CN109828364B - Vacuum intracavity confocal microscopic imaging system and method based on cage structure - Google Patents
Vacuum intracavity confocal microscopic imaging system and method based on cage structure Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 6
- 238000007789 sealing Methods 0.000 claims abstract description 91
- 239000013307 optical fiber Substances 0.000 claims abstract description 65
- 230000003287 optical effect Effects 0.000 claims abstract description 64
- 238000006073 displacement reaction Methods 0.000 claims abstract description 43
- 239000010453 quartz Substances 0.000 claims abstract description 28
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000004458 analytical method Methods 0.000 claims abstract description 15
- 238000009434 installation Methods 0.000 claims abstract description 5
- 238000013519 translation Methods 0.000 claims description 50
- 239000000835 fiber Substances 0.000 claims description 32
- 238000004624 confocal microscopy Methods 0.000 claims description 20
- 230000008878 coupling Effects 0.000 claims description 16
- 238000010168 coupling process Methods 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 11
- 238000011160 research Methods 0.000 claims description 11
- 230000005855 radiation Effects 0.000 claims description 8
- 238000004806 packaging method and process Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 238000010183 spectrum analysis Methods 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 3
- 238000011835 investigation Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- 210000001503 joint Anatomy 0.000 claims description 2
- 230000004807 localization Effects 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 230000010354 integration Effects 0.000 abstract description 4
- 238000012423 maintenance Methods 0.000 abstract description 4
- 238000005457 optimization Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 230000005693 optoelectronics Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
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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
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a vacuum intracavity confocal microscopic imaging system and method based on a cage structure.
Background
Confocal microscopy is a common scientific research device for studying fluorescence imaging, weak fluorescence signal collection, detection and signal analysis. Researchers need to study the nature of the fluorescence radiated by samples in vacuum or cryogenic environments in certain specific situations, but imaging, collecting, detecting and analyzing sample fluorescence in vacuum or cryogenic chambers often requires the user to build up a microscopic imaging system by himself.
The current commercial microscopic imaging system equipped with the vacuum cavity or the low-temperature cavity is complex in general structure, large in occupied space and volume and excessive in optical elements, so that the collection efficiency and the detection efficiency of fluorescent signals are reduced, and the cost is generally high. In order to avoid a high-cost vacuum cavity or low-temperature cavity compatible microscopic imaging system, some users choose to construct the microscopic imaging system by themselves, the time cost and the research and development cost of the microscopic imaging system are generally higher, and the invention aims to provide the vacuum cavity confocal microscopic system based on the cage structure, which reduces the use of optical elements such as a reflector and a lens to the greatest extent, simplifies the structural design of a microscope, greatly improves the fluorescence collection efficiency and the fluorescence detection efficiency, and reduces the space occupation volume and the cost of equipment. The micro objective is arranged in the vacuum cavity, and can use the micro objective with large numerical aperture and short working distance, thereby improving fluorescence collection efficiency and imaging resolution, and being compatible to use in vacuum environment and low temperature environment; the microscope system main body is arranged outside the vacuum cavity or the low-temperature cavity, adopts a stable cage structure, is assembled by a cage structure optical-mechanical assembly which is easy to install and disassemble, and can conveniently and rapidly perform optimization adjustment on scanning lasers and fluorescence of different wavelengths. The invention has the outstanding advantages of high integration level, small occupied volume, easy installation and debugging, simple and convenient operation and maintenance, etc. 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.
Disclosure of Invention
The technical solution of the invention is as follows: the vacuum cavity internal confocal microscopic imaging system and the vacuum cavity internal confocal microscopic imaging method based on the cage structure are provided, a three-dimensional electric displacement table compatible with a vacuum environment or a low-temperature vacuum environment is used for carrying out large-range scanning and research area positioning on a sample, a vibrating mirror system is arranged outside the vacuum cavity and is used for carrying out precise scanning on incident laser, and the scanning range, the step distance and the speed of the vibrating mirror can be precisely controlled through a computer; compared with an external microscope objective, the microscope objective with large numerical aperture and short working distance can be used, so that the fluorescence collection efficiency and the imaging resolution are improved, and the microscope objective can be compatible to be used in a vacuum environment and a low-temperature environment; the microscope system main body is arranged outside the vacuum cavity or the low-temperature cavity, adopts a stable cage structure, is assembled by a cage structure optical-mechanical assembly which is easy to install and disassemble, and can conveniently and rapidly perform optimization adjustment on scanning lasers and fluorescence of different wavelengths. The invention has the outstanding advantages of high integration level, small occupied volume, easy installation and debugging, simple and convenient operation and maintenance, etc. 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.
