CN117269127A - Intelligent fluorescence correlation spectrum acquisition method and device with accurate addressing - Google Patents
Intelligent fluorescence correlation spectrum acquisition method and device with accurate addressing Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The invention provides an intelligent acquisition method and device for a fluorescence correlation spectrum with accurate addressing. The method comprises the following specific steps: step one, a microscope scanning module performs one-time addressing raster scanning imaging, step two, an intelligent acquisition module is utilized to accurately address a target area where a fluorescent signal appears, coordinates of the target area are recorded, and the coordinates of the target area are input to the microscope scanning module, so that accurate acquisition of data of the target area is realized. The intelligent acquisition method and the intelligent acquisition device for the fluorescence correlation spectrum of the accurate addressing, provided by the invention, solve the problem of randomness when single-point FCS data acquisition is performed in an artificial selection area, reduce the possibility of information such as error estimation of molecular diffusion dynamics caused by address selection errors, and realize automatic acquisition of all accurate addressing coordinates by controlling a scanning galvanometer through an FPGA.
Description
Technical Field
The invention relates to the field of fluorescence microscopic imaging and detection, in particular to an intelligent acquisition method and device for a precisely addressed fluorescence correlation spectrum.
Background
Fluorescence correlation spectroscopy (Fluorescence Correlation Spectroscopy, FCS) was first proposed by Magde et al in 1972 as a single-molecule detection technique for obtaining fluorescence correlation spectroscopy curves by measuring fluctuation of fluorescence signals generated by Brownian motion of fluorescent molecules in a very small detection area based on an optical microscope and performing correlation function analysis on the fluctuation signals, and molecular dynamics, interactions and structural changes in living cell organisms can be measured by studying changes in fluorescence intensity over time or space, but due to limitations of computer technology, optical technology and optical detection technology, sensitivity of detection is not high and does not raise a wide interest, and in 1993, it was first proposed to combine laser Confocal scanning technology with FCS (Confocal FCS) to reduce irradiation volume and detection volume of samples using laser Confocal technology (to 10) -15 L, namely below 1 fL), eliminates the interference of scattered light on the test, greatly improves the signal-to-noise ratio of the FCS, and proposes a grating image correlation spectroscopy (grating image correlation spectroscopy) technology based on the traditional single-point FCS in 2005, wherein the grating image correlation spectroscopy technology is based on fluorescence signal fluctuation of fluorescent molecules due to Brownian motion during grating scanning imaging, and can analyze diffusion dynamics and the like in the traditional single-point FCS, and can improve the two-dimensional super-resolution image.
Because the traditional Confocal FCS only allows data to be collected in a small detection volume at a time, the spatial and temporal characteristics of interactions of lipids or other biological processes cannot be accurately described in a single collection process, and if the laser power is too high, phototoxicity, photobleaching and the like are not negligible problems, meanwhile, the traditional collection mode needs to consider that coordinates are selected for single-point data collection, and the accuracy of results is seriously affected by random site selection, so that the judgment of living cell biological dynamics is not facilitated.
When the traditional grating image correlation spectrum is used for grating scanning imaging, a large part of areas are free from fluorescence or interaction, but a lot of time is spent for scanning the areas, so that the time resolution of the grating image correlation spectrum technology is poor, and how to accurately find the region of interest and improve the time resolution is a problem which must be solved by the traditional grating image correlation spectrum technology.
Therefore, it is necessary to provide an intelligent acquisition method and device for fluorescence correlation spectrum with accurate addressing to solve the above technical problems.
Disclosure of Invention
The invention provides an intelligent acquisition method and device for a fluorescence correlation spectrum with accurate addressing, which solve the problems.
In order to solve the technical problems, the invention provides;
the application method of the precisely addressed fluorescence correlation spectrum intelligent acquisition device comprises the following specific steps:
step one, a microscope scanning module performs one-time addressing raster scanning imaging;
accurately addressing a target area with fluorescent signals by using an intelligent acquisition module, recording coordinates of the target area, and inputting the coordinates of the target area into a microscope scanning module to realize accurate acquisition of data of the target area;
and thirdly, the intelligent acquisition module synchronously controls the microscope detection module to transmit the acquired fluorescence signals to a computer, and analyzes the data according to a time or space autocorrelation function to obtain a Fluorescence Correlation Spectrum (FCS) curve or a grating image correlation spectrum curve.
