CN114935815A - Microscopic imaging system and method based on scanning galvanometer and nano translation stage - Google Patents
Microscopic imaging system and method based on scanning galvanometer and nano translation stage Download PDFInfo
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Abstract
The invention discloses a microscopic imaging system and a microscopic imaging method based on a scanning galvanometer and a nano translation stage, which are used for realizing three-dimensional point-by-point scanning imaging and are important components of a laser scanning confocal microscope. The invention uses the combination of the scanning galvanometer and the nano translation stage to realize the confocal three-dimensional progressive scanning, and uses the FPGA to control the precise deflection of the galvanometer and realize the synchronous signal transmission. The invention realizes the functions of communication between the galvanometer and the FPGA, setting of a galvanometer scanning mode, control and position display of the nano translation table, point-by-point scanning and synchronous acquisition of photon number, real-time display of the photon number, drawing of two-dimensional images, data storage and the like, and is very suitable for autonomous construction and related research and development application of a scanning microscope system.
Description
Technical Field
The invention relates to the technical field of microscopic scanning systems, and is suitable for high-resolution imaging of samples in medical and life science research. In particular to a microscopic imaging system and a microscopic imaging method based on a scanning galvanometer and a nano translation stage, which realize the control of elements of optoelectronic equipment and the acquisition of optical information.
Background
With the rapid development of the life science field, people put higher and higher requirements on the microscope imaging technology. In recent years, people have attracted extensive interest in obtaining information in a sub-diffraction limit region by using a super-resolution imaging technology which breaks through the optical diffraction limit. Super-resolution imaging is a far-field imaging technique that achieves far less than the optical diffraction limit through various nonlinear mechanisms. In biological imaging, the super-resolution technologies can mostly realize the transverse resolution of about 30-80nm and the longitudinal resolution of about 100nm, and the development of life science is greatly promoted. The laser scanning confocal microscope is an important technical means in biological and medical fields of cell biology, molecular biology, genetics, embryology and the like, and is also an important research and development platform for constructing super-resolution imaging. Based on a laser confocal microscope platform, a loss light path or an annular focal spot excitation light path is added, the system can be upgraded and modified into a stimulated emission loss microscope and a fluorescence radiation differential super-resolution microscope, the resolution is improved, and the research of related fields on ultra-fine scales is facilitated. Therefore, the self-constructed laser microscope confocal imaging system has important practical significance for developing scientific research of super-resolution imaging.
In the laser confocal imaging process, laser focal spots are required to realize single-point scanning and scanning-synchronous photon number recording on a three-dimensional region of a sample. Two common scanning modes are sample scanning and beam scanning. The sample scanning mode refers to that the laser focus is not moved, and the sample moves in two dimensions and three dimensions. The beam scanning mode means that the sample is not moved and the laser focus moves in two dimensions and three dimensions. The sample scanning mode is generally realized by using three-dimensional piezoelectric nano translation stage equipment, the three-dimensional movement control is easy to realize, but the speed is low, and meanwhile, the problems of position deviation and the like easily generated in the repeated imaging process are solved, so that the imaging precision is influenced. The light beam scanning mode is generally realized by using a scanning galvanometer, has higher imaging speed and smaller disturbance to a sample, and is more suitable for biological imaging of a living sample. However, the longitudinal scanning control light path of the light beam scanning mode is complex, and is mainly used for realizing two-dimensional transverse scanning imaging.
Disclosure of Invention
The invention aims to provide a microscopic imaging system and a microscopic imaging method based on a scanning galvanometer and a nano translation stage, which are used for realizing three-dimensional scanning imaging of quick galvanometer two-dimensional scanning and high-precision axial scanning based on the combination of transverse light beam scanning of the scanning galvanometer and axial movement of the nano translation stage.
In order to realize the purpose, the invention provides the following technical scheme: a microscopic imaging system based on a scanning galvanometer and a nano translation stage is divided into three parts, including an FPGA control galvanometer reciprocating scanning part, a receiving part of initial signals and a sending part of synchronous signals, a control part of an upper computer to the nano translation stage, a receiving part of the upper computer to the synchronous signals and a collecting, redrawing and storing part of photon information; the FPGA controls the scanning of the galvanometer, the receiving of the initial signal and the sending part of the synchronous signal to be written and burned to the FPGA development board by adopting Verilog language; the upper computer rewrites the control part of the nano translation stage by calling the API of the control part; the upper computer receives the synchronous signals and collects, redraws and stores the photon information by calling an API (application program interface) function and an OpenGl drawing plug-in of a time-dependent single photon counting TCSPC (TCSPC) collection card; the upper computer software system is developed based on MFC under VS2013 compiling environment.