The invention is realized by the following modes:
the invention discloses a vacuum intracavity confocal microscopic imaging system based on a cage structure, which comprises the following components: 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, a cage-type structure optical-mechanical assembly, 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 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;
cage structure ray apparatus subassembly: for fixing and connecting optical elements; at the junction of the optical components of the system;
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; is positioned on the upper side of the dichroic mirror;
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.
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.
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, and a clamping ring can be placed in the internal thread connector and used for fixing the quartz window sheet.
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.
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.
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.
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.
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.
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.
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, a cage-type structure adapter and the like; 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 the single-mode fiber, the incident direction and the spatial position of the incident laser can be adjusted through the XY two-dimensional translation adjusting frame and the two-dimensional optical adjusting frame, and the beam waist position and the beam waist size of the incident laser can be adjusted through the Z-axis translation mounting seat.
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, a cage-type structure adapter and the like; 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 optical fibers is rapidly optimized by adjusting optical-mechanical components such as a two-dimensional optical adjusting frame, a Z-axis translation mounting seat, an XY two-dimensional translation adjusting frame and the like.
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.
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.
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.
The single-mode fiber at the top of the fluorescence collection module can carry out spatial filtering on fluorescence, and stray light and partial background signals can be filtered out, so that the signal-to-noise ratio of fluorescence signals can be improved; 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.
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.
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.
The invention discloses a vacuum intracavity confocal microscopic imaging method based on a cage structure, which is realized as follows:
The sample is fixed on a three-dimensional displacement table in a 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, a vibrating mirror system is fixed on the upper side of the vacuum cavity, then the vacuum cavity is pumped into a vacuum environment through 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; 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 laser is transmitted to the confocal microscopy system through the single-mode fiberAdjusting the Z-axis translation mounting seat to control the beam waist position and the beam waist size of the incident laser; 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, the computer can control the three-dimensional displacement table to move and scan, the appearance of the sample surface is imaged in real time through the imaging CCD and the computer, and if fluorescence is required to be collected and the subsequent signal analysis is carried out, the reflector at the upper side of the imaging CCD is pulled out; the fluorescence signal enters a fluorescence collection module after laser and stray light are further filtered by the optical filter, and the coupling efficiency of the fluorescence signal coupled into the single-mode optical fiber is rapidly optimized by adjusting optical-mechanical components such as a two-dimensional optical adjusting frame, a Z-axis translation mounting seat, an XY two-dimensional translation adjusting frame and the like; 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; the position of the incident laser focused on the surface of the sample is finely scanned by controlling a galvanometer system through a computer, and then the fluorescence intensity of fluorescence under each light spot in the integral time is recorded in real time by matching with a pulse counter, 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; single mode fiber at the tail end of the fluorescence collection module couples the collected fluorescence to the user's light The spectrometer performs spectral analysis or is coupled to a custom optical or optoelectronic system for use and analysis.
