, small occupied volume, easy installation and debugging, simple and convenient operation and maintenance, and the like. The invention can be used for high-precision imaging of weak fluorescent signals in a vacuum cavity or a low-temperature cavity, high-efficiency collection of a single photon source and signal analysis.

Description

A confocal microscopy imaging system and method in a vacuum cavity based on a cage structure

Technical field

The invention relates to the field of optical microscopy imaging, and in particular to a confocal microscopy imaging system and method in a vacuum cavity based on a cage structure.

Background technique

Confocal microscope is a commonly used scientific research equipment for studying fluorescence imaging and weak fluorescence signal collection, detection and signal analysis. Researchers need to study the properties of fluorescence radiated by samples in vacuum or low-temperature environments under certain circumstances. However, imaging, collection, detection and analysis of sample fluorescence in vacuum or low-temperature chambers usually require Users build their own microscopy imaging systems.

Currently, the microscopic imaging systems equipped with commercial vacuum chambers or low-temperature chambers generally have complex structures, occupy a large space, and have too many optical components, thus reducing the collection efficiency and detection efficiency of fluorescence signals, and the cost is generally high. In order to avoid the high-cost vacuum chamber or low-temperature chamber-compatible microscopy imaging system, some users choose to build their own microscopy imaging system. The time cost and R&D cost are usually high. The present invention aims to provide a cage-based structure The in-vacuum confocal microscopy system minimizes the use of optical components such as mirrors and lenses, simplifying the structural design of the microscope, thus greatly improving the fluorescence collection efficiency and detection efficiency, while reducing the Small devices take up space and cost. The microscope objective is placed inside the vacuum cavity, allowing the use of microscope objectives with large numerical aperture and short working distance, thereby improving fluorescence collection efficiency and imaging resolution, and being compatible with use in both vacuum and low-temperature environments; microscopy system The main body is placed outside the vacuum chamber or low-temperature chamber and adopts a stable cage structure. It is assembled from cage-structured optical-mechanical components that are easy to install and disassemble. It can conveniently and quickly make optimized adjustments for scanning lasers and fluorescence of different wavelengths. The invention has outstanding advantages such as high integration, small occupied volume, easy installation and debugging, and simple operation and maintenance. The invention can be used for high-precision imaging of weak fluorescence signals inside a vacuum cavity or a low-temperature cavity, and high-efficiency collection and signal analysis of single photon sources.

Contents of the invention

The technical problem of the present invention is to overcome the shortcomings of the existing technology and provide a confocal microscopy imaging system and method in a vacuum chamber based on a cage structure, using a three-dimensional electric displacement stage compatible with a vacuum environment or a low-temperature vacuum environment to perform imaging on samples Large-scale scanning and research area positioning. There is a galvanometer system outside the vacuum cavity. The galvanometer system is used to precisely scan the incident laser. The scanning range, step distance and speed of the galvanometer can be precisely controlled by a computer; the microscope objective lens is suspended. On the underside of the sealing cover of the vacuum chamber and placed inside the vacuum chamber, compared with an external microscope objective lens, the present invention can use a microscope objective lens with a large numerical aperture and a short working distance, thereby improving fluorescence collection efficiency and imaging resolution. High efficiency and can be used in both vacuum and low-temperature environments; the main body of the microscopy system is placed outside the vacuum chamber or low-temperature chamber, adopts a stable cage structure, and is assembled from cage-structured optical-mechanical components that are easy to install and disassemble. Optimal adjustments can be made conveniently and quickly for scanning laser and fluorescence of different wavelengths. The invention has outstanding advantages such as high integration, small occupied volume, easy installation and debugging, and simple operation and maintenance. The invention can be used for high-precision imaging of weak fluorescence signals inside a vacuum cavity or a low-temperature cavity, and high-efficiency collection and signal analysis of single photon sources.

The present invention is achieved in the following ways:

The present invention is a confocal microscopy imaging system in a vacuum cavity based on a cage structure, including: a vacuum cavity, a vacuum cavity sealing cover, a quartz window, a three-dimensional electric displacement stage, a galvanometer system, and a microscope objective lens. Fluorescence microscope barrel, cage structure optical machine components, lasers, single-mode optical fiber, optical fiber beam splitter, dichroic mirror, optical filter, imaging CCD, single photon detector, pulse counter, coincident instrument and computer; among them,

Vacuum cavity: used to install the three-dimensional electric displacement stage and seal the three-dimensional electric displacement stage and the sample; located on the lower left side of the microscope imaging system and fixed on the optical platform;

Vacuum chamber sealing cover: used to seal the vacuum chamber; located on the upper side of the vacuum chamber;

Quartz window: used to seal the vacuum chamber; located in the center of the vacuum chamber sealing cover;

Three-dimensional electric displacement stage: used for large-scale scanning of samples and positioning of research areas; located on the lower side of the interior of the vacuum chamber, connected to the displacement stage controller, which is connected to the computer;

Galvanometer system: used for precise scanning of laser, with the computer accurately controlling the scanning range, scanning step distance and scanning rate; installed on the upper side of the sealing cover of the vacuum chamber, the galvanometer system is connected to the galvanometer driver, and the galvanometer driver Connect to computer;

Microscope objective: used to focus the laser beam that excites the sample, and also used to collect the fluorescence signal radiated by the sample in the vacuum cavity or low-temperature cavity and couple it into the single-mode optical fiber; hung on the underside of the vacuum cavity sealing cover and placed side by side inside the vacuum chamber;

Fluorescence microscope barrel: It consists of a vertical laser incident module and a horizontal fluorescence collection module, which enables the incident laser to be focused on the sample, and the fluorescence signal is collected and coupled into a single-mode optical fiber; located in the microscope objective The upper side is fixed on the microscope cantilever, which is fixed on the optical flat plate or package base;

Cage structure optical machine assembly: used for fixing and connecting optical components; located at the installation of optical components of the system and the connection of each optical device;