Furthermore, the scanning double-vibration mirror is used for realizing the transverse plane scanning of the sample, the nano translation stage is used for realizing the axial movement, and the double-vibration mirror and the nano translation stage are combined to realize the three-dimensional point-by-point scanning of the sample.
Furthermore, the galvanometer scanning is realized by connecting triangular wave voltage and step wave voltage output by the FPGA to the X axis and the Y axis of the galvanometer so as to control the galvanometer to rotate accurately, the scanning light beam can move accurately in a two-dimensional space, and the scanning mode is in a bow-shaped moving track.
Further, the initial signal is received through an FPGA to achieve uart serial port communication, the uart serial port detects whether a scanning signal appears, and if the scanning signal is detected, the galvanometer is controlled to start scanning according to the scanning signal.
Further, the sending of the synchronization signal is that the galvanometer scans to a specific position, that is, the X direction and the Y direction of the galvanometer reach specified voltages, and a synchronization signal is sent.
Furthermore, the control part of the nano translation stage is rewritten by calling a DLL file of the nano translation stage, the moving step length of the X, Y, Z axis is set by rewriting and calling a library function and a control of an MFC, the accurate movement of the X, Y plane of the nano translation stage and the up-and-down movement of the Z axis are realized, and the relative position of the current movement is displayed.
Further, performing synchronous reception on the photons comprises: scanning, collecting photon signals synchronously, and synchronously storing the collected photon number into a self-defined container in a program;
the photon collecting part carries out photoelectric conversion on the collected fluorescence by an Avalanche Photodiode (APD); signals of the APD are input into a time-dependent single photon counting TCSPC acquisition card, photon information of each imaging pixel point is acquired, and photon counting is achieved;
the scanning and the collected photon number are synchronous, the serial port communication function is realized by using Windows to call API, and the synchronous signal sent by FPGA is received.
Furthermore, in the image redrawing part, the collected photon number information of each pixel is stored in a user-defined vector container in a one-to-one correspondence manner according to the set scanning time interval and the spatial position of the corresponding galvanometer scanning light beam, and then the photon number in the container is extracted according to the scanning manner and is drawn in an OpenGL in a gray scale pattern manner.
The imaging method of the microscopic imaging system based on the scanning galvanometer and the nano translation stage comprises the following steps:
step 1: connecting an FPGA development board, downloading a Bit file, opening a serial port, and connecting a single photon counting TCSPC acquisition card and a nano translation stage;
step 2: clicking a parameter setting button to open a dialog box to set various parameters of the single photon counting TCSPC acquisition card;
and step 3: setting parameters of serial port number, baud rate, check bit and data bit in a control interface, and setting a scanning mode and the number of pixels according to requirements; when the scanning mode parameter is set to be 1, correspondingly scanning 32 pixels by 32 pixels; when the scanning mode parameter is 2, scanning 64 pixels by 64 pixels; when the scanning mode parameter is 3, scanning 128 pixels by 128 pixels; when the scanning mode parameter is 4, scanning 256 pixels by 256 pixels; when the scanning mode parameter is 5, performing three-dimensional chromatographic scanning, and when performing chromatographic scanning, setting the number of scanning layers through the FPGA, and setting a Z-axis moving step length on a control interface;
and 4, step 4: starting a ComBox control which can control scanning by starting scanning, selecting the XY surface scanning of a galvanometer, starting a scanning thread, and executing a corresponding imaging scanning process;
and 5: in the scanning process, a user sees a real-time photon number curve, the single imaging scanning process is finished, and a scanned image is presented in an OpenGl area of a control interface when clicking to start drawing;
step 6: after scanning is finished, setting the Z-axis step length of the nano translation stage, and enabling the sample to move up and down to obtain the best focal plane to obtain the clearest image; moving the nano translation stage to perform X-axis movement or Y-axis movement according to the scanning range, and scanning by a galvanometer to obtain an image in a larger range;
and 7: when the three-dimensional scanning is carried out, the Z-axis moving step length of the nano translation stage is set in advance, when the scanning of the galvanometer finishes one XY plane, the upper computer receives a scanning finishing signal and automatically controls the movement of the nano translation stage, and after the step length is finished, the galvanometer continues the XY plane scanning until the set number of scanning planes is finished;
and 8: and clicking an image information saving button by a user, and storing txt data of the scanned image.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses the combination of the scanning galvanometer and the nano translation stage to realize the confocal three-dimensional progressive scanning, and the system is a software system which utilizes VC + + language and MFC developed scanning imaging application program and utilizes FPGA to control the accurate deflection of the galvanometer and realize the synchronous signal transmission. The invention realizes the functions of communication between the galvanometer and the FPGA, setting of a galvanometer scanning mode, control and position display of the nano translation table, point-by-point scanning and synchronous acquisition of photon numbers, real-time display of the photon numbers, drawing of two-dimensional images, data storage and the like, and is very suitable for the independent construction and related research and development application of a scanning microscope system.