Further, the intelligent acquisition module is controlled in real time by a field programmable gate array FPGA development board, the intelligent acquisition module firstly carries out data acquisition on coordinates obtained by accurate addressing, the two acquisition modes sequentially carry out FCS analysis on single-point scanning of coordinates or grating image related spectrum analysis on grating scanning of a region, and the X axis and the Y axis of a scanning galvanometer are controlled to realize data acquisition on all target regions.
Further, after the microscope scanning system finishes addressing raster scanning, the following scanning modes are adopted for single-point scanning and raster scanning:
the first and recorded coordinates are sequentially arranged in space, the FPGA development board transmits the coordinates of the first pixel to a microscope scanning system, the deflection angles of the X axis and the Y axis of a scanning vibrating mirror are controlled, the scanning time is fixed, the data are detected by a single photon counter, after the scanning is finished, the FPGA development board transmits the second coordinates to the microscope scanning system, the process is repeated until all the coordinates accurately positioned on the whole two-dimensional pixel surface are acquired, and the acquired data are transmitted to a computer for time autocorrelation analysis;
the second, the coordinate of record is arranged in space sequentially, the FPGA development board finds out and includes 256 x 256 picture elements with the maximum addressing coordinate, carry on according to the traditional raster scanning mode, scan the first line from beginning, until the first line 256 picture elements are scanned, turn over to the second line and scan, repeat the above-mentioned process, until the 256 lines are scanned, repeat the raster scanning from the first picture element again, scan at least 10 complete picture of 256 x 256 picture elements, detect the fluorescent signal by the photodetector, the signal is sent to the computer and carried on the space autocorrelation analysis;
further, the fluctuation value δf (t) caused by the change of the intensity of the fluorescent signal at any time t in the detection micro-region can be expressed as:
δF(t)＝F(t)-<F(t)>
wherein F (t) represents the total fluorescence intensity of the system at the moment t, and the symbol represents the average value of the solving function in a certain time; correlating the fluorescence rise and fall values δf (t) within the detection microcell with the delay time, the normalized time autocorrelation function G (τ) can be expressed as:
wherein the delay time tau represents the stay time of fluorescent molecules in the detection volume, F (t+tau) represents the fluorescence intensity after the delay time tau at any moment t, G (tau) represents the change degree of the molecular motion state in the detection volume after the delay time tau, and the data acquired by the single photon counter are subjected to partition processing by using a time autocorrelation function to obtain an FCS curve, and information such as molecular dynamics in living cell biology is explored.
Further, molecular dynamics information is extracted from the raster scan image using raster image correlation spectroscopy, which has two steps:
firstly, removing stationary or slowly moving objects by background subtraction, and subtracting an average value of a group of continuous images from the obtained images needing to be analyzed by average background subtraction;
in the second step, a hidden time structure exists between any two pixels under raster scanning, if a certain correlation exists between fluorescence intensities between the two pixels, the correlation can be displayed by a spatial autocorrelation function, and the spatial autocorrelation function can be expressed as:
wherein, I (x, y) represents the fluorescence intensity on each pixel point, xi and psi respectively represent the variation in the x direction and y direction space on the raster scanning image, the symbol represents the average value, the acquired raster image is analyzed by using the space autocorrelation function to obtain the raster image correlation spectrum curve, and the information such as molecular dynamics in living cell biology is explored.
Further, the fluorescent probe on the photodetector may be any one of a quantum dot, an organic dye, or a rare earth up-conversion nanoparticle, which may be bonded to a NOBF 4 And (3) reacting to remove oleic acid ligand on the particle surface, so that the rare earth up-conversion nano particles are dissolved in water for application.
The intelligent fluorescence correlation spectrum acquisition device with accurate addressing comprises an excitation light generation module, a micro scanning module, an FPGA development board and a photoelectric detection module;
the excitation light generation module comprises an infrared continuous laser, an optical filter, a collimation beam expander, a half wave plate and a polaroid;
the microscopic scanning module comprises a scanning galvanometer, a scanning lens, a tube lens, a high-reflection low-transmission dichroic mirror and an objective lens;
the photoelectric detection module comprises a focusing lens, a photoelectric detector and a single photon counter.