Laser: used for optical excitation of samples. The laser light emitted by the laser is coupled into the laser incident module of the system through the single-mode optical fiber of the incident module; located on the optical flat panel or package base;

Single-mode optical fiber: used to couple the incident laser into the imaging system and couple the fluorescence signal at the collection end into the optical fiber beam splitter; there is a single-mode optical fiber at the end of the laser incident module and the end of the fluorescence collection module;

Optical fiber beam splitter: realizes the splitting of fluorescence signals; the single-mode optical fiber tail end connected to the fluorescence collection port is connected through the optical fiber flange and fixed on the optical platform or packaging base;

Dichroic mirror: realizes the reflection of the incident laser, reflects the incident laser to the tail end of the microscope objective, and is used to excite the fluorescence of the sample. It also realizes the transmission of the sample's radiated fluorescence, with the purpose of filtering out the incident laser; Located on the upper side of the microscope objective lens;

Optical filter: to further filter the laser signal and enhance the signal-to-noise ratio of the fluorescence signal; located on the upper side of the dichroic mirror;

Imaging CCD: realizes imaging of the surface topography of the sample, and is used to observe and locate the structural units and research areas on the sample surface; it is located on the lower side of the fluorescence microscope barrel and fixed on the upper bottom of the microscope cantilever;

Single-photon detector: realizes photon counting of weak fluorescence signals at the single-photon level; the two single-photon detectors are connected to the two tail ends of the optical fiber beam splitter and fixed on the optical platform or installation base;

Pulse counter: realizes the readout of single photon detector signals; the pulse counter is connected to one of the single photon detectors and is located on the optical platform or in the controller chassis;

Coincidence meter: realizes the coincidence measurement of the signals of two single-photon detectors, and is used to analyze the single-photon properties of the fluorescent light source; located on the optical platform or in the controller chassis;

Computer: realizes the control of the three-dimensional electric displacement stage; realizes the real-time display, recording and analysis of the photon count detected by the single-photon detector; realizes the data processing of the coincident instrument detection results, and is used to analyze the single-photon properties of the fluorescent light source.

In a low-temperature environment, the three-dimensional electric displacement stage adopts a cryogenic-compatible three-dimensional electric displacement stage with a minimum temperature of 4K, and is compatible with both vacuum environment and low-temperature environment systems.

There is a groove on the upper surface of the vacuum chamber, and the groove is used to place a sealing rubber ring. The vacuum chamber is sealed by the sealing rubber ring and the vacuum chamber sealing cover; there is a concave positioning hole on the upper edge of the vacuum chamber, and the positioning hole Used for positioning when assembling the sealing cover of the vacuum chamber; there is a light hole on the sealing cover of the vacuum chamber, which is sealed by a quartz window and a sealing rubber ring. The quartz window is used to transmit the laser beam outside the cavity and the fluorescence signal inside the cavity. ; There is an internal thread interface around the light hole on the upper side of the vacuum chamber sealing cover. A snap ring can be placed inside the internal thread interface, and the snap ring is used to fix the quartz window.

There are multiple threaded holes on the upper side of the vacuum chamber sealing cover. The threaded holes on the upper side of the vacuum chamber sealing cover are used to fix the galvanometer system; there are positioning holes on the upper side of the vacuum chamber sealing cover for assembling the vacuum chamber seal. positioning when the vacuum chamber sealing cover is installed; there is a convex positioning hole on the lower side of the vacuum chamber sealing cover, which is used for positioning when assembling the vacuum chamber sealing cover.

There are multiple threaded holes on the underside of the vacuum chamber sealing cover. The threaded holes on the underside of the vacuum chamber sealing cover are used to fix the mounting plate. There are cage-type assembly struts on the mounting plate, and there are cage-type assembly struts on the cage-type assembly struts. The mounting plate has a microscope objective lens fixed on the cage mounting plate, and the height of the microscope objective lens can be adjusted by adjusting the relative position of the cage mounting plate relative to the cage assembly support rod.

The microscopic objective lens is suspended under the sealing cover of the vacuum chamber, that is, the microscopic objective lens is placed inside the vacuum chamber, and a microscopic objective lens with a large numerical aperture, that is, a maximum NA=0.9, and a short working distance, with a minimum length of 1 mm, is used to improve For fluorescence collection efficiency and imaging resolution, the microscope objective lens is used to focus the laser beam that excites the sample, and is also used to collect the fluorescence signal radiated by the sample in the vacuum cavity or cryogenic cavity.

The three-dimensional electric displacement stage is located inside the vacuum chamber, and the control cable is led out to the outside of the vacuum chamber through the vacuum sealing flange. The three-dimensional electric displacement stage is connected to the displacement stage controller through the control cable, and the displacement stage controller is connected to the computer through a USB cable. , used for large-scale scanning of samples and positioning of research areas.

The galvanometer system located on the upper side of the vacuum cavity is connected to the galvanometer driver through a control cable. The galvanometer driver is connected to the computer through a USB cable. The computer accurately controls the scanning range, scanning step and scanning rate of the galvanometer system with control accuracy. The advantages of high efficiency and high stability.

The fluorescence microscope barrel is composed of a vertical laser incident module and a horizontal fluorescence collection module, which are perpendicular to each other and can be adjusted independently; the laser emitted by the laser is coupled into the laser incident module through a single-mode optical fiber and passes through a dichroic mirror. It is reflected to the XY two-dimensional scanning galvanometer, and then reflected to the tail end of the microscope objective lens by the mirror galvanometer. The incident laser is focused on the sample in the vacuum chamber or cryogenic chamber through the microscope objective lens; the fluorescence signal radiated by the sample passes through the same display The micro-objective lens forms nearly parallel light, and the emitted fluorescence passes through the dichroic mirror and filter, and is finally coupled into the single-mode optical fiber through the fluorescence collection module.