Drawings
FIG. 1 is a system interface diagram of the present invention;
FIG. 2 is a simulation diagram of the FPGA controlling the galvanometer and receiving and sending signals;
FIG. 3 is a system flow diagram of the present invention;
in FIG. 4, (a) is two-dimensional galvanometer scanning imaging of a 40 nm diameter fluorescent bead sample; (b) an enlarged view of the two-dimensional galvanometer scan imaging data corresponding to FIG. 4 (a);
FIG. 5 shows the experimental results of three-dimensional scanning imaging with the combination of two-dimensional transverse scanning imaging of the galvanometer and axial movement of the nano translation stage, applied to a confocal laser scanning imaging system.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further explained with reference to the accompanying drawings. The embodiments described herein are only for explaining the technical solution of the present invention and are not limited to the present invention.
A microscopic imaging system based on a scanning galvanometer and a nano translation stage mainly comprises a galvanometer scanning and initial signal receiving and synchronous signal sending part under the control of an FPGA, a serial port communication part and a nano translation stage accurate control part under the control of upper computer software, a photon acquisition card parameter setting part, various state display parts of the system, photon number equal time interval display parts, a photon information acquisition part synchronous with scanning, and a photon information processing, displaying, drawing and storing part.
The galvanometer scanning under the control of the FPGA is realized by converting digital signals of triangular waves and step waves output by the FPGA into analog voltages through a digital-to-analog conversion module and connecting the analog voltages to the galvanometer so as to control the galvanometer to rotate accurately, so that laser spots can move accurately in a two-dimensional space, and the scanning mode is reciprocating scanning. The receiving of the initial signal is that the FPGA receives a scanning signal sent by an upper computer through UART serial port communication, the UART serial port detects whether the initial signal appears, determines a scanning mode according to the received scanning signal and starts to control galvanometer scanning;
the control part of the nano translation stage is rewritten by calling an API (application program interface) of the nano translation stage, the moving step length of an X, Y, Z axis is set, accurate movement of a X, Y axis of the nano translation stage and up-and-down movement of a Z axis are realized, and the movement of the nano translation stage and the FPGA control galvanometer are combined to perform three-dimensional chromatography scanning;
calling API of TCSPC card to use the photon collection card, wherein the key point is that the time of scanning a step length is consistent with the time of collecting the step length by the collection card;
displaying the current TCSPC card state, the nano translation stage moving state, the serial port communication setting state and the like for each state display part of the system by using a Listbox Control;
timing a photon number equal time interval display part through an OnTimer function of a timer in an MFC, and displaying in a Teechart control in a mode of collecting the number of photons every 10 ms; this function will facilitate the adjustment of CFD etc. in the optical path;
in the part for synchronously acquiring the photon information and the scanning pixels, when a synchronous signal sent by an FPGA is received, a TCSPC photon counting card automatically calls a self-defined photon number acquisition function APD _ READ (), starts to acquire the photon information, stores the acquired photon information into a self-defined Vector container, extracts the photon information from the Vector container after scanning is finished, and displays the photon information in an OpenGl control in a gray value mode. And storing the photon number information in a TXT file form for processing;
the imaging method of the microscopic imaging system based on the scanning galvanometer and the nano translation stage comprises the following steps:
(1) connecting an FPGA development board, downloading a Bit file, opening a serial port, and connecting a photon counting card and a nano translation table.
(2) Clicking a set parameter button, and opening a dialog box to set various parameters of the TCSPC counting card.
(3) And setting parameters of serial port number, baud rate, check bit and data bit in the control interface. And setting a scanning mode and the number of pixels according to needs. In the invention, when the scanning mode parameter is set to be 1, 32 pixels are correspondingly scanned by 32 x 32 pixels; when the scanning mode parameter is 2, scanning 64 pixels by 64 pixels; when the scanning mode parameter is 3, scanning 128 × 128 pixels; when the scanning mode parameter is 4, scanning 256 pixels by 256 pixels; and when the scanning mode parameter is 5, performing three-dimensional chromatographic scanning, and when performing the chromatographic scanning, setting the number of scanning layers through the FPGA, and setting the Z-axis moving step length on a control interface.