Compared with the prior art, the invention has the advantages that: the vacuum intracavity confocal microscopic system based on the cage structure provided by the invention reduces the use of optical elements such as a reflecting mirror and a lens to the greatest extent, simplifies the structural design of a microscope, greatly improves the collection efficiency and the detection efficiency of fluorescence, and reduces the space occupation volume of equipment. Compared with an external microscope objective, the microscope objective with large numerical aperture and short working distance can be used, so that the fluorescence collection efficiency and the imaging resolution are improved, and the microscope objective can be compatible to be used in a vacuum environment and a low-temperature environment; the microscope system main body is arranged outside the vacuum cavity or the low-temperature cavity, adopts a stable cage structure, is assembled by a cage structure optical-mechanical assembly which is easy to install and disassemble, and can conveniently and rapidly perform optimization adjustment on scanning lasers and fluorescence of different wavelengths. The invention has the outstanding advantages of high integration level, small occupied volume, easy installation and debugging, simple and convenient operation and maintenance, etc. 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.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, it being obvious that the drawings described below are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic view of a device according to an embodiment of the present invention;
FIG. 2 is a schematic view of a vacuum chamber according to an embodiment of the present invention;
FIG. 3 is a schematic top view of a vacuum chamber seal cover according to an embodiment of the present invention;
FIG. 4 is a schematic view of a clasp and quartz window according to one embodiment of the present invention;
FIG. 5 is a schematic view of the underside of a vacuum chamber seal cap according to an embodiment of the present invention;
FIG. 6 is a flow chart of fluorescent signal analysis according to an embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
The invention is further illustrated with reference to fig. 1-6.
Fig. 1 is a schematic diagram of an apparatus according to an embodiment of the invention.
The upper right side of the microscope objective is provided with a fluorescence microscope tube with a cage structure, and the microscope tube consists of a laser incidence module in the vertical direction and a fluorescence collection module in the horizontal direction, which are mutually vertical and can be independently adjusted; the laser emitted by the laser is coupled to a laser incidence module by a single-mode fiber 1, reflected to an XY two-dimensional scanning galvanometer 14 by a dichroic mirror 10, reflected to the tail end of a microscope objective 24 by the galvanometer 14, and the incident laser 8 is focused on a sample in a vacuum or low-temperature cavity by the microscope objective 24; the fluorescence signal 31 radiated by the sample forms nearly parallel light through the same microscope objective 24, the emergent fluorescence passes through the dichroic mirror 10 and the optical filter 30, and finally is coupled into the single-mode fiber 37 through the fluorescence collection module;
the laser incidence module is assembled by a single-mode fiber 1, an optical fiber adapter 2, an XY two-dimensional translation adjusting frame 3, an aspheric lens 4, a Z-axis translation mounting seat 5, a cage-type assembly supporting rod 6, a two-dimensional optical adjusting frame 7, a cage-type structure adapter and the like; the single-mode optical fiber 1 is connected with the optical fiber adapter 2, the optical fiber adapter 2 is fixed on the XY two-dimensional translation adjusting frame 3, and the aspheric lens 4 is fixed on the Z-axis translation mounting seat 5; the incidence direction and the space position of the incident laser 8 can be adjusted through the XY two-dimensional translation adjusting frame 3 and the two-dimensional optical adjusting frame 7, and the beam waist position and the beam waist size of the incident laser 8 can be adjusted through the Z-axis translation mounting seat 5; the XY two-dimensional translation adjusting frame 3, the Z-axis translation mounting seat 5 and the two-dimensional optical adjusting frame 7 are connected and fixed through the cage-type assembly supporting rod 6;
The laser incidence module is fixed on the upper side of the cage-type cube 9;
the dichroic mirror 10 is fixed inside the cage cube 9;
the cage-type cube 9 is fixed on the upper side of the microscope cantilever 12 through a mounting frame 11;
the microscope cantilever 12 is fixed on a supporting rod 13 by bolts, and the supporting rod 13 is fixed on a workbench or a packaging base;