The laser incident module consists of a single-mode optical fiber, an optical fiber adapter, an XY two-dimensional translation adjustment frame, an aspheric lens, a Z-axis translation mount, a cage assembly support rod, a two-dimensional optical adjustment frame, and a cage structure adapter. The single-mode optical fiber is connected to the optical fiber adapter, the optical fiber adapter is fixed on the XY two-dimensional translation adjustment frame, and the aspheric lens is fixed on the Z-axis translation mounting base; the XY two-dimensional translation adjustment frame, The Z-axis translation mount and the two-dimensional optical adjustment mount are connected and fixed through cage-type assembly struts; the incident laser is coupled into the microscope imaging system through a single-mode optical fiber, and the incident direction and spatial position of the incident laser can be determined through the XY two-dimensional translation adjustment mount Adjusted with a two-dimensional optical adjustment mount, the beam waist position and size of the incident laser can be adjusted through the Z-axis translation mount.

The fluorescence collection module consists of a two-dimensional optical adjustment frame, a cage assembly strut, a Z-axis translation mount, an achromatic aspheric lens, an XY two-dimensional translation adjustment frame, an optical fiber adapter, a single-mode optical fiber, and a cage structure adapter. It is assembled with connectors, etc.; the single-mode optical fiber is connected to the optical fiber adapter, the optical fiber adapter is fixed on the XY two-dimensional translation adjustment frame, and the achromatic aspheric lens is fixed on the Z-axis translation mounting base; by adjusting the two Opto-mechanical components such as dimensional optical adjustment mount, Z-axis translation mount, and XY two-dimensional translation adjustment mount are used to quickly optimize the coupling efficiency of fluorescence signals into single-mode optical fibers.

The microscope barrel is located outside the vacuum cavity. The main body of the laser scanning confocal microscopy system adopts a stable cage structure and is assembled from cage-structured optical-mechanical components that are easy to install and disassemble. It can quickly and easily target different wavelengths. Optimally adjust the excitation laser and fluorescence signals.

The microscope tube is fixed on the microscope cantilever, and the height of the microscope tube is adjusted according to the height of the vacuum chamber.

There is an imaging CCD on the lower side of the fluorescence microscope barrel. The imaging CCD is fixed on the microscope cantilever. There is an optional reflector on the upper side of the imaging CCD. When it is necessary to image the sample shape or locate the research area, just insert the reflector; When coupling fluorescence signals, just pull out the reflector.

The single-mode optical fiber at the top of the fluorescence collection module can spatially filter the fluorescence and filter out stray light and part of the background signal to improve the signal-to-noise ratio of the fluorescence signal; at the same time, the single-mode optical fiber coupling output is more convenient for subsequent fluorescence intensity detection. Spectral analysis, single photon source properties analysis and single photon source applications.

The single-mode optical fiber at the end of the fluorescence collection module is connected to a fiber splitter. The two output ports of the fiber splitter are respectively connected to two single-photon detectors. The signal output port of one of the single-photon detectors is connected through a BNC. The pulse counter is connected to the signal input port of the pulse counter, and the pulse counter is connected to the computer through a USB cable for real-time detection and display of the fluorescence intensity in the vacuum chamber or low-temperature chamber.

The signal output ports of the two single photon detectors are respectively connected to the two signal input ports of the conformer through BNC connecting lines. The conforming instrument is connected to the computer through a USB connecting line for measuring and analyzing the vacuum chamber or cryogenic chamber. Properties of single photon sources within.

A confocal microscopy imaging method in a vacuum cavity based on a cage structure of the present invention is implemented as follows:

The sample is fixed on the three-dimensional displacement stage in the vacuum chamber. The vacuum chamber sealing cover with the microscope objective lens and quartz window fixed on it is placed on the upper side of the vacuum chamber. It is sealed with a sealing rubber ring and the galvanometer system is fixed on the vacuum chamber. The upper side of the vacuum cavity is then evacuated into a vacuum environment through a vacuum pump. If a low-temperature environment is required, the sample stage is cooled after the pressure in the cavity is lower than 10 ^-1 Pa; the laser emitted by the laser passes through the tail of the laser incident module The single-mode optical fiber at the end is coupled to the confocal microscope system, and the incident direction and spatial position of the incident laser are controlled by adjusting the XY two-dimensional translation adjustment frame and the two-dimensional optical adjustment frame in the vertical laser incident module. By adjusting the Z axis Translate the mounting base to control the beam waist position and size of the incident laser; the laser is reflected by the dichroic mirror on the lower side to the galvanometer system on the left, and then reflected by the two mirrors in the galvanometer system before passing through the quartz window The film reaches the end of the microscope objective lens, and finally the laser beam is focused by the microscope objective onto the surface of the sample fixed on the three-dimensional electric displacement stage; the fluorescence radiated by the sample is collected by the microscope objective and converted into nearly parallel light, and then passes upward through the quartz window to the outside of the vacuum chamber, and then reflected by the galvanometer and then passed through the dichroic mirror. The fluorescence direction is the horizontal direction; if it is necessary to characterize the topography of the sample surface or locate the research area, insert the reflector on the upper side of the imaging CCD, and the computer The three-dimensional displacement stage can be controlled to move and scan, and the surface morphology of the sample is imaged in real time through the imaging CCD and computer. If you need to collect fluorescence and do subsequent signal analysis, pull out the reflector on the upper side of the imaging CCD; the fluorescence signal passes through the filter After further filtering out the laser and stray light, it enters the fluorescence collection module. By adjusting the two-dimensional optical adjustment mount, Z-axis translation mount, XY two-dimensional translation adjustment mount and other optical-mechanical components, the coupling of the fluorescence signal into the single-mode fiber is quickly optimized. Efficiency; the other end of the single-mode optical fiber on the fluorescence collection module is connected to the optical fiber splitter through the optical fiber flange. The beam is split into two. The two output ports of the optical fiber splitter are connected to two single-photon detections respectively. Among them The signal output port of a single light detector is connected to a pulse counter, and the pulse counter is connected to a computer. The fluorescence intensity signal radiated by the sample is displayed and analyzed in real time through the computer, and the changes in fluorescence intensity can be recorded and stored; the galvanometer system is controlled by the computer through the computer. Control to precisely scan the position where the incident laser is focused on the sample surface, and then cooperate with the pulse counter to record the fluorescence intensity of the fluorescence under each spot within the integration time in real time, thereby achieving two-dimensional high-precision imaging of the fluorescence on the sample surface; two single-unit The photon detectors are respectively connected to the two signal input ports of the conformer, and the conformer is connected to the computer. The single-photon properties of the fluorescent light source are analyzed through the conformer and the computer. The single-mode optical fiber at the end of the fluorescence collection module couples the collected fluorescence to the user. spectrometer for spectral analysis, or coupled to a user-defined optical or optoelectronic system for use and analysis.