(4) Starting scanning, clicking a Button Control for controlling scanning, selecting the XY surface scanning of the galvanometer, starting a scanning thread, and executing a corresponding imaging scanning process.
(5) During the scanning process, the user can see the real-time photon number curve. When the single imaging scanning process is finished and the drawing is started by clicking, a scanning image is displayed in an OpenGl area of the control interface.
(6) After scanning is finished, the Z-axis step length of the nano translation stage can be set, and the sample can move up and down to obtain the best focal plane to obtain the clearest image. The nano translation stage can also be moved along the X axis or the Y axis according to the scanning range, and then the image in a larger range can be obtained through scanning by the galvanometer.
(7) When three-dimensional scanning is carried out, the initial position and the moving step length of the Z axis of the nano translation stage are set in advance. At the set axial initial position, the galvanometer starts a transverse scanning imaging of an XY plane. When the galvanometer finishes scanning and imaging an XY plane, the upper computer receives a scanning finishing signal and automatically controls the Z axis of the nano translation stage to move by a step length, and after the step length is finished, the galvanometer finishes scanning the XY plane at the axial position. And repeating the processes until the set scanning layer number is scanned, and realizing three-dimensional scanning.
(8) The user can click the save image information button to store the txt data of the image scanned this time.
Examples
Fig. 1 is a system interface diagram of the present invention, in which a ListBox control is arranged at the top left corner, and is used to display the current state of the system, the scanning mode, the nano translation stage moving mode, and other situations. The TeeChart control is arranged in the middle-upper part, and the photon number change is displayed in real time by connecting the photon numbers of different pixel points into a line. The black frame at the upper right corner is an OpenGl interface, and photon numbers in the Vector container are extracted and converted into pixel values to be used for displaying a two-dimensional image and a three-dimensional image. The parameters of the nano translation stage, the movement mode and the current relative position of the nano translation stage are set under the OpenGl interface. The real-time display of parameters such as counting rate of the acquisition card is arranged at the lower left, and a clearer image can be obtained by setting various parameters to adjust TCSPC. The middle area is used for displaying the maximum and minimum photon numbers, receiving synchronous signals and displaying the photon number acquisition times. The middle-lower area is a start and initialization button and comprises TCSPC start, nanometer translation stage start, serial port opening, real-time photon curve display, image information storage and the like. The lower right area is a setting area for parameters such as system serial port communication mode setting, number of scanning pixels and the like; the lowest part is set and quit.
Based on the C + + object-oriented characteristic, modules with different functions are packaged into different classes, wherein the classes comprise an dialogue frame class, a parameter setting class, a drawing class, a serial port communication class and a management class of the whole application program.
Fig. 2 is a simulation diagram of controlling the galvanometer and receiving and transmitting signals by the FPGA. The figure is a simulation figure of three-dimensional chromatographic scanning waveform and serial port receiving and sending, namely, the FPGA receives a signal 5 through a uart serial port, 5 times of axial chromatographic scanning are carried out, and XY transverse scanning of a galvanometer is completed on each layer. Triangular wave data are written into a ROM of the FPGA, and then the data are read out in AN address reading mode according to a scanning time sequence and are transmitted to AN AN9767 digital-to-analog conversion module, digital signals are converted into analog signals and are transmitted to a galvanometer, the X-axis control voltage of the galvanometer is connected with the triangular wave data, and the Y-axis control voltage is connected with the step wave data. When the triangular wave is completed by half, the step wave is increased by one step. The two-dimensional scanning of the system adopts a point scanning mode, the address is 0 at the beginning of scanning, the X axis and the Y axis are at the initial positions, and the computer sends a command to the FPGA development board. The FPGA controls the galvanometer to move along an X axis firstly, when a pixel is moved, the FPGA sends a synchronous signal to the upper computer through a serial port, the upper computer receives the synchronous signal and calls the counting card to count an acquisition time, and the number of photons in the time is stored in a self-defined Vector container. When the X axis finishes scanning one line, the Y axis automatically increases one step length, and the scanning is repeated to form an arc-shaped scanning path.