Compared with the related art, the intelligent acquisition method and the intelligent acquisition device for the precisely addressed fluorescence related spectrum have the following beneficial effects:
1. compared with the traditional Confocal FCS technology, the invention utilizes one-time addressing raster scanning imaging, accurately addresses a target area with fluorescence, and sequentially acquires the data of the target area through the intelligent acquisition module, which is helpful for solving the randomness when the single-point FCS data acquisition is performed on the manually selected area, the accurate addressing reduces the possibility of information such as error of address selection and error estimation of molecular diffusion dynamics, and simultaneously the FPGA controls the scanning galvanometer to realize automatic acquisition of all accurate addressing coordinates.
2. Compared with the traditional raster image related spectrum technology, the invention utilizes one-time raster scanning imaging to accurately address the fluorescent target area, and carries out raster scanning in the positioned area through the intelligent acquisition module, thereby shortening the imaging time, and simultaneously shortening the time for post-processing data in the smaller target area. This helps to shorten the imaging time of the region of no interest during the scanning process of the conventional grating image correlation spectroscopy technique, thereby improving the time resolution.
3. Compared with the traditional fluorescence correlation spectroscopy technology, the device provided by the invention solves some defects of the traditional Confocal FCS and the traditional grating image correlation spectroscopy technology through FPGA control, and meanwhile, the device can directly switch two acquisition modes without adjusting and switching optical paths and instruments and equipment, and the device only realizes intelligent acquisition through the FPGA, so that the cost is lower and the feasibility is higher.
Drawings
Fig. 1 is a schematic structural view of the device of the present invention.
FIG. 2 is a schematic diagram of an FCS intelligent acquisition method in the invention;
FIG. 3 is a schematic diagram of a method for intelligently collecting a grating image correlation spectrum in the invention;
FIG. 4 is a set of Time-Trace graphs obtained by FCS intelligent acquisition in the present invention;
FIG. 5 is a diagram of FCS trace obtained by intelligent FCS acquisition in the present invention;
FIG. 6 is a two-dimensional laser scanning fluorescence image obtained by intelligent acquisition of a grating image correlation spectrum in the invention;
fig. 7 is a three-dimensional trace diagram of a grating image correlation spectrum obtained by intelligent acquisition of the grating image correlation spectrum.
Reference numerals in the drawings: 1. the infrared continuous laser comprises an infrared continuous laser, 2, an optical filter, 3, a collimation beam expander, 4, a half wave plate, 5, a polaroid, 6, a scanning galvanometer, 8, a scanning lens, 9, a tube lens, 11, an objective lens, 12, a high-reflection low-transmission dichroic mirror, 13, a focusing lens, 14, a photoelectric detector, 15, a single photon counter, 16 and an FPGA development plate.
Detailed Description
The invention will be further described with reference to the drawings and embodiments.
Referring to fig. 1-7 in combination, a precisely addressed fluorescence correlation spectrum intelligent acquisition device includes an excitation light generation module, a microscanning module, an FPGA development board 16, and a photoelectric detection module;
the excitation light generation module comprises an infrared continuous laser 1, an optical filter 2, a collimation beam expander 3, a half wave plate 4 and a polaroid 5;
the microscopic scanning module comprises a scanning galvanometer 6, a scanning lens 8, a tube lens 9, a high-reflection low-transmission dichroic mirror 12 and an objective lens 11;
the photo detection module comprises a focusing lens 13, a photo detector 14 and a single photon counter 15.
The laser generating module is used for generating continuous near-infrared steady laser beams serving as excitation light, the infrared continuous laser 1 emits a stable laser beam, the laser beam passes through the optical filter 2 to filter laser beams with other wavelengths, the collimating and beam expanding lens 3 is used for carrying out pinhole filtering on the collimating and beam expanding lens, the laser beam enters the microscope scanning system after being shaped by the collimating and beam expanding lens 3 and is focused by the objective lens 11 to obtain Gaussian light spots reaching diffraction limit size, the microscope scanning module carries out raster scanning imaging, the focused Gaussian light spots excite the fluorescent nanoprobe, the photoelectric detection module receives fluorescent signals to generate a two-dimensional laser scanning image, and the fluorescent region in the scanning image is accurately positioned;
the near-infrared laser 1 generates continuous near-infrared steady laser beam output, the laser beam is filtered and collimated by the optical filter 2 and the collimating beam expander 3, and the power of the laser beam is adjusted by the cooperation of the half-wave plate 4 and the polaroid 5.