The advantage of the present invention compared with the prior art is that the cage-structure-based intra-vacuum confocal microscopy system provided by the present invention minimizes the use of optical elements such as mirrors and lenses, and simplifies The structural design of the microscope is improved, thereby greatly improving the fluorescence collection efficiency and detection efficiency, while reducing the space occupied by the equipment. The microscope objective lens is suspended from the underside of the sealing cover of the vacuum chamber and placed inside the vacuum chamber. Compared with an external microscope objective lens, the present invention can use a microscope objective lens with a large numerical aperture and a short working distance, thereby improving fluorescence collection. The efficiency and imaging resolution can be compatible with both vacuum and low-temperature environments; the main body of the microscopy system is placed outside the vacuum chamber or low-temperature chamber, using a stable cage structure and composed of cage-structured optical-mechanical components that are easy to install and disassemble. It is assembled and can be easily and quickly optimized for different wavelengths of scanning laser and fluorescence. The invention has outstanding advantages such as high integration, small occupied volume, easy installation and debugging, and simple operation and maintenance. The invention can be used for high-precision imaging of weak fluorescence signals inside a vacuum cavity or a low-temperature cavity, and high-efficiency collection and signal analysis of single photon sources.

Description of the drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below. It is obvious that the drawings described below are only some embodiments of the present invention and are not useful in this field. For ordinary technical personnel, other drawings can also be obtained based on these drawings without exerting creative work.

Figure 1 is a schematic diagram of a device structure according to an embodiment of the present invention;

Figure 2 is a schematic diagram of a vacuum chamber according to an embodiment of the present invention;

Figure 3 is a schematic diagram of the upper side of the vacuum chamber sealing cover provided by an embodiment of the present invention;

Figure 4 is a schematic diagram of a retaining ring and a quartz window provided by an embodiment of the present invention;

Figure 5 is a schematic view of the lower side of the vacuum chamber sealing cover provided by an embodiment of the present invention;

Figure 6 is a flow chart of fluorescence signal analysis according to an embodiment of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

The present invention will be further described according to Figures 1-6.

Figure 1 is a schematic structural diagram of a device provided by an embodiment of the present invention.

The upper right side of the microscope objective lens is a cage-structured fluorescence microscope tube. The microscope tube is composed of a vertical laser incident module and a horizontal fluorescence collection module. The two are perpendicular to each other and can be adjusted independently; the laser emitted by the laser is The single-mode optical fiber 1 is coupled to the laser incident module, reflected to the XY two-dimensional scanning galvanometer 14 through the dichroic mirror 10, and then reflected to the tail end of the microscope objective lens 24 by the mirror galvanometer 14. The incident laser 8 passes through the microscope objective lens 24 Focused on the sample in a vacuum or low-temperature chamber; the fluorescence signal 31 radiated by the sample forms nearly parallel light through the same microscope objective lens 24, and the outgoing fluorescence passes through the dichroic mirror 10 and filter 30, and is finally coupled through the fluorescence collection module Into single mode fiber 37;

The laser incident module consists of a single-mode optical fiber 1, an optical fiber adapter 2, an XY two-dimensional translation adjustment frame 3, an aspheric lens 4, a Z-axis translation mounting base 5, a cage assembly support 6, and a two-dimensional optical adjustment frame 7 , cage structure adapters, etc.; the single-mode fiber 1 is connected to the fiber adapter 2, the fiber adapter 2 is fixed on the XY two-dimensional translation adjustment frame 3, and the aspheric lens 4 is fixed on the Z-axis translation On the mounting base 5; the incident direction and spatial position of the incident laser 8 can be adjusted through the XY two-dimensional translation adjustment frame 3 and the two-dimensional optical adjustment frame 7, and the beam waist position and size of the incident laser 8 can be adjusted through the Z-axis translation mounting base 5 Make adjustments;

The laser incident module is fixed on the upper side of the cage cube 9;

The dichroic mirror 10 is fixed inside the cage cube 9;

The cage cube 9 is fixed on the upper side of the microscope cantilever 12 through the mounting bracket 11;

The microscope cantilever 12 is fixed on the support rod 13 with bolts, and the support rod 13 is fixed on the workbench or packaging base;

On the left side of the cage cube 9 and the dichroic mirror 10 is an XY two-dimensional scanning galvanometer 14. The XY two-dimensional scanning galvanometer 14 is equipped with a reflector 15 and a reflector 16. The two reflectors are composed of two mirrors. Motor drive, the motor is connected to the driver, and the driver is connected to the computer, which can accurately control the scanning range and scanning step of the galvanometer;

The galvanometer 14 is fixed on the lower vacuum chamber sealing cover 18 through the mounting bracket 17;

There is a quartz window piece in the center of the vacuum chamber sealing cover 18, and the vacuum chamber sealing cover 18 and the quartz window piece are sealed by a sealing rubber ring;

Furthermore, the quartz window can be coated with a high-permeability film according to the fluorescence wavelength range of the sample, thereby reducing the transmission loss of fluorescence;

The lower side of the vacuum chamber sealing cover 18 is the vacuum chamber 19, and a sealing rubber ring is used to seal the vacuum chamber sealing cover 18 and the vacuum chamber 19;