In fig. 2, after the XY scan of one image is completed, the address is taken to be 0, the r _ stop signal is pulled high, and the r _ cnt _ wait signal starts to count for a while. In the period of time, the upper computer receives a signal for finishing transverse scanning of an image, automatically starts the nano translation stage, and moves along the Z axis according to the movement step length set by the user. The Z axis moves by one step, and the galvanometer starts XY surface scanning again. This operation is repeated until the set number of axial scanning layers is completed.
As shown in fig. 3, which is a flow chart of the whole system, initialization is first performed, including connecting an FPGA development board, downloading a Bit file, opening a serial port, connecting a photon acquisition card and a nano translation stage, and zeroing the nano translation stage; if the initialization is not successful, the initialization is performed again. After the initialization is completed, the relevant parameters are set, the parameters of TCSPC are set first, the CFD threshold is set, and then the nano translation stage is set. If the step length of Z-axis scanning needs to be set for three-dimensional tomography scanning, the step length setting range is 0.01um to 1 um. After the parameter setting is finished, the scanning mode of the galvanometer scanning serial port communication part is selected, and two-dimensional scanning or three-dimensional tomographic scanning of 32 pixels by 32 pixels or 64 pixels by 64 pixels or 128 pixels by 128 pixels is set. When the scanning mode is three-dimensional chromatography, firstly, the XY surface scanning of the galvanometer is started, after the galvanometer scans a plane, the upper computer automatically controls the nano translation table to move along the Z axis, and after the Z axis movement is finished, the galvanometer scanning is automatically carried out, and the required axial layer number is always scanned. And when the scanning required layer number is finished, carrying out drawing operation, extracting the photon number stored in the Vector container, and drawing the photon information in a drawing window according to a scanning mode. And clicking to store after drawing is finished, and storing information of photon number. After the scanning is finished, if the scanning is to be continued around the current scanning range, the X axis and the Y axis can be moved by the nano translation stage, and then the scanning is carried out.
Fig. 4(a) shows two-dimensional galvanometer scanning imaging of a 40 nm diameter fluorescent bead sample, with 64 × 64 pixels. Fig. 4 (b) is an enlarged view of the corresponding two-dimensional galvanometer-scanned imaging data.
Fig. 5 is an example of an interface diagram of a three-dimensional tomographic scanning imaging operation performed on a gold particle sample with a diameter of 90 nm, where the number of axial scanning layers is set to 5 by the FPGA, the Z-axis step size is set to 1um in the pull-down frame, and the number of pixels per layer is 32 × 32. In the interface, the ListBox control at the upper left corner displays the XY scanning progress and the Z-axis moving progress in real time. The initial position of the scanning surface in the axial direction may be set by controlling the Z-axis position. In this example, the nano translation stage starts from the (0, 0, 0) position, the Z axis moves by 1um every time one image is scanned, the final position of the nano translation stage at the end of the scanning is (0, 0, 4), and the Z axis position is also displayed in the right text box at this time to be 4 um.
It will be apparent to those skilled in the art that our system is actually feasible, that the foregoing represents only the preferred embodiment of the invention, that the description is more specific and detailed, and that modifications to the foregoing embodiment or equivalent alternatives thereto may be made without departing from the spirit of the invention and these modifications or improvements are within the scope of the invention as claimed.
Claims (9)
1. A microscopic imaging system based on a scanning galvanometer and a nano translation stage is characterized in that: the system is divided into three parts, including an FPGA control galvanometer reciprocating scanning and initial signal receiving and synchronous signal sending part, an upper computer control part for a nanometer translation stage, and an upper computer receiving and photon information collecting, redrawing and storing part; the FPGA controls the scanning of the galvanometer, the receiving of the initial signal and the sending part of the synchronous signal to be written and burned to the FPGA development board by adopting Verilog language; the upper computer rewrites the control part of the nano translation stage by calling the API of the control part; the upper computer receives the synchronous signals and collects, redraws and stores the photon information by calling an API function and an OpenGl drawing plug-in of a time-dependent single photon counting TCSPC collection card; the upper computer software system is developed based on MFC under VS2013 compiling environment.
2. A scanning galvanometer and nano translation stage based microscopy imaging system as claimed in claim 1, wherein: the method is characterized in that the transverse plane scanning of a sample is realized by using a scanning double-vibrating mirror, the axial movement is realized by using a nano translation stage, and the three-dimensional point-by-point scanning of the sample is realized by combining the double-vibrating mirror and the nano translation stage.