the left sides of the cage-type cube 9 and the dichroic mirror 10 are provided with XY two-dimensional scanning galvanometer 14, a reflecting mirror 15 and a reflecting mirror 16 are arranged in the XY two-dimensional scanning galvanometer 14, the two reflecting mirrors are respectively driven by two motors, the motors are connected with a driver, and the driver is connected with a computer, so that the scanning range and the scanning step distance of the galvanometer can be accurately controlled;
the vibrating mirror 14 is fixed on a vacuum cavity sealing cover 18 at the lower side through a mounting frame 17;
the center of the vacuum cavity sealing cover 18 is provided with a quartz window sheet, and the vacuum cavity sealing cover 18 and the quartz window sheet are sealed by a sealing rubber ring;
furthermore, the quartz window sheet can be plated with a high-permeability film according to the fluorescence wavelength range of the sample, so that the transmission loss of fluorescence is reduced;
the lower side of the vacuum cavity sealing cover 18 is provided with a vacuum cavity 19, and a sealing rubber ring is used for sealing between the vacuum cavity sealing cover 18 and the vacuum cavity 19;
The inside of the vacuum cavity 19 is provided with a fluorescence imaging important element microscope objective 24, the microscope objective 24 is fixed on the lower side of a cage type mounting plate 23, the cage type mounting plate 23 is fixed on a cage type mounting support rod 22, the height of the microscope objective 24 can be realized by adjusting the vertical position of the cage type mounting plate 23, the cage type mounting support rod 22 is fixed on the lower side of a mounting plate 20, and the mounting plate 20 is fixed on the lower side of a vacuum cavity sealing cover 18 through a bolt 21; the micro objective 24 is used for focusing the laser beam 8 exciting the sample and also for collecting the fluorescent signal 31 of the sample radiation;
the lower side of the microscope objective 24 is provided with a sample mounting platform 25 and an electric displacement platform 26, and the electric displacement platform 26 is an electric displacement platform compatible with a vacuum environment;
further, if the vacuum chamber is in a low-temperature environment, the electric displacement table 26 can select an electric displacement table compatible with the low-temperature environment and the vacuum environment at the same time;
the lower right side of the cage 9 and dichroic mirror 10 is an imaging CCD 28, the imaging CCD 28 being used for imaging the topography of the sample surface in order to observe and locate the structural units and investigation region of the sample surface; the imaging CCD 28 is fixed on the upper side of the microscope cantilever 12 through a supporting rod;
an optional 45-degree reflecting mirror 27 is arranged on the upper side of the imaging CCD 28, the reflecting mirror 27 is inserted into a microscope tube when the surface morphology of a sample is required to be observed or a sample surface structural unit is required to be observed and positioned, and the reflecting mirror 27 is pulled out when fluorescent imaging and collection are required;
The right side of the cage-type cube 9 and the 45-degree reflecting mirror 27 is a fluorescence collection module;
the fluorescence collection module is assembled by a two-dimensional optical adjusting frame 29, a cage-type assembly support rod 32, a Z-axis translation mounting seat 33, an achromatic aspheric lens 34, an XY two-dimensional translation adjusting frame 35, an optical fiber adapter 36, a single-mode optical fiber 37, a cage-type structure adapter and the like; the single-mode optical fiber 37 is connected with the optical fiber adapter 36, the optical fiber adapter 36 is fixed on the XY two-dimensional translation adjusting frame 35, and the achromatic aspheric lens 34 is fixed on the Z-axis translation mounting seat 33; the coupling efficiency of the fluorescent signal 31 into the single-mode fiber 37 can be optimized rapidly by adjusting the two-dimensional optical adjusting frame 29, the Z-axis translation mounting seat 33 and the XY two-dimensional translation adjusting frame 35;
the right side of the two-dimensional optical adjusting frame 29 is provided with an optical filter 30 for filtering laser signals;
further, the filter 30 may be a combination of a long-pass filter, a band-pass filter, and a short-pass filter, filtering out fluorescent signals in a wavelength range of interest;
fig. 2 is a schematic view of a vacuum chamber according to an embodiment of the present invention. As shown in the figure 2 of the drawings,
the upper surface 201 of the vacuum cavity is a smooth plane, the upper surface of the vacuum cavity is provided with a groove 202 for placing a rubber sealing ring, the rubber sealing ring is in contact with the vacuum cavity and a vacuum cavity sealing cover for sealing the vacuum cavity, and the edge of the upper surface of the vacuum cavity is provided with a concave positioning hole 203 for positioning when the vacuum cavity sealing cover is assembled;
Fig. 3 is a schematic top view of a vacuum chamber seal cover according to an embodiment of the invention. As shown in the figure 3 of the drawings,