There is a microscope objective 24, an important component of fluorescence imaging, inside the vacuum chamber 19. The microscope objective 24 is fixed on the lower side of the cage mounting plate 23. The cage mounting plate 23 is fixed on the cage assembly support rod 22. The microscope objective 24 The height of 24 can be achieved by adjusting the vertical position of the cage mounting plate 23. The cage assembly support rod 22 is fixed on the lower side of the mounting plate 20, and the mounting plate 20 is fixed on the lower side of the vacuum chamber sealing cover 18 through bolts 21; The microscope objective 24 is used to focus the laser beam 8 that excites the sample, and is also used to collect the fluorescence signal 31 radiated by the sample;

On the lower side of the microscope objective 24 are a sample mounting platform 25 and an electric displacement stage 26. The electric displacement stage 26 is an electric displacement stage compatible with vacuum environments;

Furthermore, if the vacuum chamber is in a low-temperature environment, the electric displacement stage 26 can choose an electric displacement stage that is compatible with both the low-temperature environment and the vacuum environment;

The lower right side of the cage cube 9 and the dichroic mirror 10 is an imaging CCD 28. The imaging CCD 28 is used for imaging the surface topography of the sample in order to observe and locate the structural units and research areas on the sample surface; the imaging CCD 28 supports The rod is fixed on the upper side of the microscope cantilever 12;

The upper side of the imaging CCD 28 is an optional 45° reflector 27. When it is necessary to observe the surface morphology of the sample or observe and locate the structural units of the sample surface, the reflector 27 is inserted into the microscope barrel and pulled out when fluorescence imaging and collection are required. Reflector 27;

On the right side of the cage cube 9 and the 45° reflector 27 is the fluorescence collection module;

The fluorescence collection module consists of a two-dimensional optical adjustment frame 29, a cage assembly support 32, a Z-axis translation mount 33, an achromatic aspheric lens 34, an XY two-dimensional translation adjustment frame 35, an optical fiber adapter 36, a single-mode The optical fiber 37, cage structure adapter, etc. are assembled; the single-mode optical fiber 37 is connected to the optical fiber adapter 36, the optical fiber adapter 36 is fixed on the XY two-dimensional translation adjustment frame 35, and the achromatic aspheric lens 34 is fixed on on the Z-axis translation mount 33; by adjusting the two-dimensional optical adjustment mount 29, the Z-axis translation mount 33, and the XY two-dimensional translation mount 35, the coupling efficiency of the fluorescence signal 31 into the single-mode optical fiber 37 can be quickly optimized;

On the right side of the two-dimensional optical adjustment frame 29 is a filter 30 for filtering out laser signals;

Further, the filter 30 can be combined with a long-pass filter, a band-pass filter, and a short-pass filter to filter out the fluorescence signal within the wavelength range of interest;

FIG. 2 is a schematic diagram of a vacuum chamber according to an embodiment of the present invention. as shown in picture 2,

The upper surface 201 of the vacuum chamber is a smooth plane, and there is a groove 202 on the upper surface of the vacuum chamber for placing a rubber sealing ring. The rubber sealing ring is in contact with the vacuum chamber and the vacuum chamber sealing cover and is used to seal the vacuum chamber. body, there is a concave positioning hole 203 on the edge of the upper surface of the vacuum chamber, which is used for positioning when assembling the sealing cover of the vacuum chamber;

Figure 3 is a schematic diagram of the upper side of the vacuum chamber sealing cover provided by an embodiment of the present invention. As shown in Figure 3,

There is a groove 301 on the upper side of the vacuum chamber sealing cover, which is used to place a rubber sealing ring. There is a hollow baffle 302 on the upper side of the vacuum chamber sealing cover. A quartz window piece is placed above the groove 301 and the baffle 302;

There is an internal thread interface 303 on the outside of the upper hollow baffle 302 of the vacuum chamber sealing cover, and a snap ring can be placed in the internal thread interface 303 to fix the quartz window piece;

There are a plurality of threaded holes 304 on the upper side of the vacuum chamber sealing cover, and the threaded holes 304 on the upper side of the vacuum chamber sealing cover are used to fix the galvanometer system;

There is a positioning hole 305 on the upper side of the vacuum chamber sealing cover. The positioning hole 305 is used for positioning when assembling the vacuum sealing cover;

Figure 4 is a schematic diagram of a retaining ring and a quartz window provided by an embodiment of the present invention. As shown in Figure 4,

The snap ring is used to fix the quartz window piece on the sealing cover of the vacuum chamber;

There is a rotation hole 401 on the snap ring, and the rotation hole 401 is conducive to rotation when fixing the snap ring;

There are external threads 402 on the snap ring, and the external threads 402 match the internal threads 303 on the upper side of the vacuum chamber sealing cover. The external threads 402 of the snap ring are used to fix the quartz window piece on the vacuum chamber sealing cover;

The quartz window piece 403 is on the sealing cover of the vacuum chamber and fixed with a snap ring. The quartz window piece is used for the transmission of laser and fluorescence inside and outside the vacuum cavity or low-temperature cavity;

Figure 5 is a schematic view of the lower side of the sealing cover of the vacuum chamber provided by an embodiment of the present invention. As shown in Figure 5,

There is a positioning hole 501 on the lower side of the vacuum chamber sealing cover. The positioning hole 501 is used for positioning when assembling the vacuum sealing cover;

There are a plurality of threaded holes 502 on the underside of the vacuum chamber sealing cover. The threaded holes 502 on the underside of the vacuum chamber sealing cover are used to fix the mounting plate. There are cage-type assembly struts on the mounting plate, and there are cage-type assembly struts on the cage-type assembly struts. Cage mounting plate, a microscope objective lens is fixed on the cage mounting plate, and the height of the microscope objective lens can be adjusted by adjusting the relative position of the cage mounting plate relative to the cage assembly support rod;

There is a light hole 503 in the center of the sealing cover of the vacuum chamber. The light hole 503 is used to transmit the laser that excites the sample to the inside of the vacuum cavity or cryogenic chamber. The light hole 503 is also used to radiate fluorescence through the sample to the vacuum cavity. or outside the cryogenic chamber;