3. A scanning galvanometer and nano translation stage based microscopic imaging system according to claim 1, wherein: the galvanometer scanning is that triangular wave voltage and step wave voltage output by the FPGA are connected to an X axis and a Y axis of the galvanometer so as to control the galvanometer to rotate accurately, so that scanning beams can move accurately in a two-dimensional space, and the scanning mode is in a bow-shaped moving track.
4. A scanning galvanometer and nano translation stage based microscopy imaging system as claimed in claim 1, wherein: the receiving of the initial signal is realized by FPGA, the uart serial port detects whether a scanning signal appears, and if the scanning signal is detected, the galvanometer is controlled to start scanning according to the scanning signal.
5. A scanning galvanometer and nano translation stage based microscopy imaging system as claimed in claim 1, wherein: the synchronous signal is sent by scanning the galvanometer to a specific position, namely, the X direction and the Y direction of the galvanometer reach specified voltages and sending a synchronous signal.
6. A scanning galvanometer and nano translation stage based microscopic imaging system according to claim 1, wherein: the control part of the nano translation stage is rewritten by calling a DLL file of the nano translation stage, the movement step length of an X, Y, Z axis is set by rewriting and calling a library function and a control of an MFC, the accurate movement of the X, Y plane of the nano translation stage and the up-and-down movement of a Z axis are realized, and the relative position of the current movement is displayed.
7. A scanning galvanometer and nano translation stage based microscopic imaging system according to claim 1, wherein: performing synchronous reception on the photons comprises: scanning, collecting photon signals synchronously, and synchronously storing the collected photon number into a self-defined container in a program;
the photon collecting part carries out photoelectric conversion on the collected fluorescence by an Avalanche Photodiode (APD); signals of the APD are input into a time-dependent single photon counting TCSPC acquisition card, photon information of each imaging pixel point is acquired, and photon counting is achieved;
the scanning and the collected photon number are synchronous, the serial port communication function is realized by using Windows to call API, and the synchronous signal sent by FPGA is received.
8. A scanning galvanometer and nano translation stage based microscopy imaging system as claimed in claim 1, wherein: in the image redrawing part, the collected photon number information of each pixel is stored in a user-defined vector container in a one-to-one correspondence manner according to the set scanning time interval and the spatial position of the corresponding galvanometer scanning light beam, and then the photon number in the container is extracted according to the scanning manner and is drawn in an OpenGL in a gray-scale image manner.
9. An imaging method of a scanning galvanometer and nano translation stage based microscopy imaging system according to any one of claims 1 to 8, characterized in that: the method comprises the following steps:
step 1: connecting an FPGA development board, downloading a Bit file, opening a serial port, and connecting a single photon counting TCSPC acquisition card and a nano translation stage;
step 2: clicking a parameter setting button to open a dialog box to set various parameters of the single photon counting TCSPC acquisition card;
and step 3: setting parameters of serial port number, baud rate, check bit and data bit in a control interface, and setting a scanning mode and the number of pixels according to requirements; when the scanning mode parameter is set to be 1, correspondingly scanning 32 pixels by 32 pixels; when the scanning mode parameter is 2, scanning 64 pixels by 64 pixels; when the scanning mode parameter is 3, scanning 128 pixels by 128 pixels; when the scanning mode parameter is 4, scanning 256 pixels by 256 pixels; when the scanning mode parameter is 5, performing three-dimensional chromatographic scanning, and when performing chromatographic scanning, setting the number of scanning layers through the FPGA, and setting a Z-axis moving step length on a control interface;
and 4, step 4: starting a ComBox control which can control scanning by starting scanning, selecting the XY surface scanning of a galvanometer, starting a scanning thread, and executing a corresponding imaging scanning process;
and 5: in the scanning process, a user sees a real-time photon number curve, the single imaging scanning process is finished, and a scanned image is presented in an OpenGl area of a control interface when clicking to start drawing;
step 6: after scanning is finished, the Z-axis step length of the nano translation stage is set, and the sample is moved up and down to obtain the best focal plane and obtain the clearest image; moving the nano translation stage to perform X-axis movement or Y-axis movement according to the scanning range, and scanning by a galvanometer to obtain an image in a larger range;
and 7: when the three-dimensional scanning is carried out, the Z-axis moving step length of the nano translation stage is set in advance, when the scanning of the galvanometer finishes one XY plane, the upper computer receives a scanning finishing signal and automatically controls the movement of the nano translation stage, and after the step length is finished, the galvanometer continues the XY plane scanning until the set number of scanning planes is finished;
and 8: and clicking an image information saving button by a user, and storing txt data of the scanned image.
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