a groove 301 is formed in the upper side of the vacuum cavity sealing cover and is used for placing a rubber sealing ring, a hollow baffle plate 302 is arranged on the upper side of the vacuum cavity sealing cover, and quartz window sheets are placed above the groove 301 and the baffle plate 302;
an internal thread interface 303 is arranged on the outer side of the hollow baffle 302 on the upper side of the vacuum cavity sealing cover, and a clamping ring can be placed in the internal thread interface 303 and used for fixing a quartz window sheet;
the upper side of the vacuum cavity sealing cover is provided with a plurality of threaded holes 304, and the threaded holes 304 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 305, and the positioning hole 305 is used for positioning when the vacuum sealing cover is assembled;
fig. 4 is a schematic view of a clasp and quartz window according to an embodiment of the present invention. As shown in figure 4 of the drawings,
the clamping ring is used for fixing the quartz window sheet on the vacuum cavity sealing cover;
the clamping ring is provided with a rotating hole 401, and the rotating hole 401 is beneficial to rotating when the clamping ring is fixed;
the clamping ring is provided with external threads 402, the external threads 402 are matched with the internal threads 303 on the upper side of the vacuum cavity sealing cover, and the external threads 402 of the clamping ring are used for fixing the quartz window sheet on the vacuum cavity sealing cover;
The quartz window plate 403 is arranged on the vacuum cavity sealing cover and fixed by a clamping ring, and is used for transmitting laser and fluorescence inside and outside the vacuum cavity or the low-temperature cavity;
fig. 5 is a schematic diagram of the underside of a vacuum cavity seal cover according to an embodiment of the present invention. As shown in figure 5 of the drawings,
a positioning hole 501 is formed in the lower side of the vacuum cavity sealing cover, and the positioning hole 501 is used for positioning when the vacuum sealing cover is assembled;
the lower side of the vacuum cavity sealing cover is provided with a plurality of threaded holes 502, the threaded holes 502 at the lower side of the vacuum cavity sealing cover are used for fixing a mounting plate, a cage type assembly supporting rod is arranged on the mounting plate, a cage type mounting plate is arranged on the cage type assembly supporting rod, a microscope objective is fixed on the cage type mounting plate, 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;
the center of the vacuum cavity sealing cover is provided with a light-passing hole 503, the light-passing hole 503 is used for transmitting laser exciting a sample to the inside of the vacuum cavity or the low-temperature cavity, and the light-passing hole 503 is also used for transmitting sample radiation fluorescence to the outside of the vacuum cavity or the low-temperature cavity;
FIG. 6 is a flow chart of fluorescent signal analysis according to an embodiment of the present invention. As shown in figure 6 of the drawings,
the three-dimensional electric displacement table 601 is connected with a three-dimensional electric displacement table driver 602, the driver 602 is connected with a computer 603, and the scanning step distance, the scanning range and the scanning speed of the three-dimensional electric displacement table 601 can be accurately controlled by the computer 603;
The galvanometer 604 is connected with a galvanometer driver 605, the galvanometer driver 605 is connected with a computer 603, and the scanning range, the scanning step distance and the scanning rate of the galvanometer 604 can be accurately controlled by the computer 603;
the three-dimensional electric displacement table 601 generally adopts a displacement table with a large stroke, is used for scanning a sample in a large range and positioning a research area, and the galvanometer 604 is used for finely scanning a laser beam;
the single-mode fiber at the right end of the fluorescence collection module is connected with the optical fiber beam splitter 606, and two output ports of the optical fiber beam splitter 606 are respectively connected with the single-photon detector 607 and the single-photon detector 608;
the signal output port of the single photon detector 607 is connected with the signal input port of the pulse counter 609, and the pulse counter 609 is connected with the computer 603 through a USB for detecting and displaying fluorescence intensity in real time;
the signal output ports of the single photon detector 607 and the single photon detector 608 are respectively connected with two signal input ports of the coincidence instrument 610, and the coincidence instrument 610 is connected with 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.
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| CN113376138A (en) * | 2020-08-11 | 2021-09-10 | 中国科学院物理研究所 | Microscope three-dimensional moving focusing system and application |
| CN115113383B (en) * | 2022-05-30 | 2023-11-28 | 中国人民解放军国防科技大学 | Long lens barrel vacuum microscopic imaging lens for observing observed object in ultrahigh vacuum |
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