Figure 6 is a flow chart of fluorescence signal analysis according to an embodiment of the present invention. As shown in Figure 6,

The three-dimensional electric displacement stage 601 is connected to the three-dimensional electric displacement stage driver 602, and the driver 602 is connected to the computer 603. The scanning step, range and rate of the three-dimensional electric displacement stage 601 can be accurately controlled through the computer 603;

The galvanometer 604 is connected to the galvanometer driver 605, and the galvanometer driver 605 is connected to the computer 603. The scanning range, scanning step and scanning rate of the galvanometer 604 can be accurately controlled through the computer 603;

The three-dimensional electric displacement stage 601 generally adopts a large-stroke displacement stage, which is used for scanning a large range of samples and positioning the research area, and the galvanometer 604 is used for fine scanning of the laser beam;

The single-mode optical fiber at the right end of the fluorescence collection module is connected to the optical fiber splitter 606, and the two output ports of the optical fiber splitter 606 are connected to the single-photon detector 607 and the single-photon detector 608 respectively;

The signal output port of the single photon detector 607 is connected to the signal input port of the pulse counter 609. The pulse counter 609 is connected to the computer 603 via USB for real-time detection and display of fluorescence intensity;

The signal output ports of the single photon detector 607 and the single photon detector 608 are respectively connected to the two signal input ports of the coincidence meter 610. The coincidence meter 610 is connected to the computer 603 for measuring and analyzing the properties of the single photon source.

Claims (18)

1. A vacuum intracavity confocal microscopic imaging system based on a cage structure is characterized in that a microscopic system main body is of a cage structure and comprises: the device comprises a vacuum cavity, a vacuum cavity sealing cover plate, a quartz window sheet, a three-dimensional electric displacement table, a galvanometer system, a microscope objective, a fluorescent microscope tube, an optical machine component, a laser, a single-mode optical fiber, an optical fiber beam splitter, a dichroic mirror, an optical filter, an imaging CCD, a single-photon detector, a pulse counter, a coincidence instrument and a computer; wherein,

Vacuum cavity: the device is used for installing the three-dimensional electric displacement table and sealing the three-dimensional electric displacement table and the sample; the microscopic imaging system is positioned at the left lower side and fixed on the optical platform;

vacuum cavity sealing cover plate: sealing the vacuum cavity; the vacuum chamber is positioned on the upper side of the vacuum chamber;

quartz window sheet: sealing the vacuum cavity; the vacuum cavity sealing cover plate is positioned in the center of the vacuum cavity sealing cover plate;

three-dimensional electric displacement platform: for large-scale scanning of samples and localization of investigation regions; the displacement table controller is connected with the computer;

vibrating mirror system: the laser scanning device is used for finely scanning laser, and a computer precisely controls the scanning range, the scanning step distance and the scanning speed; the vibrating mirror system is arranged on the upper side of the sealing cover plate of the vacuum cavity, and is connected with a vibrating mirror driver which is connected with a computer;

microscope objective: the laser beam is used for focusing and exciting the sample, and is also used for collecting fluorescent signals radiated by the sample in the vacuum cavity or the low-temperature cavity and coupling the fluorescent signals into the single-mode optical fiber; the vacuum cavity sealing cover is hung on the lower side of the vacuum cavity sealing cover and is arranged in the vacuum cavity;

fluorescent microscope tube: the device consists of a laser incidence module in the vertical direction and a fluorescence collection module in the horizontal direction, so that the incident laser is focused on a sample, and fluorescence signals are collected and coupled into a single-mode fiber; the microscope cantilever is fixed on an optical flat or a packaging base;

Optical machine assembly: for fixing and connecting optical elements; the optical element installation part is positioned at the connecting part of each optical element of the system; the optical element includes: the device comprises a vacuum cavity sealing cover plate, a quartz window sheet, a galvanometer system, a microscope objective, a fluorescent microscope tube, a single-mode optical fiber, a dichroic mirror, an optical filter and an imaging CCD; a laser: the laser incidence module is used for optically exciting the sample, and coupling laser emitted by the laser into the system through a single-mode fiber of the incidence module; is positioned on the optical flat or the packaging base;

single mode optical fiber: the optical fiber beam splitter is used for coupling the incident laser into the imaging system and coupling fluorescent signals of the collecting end to enter the optical fiber beam splitter; the tail end of the laser incidence module and the tail end of the fluorescence collection module are respectively provided with a single mode fiber;

optical fiber beam splitter: splitting the fluorescent signal is realized; the tail end of the single-mode fiber is connected with the tail end of the fluorescence collection port through an optical fiber flange and is fixed on an optical platform or a packaging base;

dichroic mirror: the reflection of the incident laser is realized, the incident laser is reflected to the tail end of the microscope objective for exciting the fluorescence of the sample, and meanwhile, the transmission of the radiation fluorescence of the sample is also realized, so that the purpose of filtering the incident laser is realized; the lens is positioned on the upper side of the microscope objective;

An optical filter: the laser signals are further filtered, and the signal to noise ratio of the fluorescent signals is enhanced; the optical filter is positioned between the dichroic mirror and the coupling collecting end and is vertical to the light beam propagation direction of the collecting end;

imaging CCD: imaging of the surface morphology of the sample is achieved, and the imaging device is used for observing and positioning a structural unit and a research area of the surface of the sample; the fluorescent microscope tube is positioned at the lower side of the fluorescent microscope tube and is fixed at the bottom of the upper side of the microscope cantilever;

single photon detector: photon counting of weak fluorescent signals at a single photon level is realized; the two single photon detectors are respectively connected with the two tail ends of the optical fiber beam splitter and fixed on the optical platform or the mounting base;

pulse counter: realizing the reading of the signal of the single photon detector; the pulse counter is connected with one of the single photon detectors and is positioned on the optical platform or in the controller case;

coincidence instrument: the coincidence measurement of the signals of the two paths of single photon detectors is realized, and the signal detection device is used for analyzing the single photon property of a fluorescent light source; is positioned on the optical platform or in the controller cabinet;

and (3) a computer: control of the three-dimensional electric displacement table is realized; realizing real-time display, record and analysis of photon counts detected by the single photon detector; the method realizes the data processing of the detection result of the coincidence instrument and is used for analyzing the single photon property of the fluorescent light source.

2. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: in a low-temperature environment, the three-dimensional electric displacement table adopts a low-temperature compatible three-dimensional electric displacement table with the lowest temperature of 4K, and is compatible with a vacuum environment and a low-temperature environment system.

3. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the upper surface of the vacuum cavity is provided with a groove, the groove is used for placing a sealing rubber ring, and the vacuum cavity is sealed by the sealing rubber ring and a vacuum cavity sealing cover; the upper side edge of the vacuum cavity is provided with a concave positioning hole which is used for positioning when the vacuum cavity sealing cover is assembled; the vacuum cavity sealing cover is provided with a light through hole, and is sealed by a quartz window sheet and a sealing rubber ring, wherein the quartz window sheet is used for transmitting laser beams outside the cavity and fluorescent signals in the cavity; the periphery of the light passing hole on the upper side of the vacuum cavity sealing cover is provided with an internal thread connector, a clamping ring is arranged in the internal thread connector, and the clamping ring is used for fixing the quartz window sheet.

4. A cage-based in-vacuum-cavity confocal microscopy imaging system according to claim 1 or 3, characterized in that: the upper side of the vacuum cavity sealing cover is provided with a plurality of threaded holes, and the threaded holes on the upper side of the vacuum cavity sealing cover are used for fixing the vibrating mirror system; the upper side of the vacuum cavity sealing cover is provided with a positioning hole for positioning when the vacuum cavity sealing cover is assembled; the lower side of the vacuum cavity sealing cover is provided with a downward convex positioning hole for positioning during assembling the vacuum cavity sealing cover.

5. A cage-based in-vacuum-cavity confocal microscopy imaging system according to claim 1 or 3, characterized in that: the lower side of the vacuum cavity sealing cover is provided with a plurality of threaded holes, the threaded holes on the lower side of the vacuum cavity sealing cover are used for fixing a mounting plate, the mounting plate is provided with a cage type assembly supporting rod, the cage type assembly supporting rod is provided with a cage type mounting plate, the cage type mounting plate is fixedly provided with a microscope objective, and the height of the microscope objective is adjusted by adjusting the relative position of the cage type mounting plate to the cage type assembly supporting rod.

6. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the microscope objective is hung on the lower side of the vacuum cavity sealing cover, namely the microscope objective is arranged in the vacuum cavity, a large numerical aperture, namely the maximum NA=0.9 and a short working distance are used, and the microscope objective with the minimum length of 1 mm is used for improving the collection efficiency and imaging resolution of fluorescence, and is used for focusing a laser beam for exciting a sample and collecting a fluorescence signal of sample radiation in the vacuum cavity or a low-temperature cavity.

7. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the three-dimensional electric displacement platform is positioned in the vacuum cavity, the control cable is led out of the vacuum cavity through the vacuum sealing flange, the three-dimensional electric displacement platform is connected with the displacement platform controller through the control cable, and the displacement platform controller is connected with the computer through a USB (universal serial bus) line and is used for large-scale scanning of samples and positioning of a research area.

8. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the vibrating mirror system positioned on the upper side of the vacuum cavity is connected with the vibrating mirror driver through the control cable, the vibrating mirror driver is connected with the computer through the USB line, and the scanning range, the scanning step distance and the scanning speed of the vibrating mirror system are accurately controlled through the computer, so that the device has the advantages of high control precision and high stability.

9. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the fluorescent microscope tube consists of a laser incidence module in the vertical direction and a fluorescent collection module in the horizontal direction, which are mutually vertical and can be independently adjusted; the laser emitted by the laser is coupled into a laser incidence module by a single-mode fiber, reflected to an XY two-dimensional scanning galvanometer by a dichroic mirror, reflected to the tail end of a microscope objective by the galvanometer, and the incident laser is focused on a sample in a vacuum cavity or a low-temperature cavity by the microscope objective; the fluorescent signal radiated by the sample forms near parallel light through the same microscope objective, the emergent fluorescent light passes through the dichroic mirror and the optical filter, and finally is coupled into the single-mode optical fiber through the fluorescent collection module.

10. The cage-based intra-vacuum-cavity confocal microscopy imaging system of claim 1 or 9, wherein: the laser incidence module is assembled by a single-mode fiber, an optical fiber adapter, an XY two-dimensional translation adjusting frame, an aspheric lens, a Z-axis translation mounting seat, a cage-type assembly supporting rod, a two-dimensional optical adjusting frame and a cage-type structure adapter; the single-mode optical fiber is connected with an optical fiber adapter, the optical fiber adapter is fixed on an XY two-dimensional translation adjusting frame, and the aspheric lens is fixed on a Z-axis translation mounting seat; the XY two-dimensional translation adjusting frame, the Z-axis translation mounting seat and the two-dimensional optical adjusting frame are connected and fixed through cage-type assembly support rods; the incident laser is coupled into the microscopic imaging system through a single-mode fiber, the incident direction and the spatial position of the incident laser are adjusted through an XY two-dimensional translation adjusting frame and a two-dimensional optical adjusting frame, and the beam waist position and the beam waist size of the incident laser are adjusted through a Z-axis translation mounting seat.

11. The cage-based intra-vacuum-cavity confocal microscopy imaging system of claim 1 or 9, wherein: the fluorescence collection module is assembled by a two-dimensional optical adjusting frame, a cage-type assembling support rod, a Z-axis translation mounting seat, an achromatic aspheric lens, an XY two-dimensional translation adjusting frame, an optical fiber adapter, a single-mode optical fiber and a cage-type structure adapter; the single-mode optical fiber is connected with an optical fiber adapter, the optical fiber adapter is fixed on an XY two-dimensional translation adjusting frame, and the achromatic aspheric lens is fixed on a Z-axis translation mounting seat; the coupling efficiency of fluorescent signals coupled into the single-mode fiber is rapidly optimized by adjusting the two-dimensional optical adjusting frame, the Z-axis translation mounting seat and the XY two-dimensional translation adjusting frame.

12. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the microscope tube is positioned outside the vacuum cavity, the main body of the laser scanning confocal microscope system adopts a stable cage structure, and is assembled by a cage structure optical machine component which is easy to install and disassemble, so that excitation lasers and fluorescent signals with different wavelengths can be conveniently and rapidly optimized and adjusted.

13. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the microscope tube is fixed on the microscope cantilever, and the height of the microscope tube is adjusted according to the height of the vacuum cavity.

14. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: an imaging CCD is arranged at the lower side of the fluorescent microscope tube, an optional reflecting mirror is arranged at the upper side of the imaging CCD which is fixed on the microscope cantilever, and the reflecting mirror is inserted when the appearance imaging or the positioning of the research area is required to be carried out on the sample; when the fluorescent signal needs to be coupled, the reflector is pulled out.

15. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the single-mode fiber at the top of the fluorescence collection module performs spatial filtering on fluorescence, and stray light and partial background signals are filtered out so as to improve the signal-to-noise ratio of fluorescence signals; meanwhile, the single-mode fiber coupling output is more convenient for the subsequent fluorescence intensity detection, spectrum analysis, single photon source property analysis and single photon source application.

16. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the single-mode fiber at the tail of the fluorescence collection module is connected with the optical fiber beam splitter, two output ports of the optical fiber beam splitter are respectively connected with two single-photon detectors, a signal output port of one single-photon detector is connected with a signal input port of a pulse counter through a BNC connecting wire, and the pulse counter is connected with a computer through a USB connecting wire and is used for detecting and displaying the fluorescence intensity in a vacuum cavity or a low-temperature cavity in real time.

17. A cage-based vacuum in-cavity confocal microscopy imaging system according to claim 1, wherein: the signal output ports of the two single photon detectors are respectively connected with the two signal input ports of the coincidence instrument through BNC connecting wires, and the coincidence instrument is connected with the computer through a USB connecting wire and is used for measuring and analyzing the properties of the single photon source in the vacuum cavity or the low-temperature cavity.

18. The vacuum intracavity confocal microscopic imaging method based on the cage structure is characterized by comprising the following steps of:

the sample is fixed on a three-dimensional displacement table in the vacuum cavity, a vacuum cavity sealing cover fixed with a microscope objective and a quartz window sheet is covered on the upper side of the vacuum cavity, the vacuum cavity is sealed through a sealing rubber ring, and a vibrating mirror system is fixed on the upper side of the vacuum cavity Then the vacuum cavity is pumped into a vacuum environment by a vacuum pump, and if a low-temperature environment is needed, the pressure in the cavity is lower than 10 ^-1 Cooling the sample table after Pa; the laser emitted by the laser is coupled to a confocal microscopy system through a single-mode fiber at the tail end of the laser incidence module, the incidence direction and the spatial position of the incident laser are controlled by adjusting an XY two-dimensional translation adjusting frame and a two-dimensional optical adjusting frame in the laser incidence module in the vertical direction, and the beam waist position and the beam waist size of the incident laser are controlled by adjusting a Z-axis translation mounting seat; the laser is reflected to the left vibrating mirror system through the dichroic mirror at the lower side, then is reflected by the two reflecting mirrors in the vibrating mirror system, and then reaches the tail end of the microscope objective through the quartz window sheet, and finally the laser beam is focused to the surface of the sample fixed on the three-dimensional electric displacement table by the microscope objective; the fluorescence radiated by the sample is collected by the microscope objective and converted into near-parallel light, then upwards penetrates through the quartz window sheet to the outside of the vacuum cavity, and then penetrates through the dichroic mirror after being reflected by the galvanometer, and the fluorescence direction is the horizontal direction; if the appearance of the sample surface is required to be represented or a research area is positioned, a reflector at the upper side of the imaging CCD is inserted, a computer controls a three-dimensional displacement table to move and scan, the appearance of the sample surface is imaged in real time through the CCD and the computer, and if fluorescence is required to be collected and subsequent signal analysis is carried out, the reflector at the upper side of the imaging CCD is pulled out; the fluorescent signal enters a fluorescent collection module after laser and stray light are further filtered by the optical filter, and the coupling efficiency of the fluorescent signal coupled into the single-mode optical fiber is rapidly optimized by adjusting a two-dimensional optical adjusting frame, a Z-axis translation mounting seat and an XY two-dimensional translation adjusting frame optical machine component; the other end of the single-mode fiber on the fluorescence collection module is in butt joint with the optical fiber beam splitter through the optical fiber flange, the light beam is divided into two parts, two output ports of the optical fiber beam splitter are respectively connected to two single-photon detections, a signal output port of one single-photon detector is connected with a pulse counter, the pulse counter is connected with a computer, and the computer is used for displaying and analyzing fluorescent intensity signals of sample radiation in real time and recording and storing changes of fluorescent intensity; finely scanning the position of the incident laser focused on the surface of the sample by controlling a galvanometer system through a computer, and matching with pulse The impulse counter records the fluorescence intensity of the fluorescence under each light spot in real time in the integral time, so that the two-dimensional high-precision imaging of the surface fluorescence of the sample is realized; the two single photon detectors are respectively connected with two signal input ports of the coincidence instrument, the coincidence instrument is connected with the computer, and the single photon properties of the fluorescent light source are analyzed through the coincidence instrument and the computer; the single-mode fiber at the tail end of the fluorescence collection module couples the collected fluorescence to a spectrometer of a user for spectral analysis, or to a user-defined optical or photoelectric system for use and analysis.