The scanning galvanometer 6 controls the light path deflection of the laser beam to scan the sample in two dimensions, the scanning lens 8 and the field lens focus and collimate the laser beam emitted by the scanning galvanometer 6, the high-reflection low-transmission dichroic mirror 12 can reflect near infrared excitation light and transmit sample fluorescence for separating the excitation light and the fluorescence, and the laser beam is focused on the sample through the objective lens 11.
The FPGA development board 16 controls the deflection angles of the X-axis and the Y-axis of the scanning galvanometer 6 when FCS data is acquired, and supplies waveform data to the X-axis and the Y-axis of the scanning galvanometer 6 to control the scanning of the scanning galvanometer 6 when spectral data related to a raster image is acquired.
The photoelectric detection module comprises a focusing lens 13, a photoelectric detector 14 and a single photon counter 15 which are coaxially arranged in sequence, the focusing lens 13 and the photoelectric detector 14 are arranged in the advancing direction of fluorescence collected by the objective lens 11, the objective lens 11 collects a part of fluorescence signals, the signals are received by the photoelectric detector 14 through the high-reflection low-transmission dichroic mirror 12 and the focusing lens 13, after each time the signals detected by the photoelectric detector 14 are received, the signals are sent to the FPGA development board 16, the FPGA development board 16 controls the scanning galvanometer 6 to rotate through waveform input, the focusing light spot is moved to scan the next pixel, thus obtaining a two-dimensional laser scanning image, the single photon counter 15 records the arrival time of each photon, and transmits the signals to the computer through the FPGA development board 16, the computer can obtain a curve of the change of fluorescence intensity along with time according to the arrival time of the photons, after a group of data is collected, the FPGA controls the deflection angles of the X axis and the Y axis of the scanning galvanometer, the acquisition is continued for the next pixel position obtained through accurate addressing until all the accurate addressing areas are collected
The application method of the precisely addressed fluorescence correlation spectrum intelligent acquisition device comprises the following specific steps:
step one, a microscope scanning module performs one-time addressing raster scanning imaging;
accurately addressing a target area with fluorescent signals by using an intelligent acquisition module, recording coordinates of the target area, and inputting the coordinates of the target area into a microscope scanning module to realize accurate acquisition of data of the target area;
and thirdly, the intelligent acquisition module synchronously controls the microscope detection module to transmit the acquired fluorescence signals to a computer, and analyzes the data according to a time or space autocorrelation function to obtain a Fluorescence Correlation Spectrum (FCS) curve or a grating image correlation spectrum curve.
The intelligent acquisition module is controlled in real time by the field programmable gate array FPGA development board 16, the intelligent acquisition module firstly carries out data acquisition on coordinates obtained by accurate addressing, the two acquisition modes sequentially carry out FCS analysis on single-point scanning of coordinates or grating image related spectrum analysis on grating scanning of a region, and the X axis and the Y axis of a scanning galvanometer are controlled to realize data acquisition on all target regions.
After the microscope scanning system finishes addressing raster scanning, the single-point scanning and raster scanning adopt the following scanning modes:
the first and recorded coordinates are sequentially arranged in space, the FPGA development board 16 transmits the coordinates of the first pixel to a microscope scanning system, the deflection angles of the X axis and the Y axis of a scanning vibrating mirror are controlled, the scanning time is fixed, the data are detected by the single photon counter 15, after the scanning is finished, the FPGA development board 16 transmits the second coordinates to the microscope scanning system, the process is repeated until all the coordinates accurately positioned on the whole two-dimensional pixel surface are acquired, and the acquired data are transmitted to a computer for time autocorrelation analysis;
the second, recorded coordinates are arranged in sequence in space, the FPGA development board 16 finds 256 x 256 pixels containing the most addressing coordinates, scans the first line from scratch until the 256 th pixel of the first line is scanned, then transfers to the second line scan, repeats the above process until the 256 lines are scanned, repeats the raster scan from the first pixel, scans at least 10 complete images of 256 x 256 pixels, detects fluorescent signals by the photodetector 14, and sends the signals to the computer for spatial autocorrelation analysis;
the fluctuation value delta F (t) caused by the change of the intensity of the fluorescent signal in the detection micro-area at any time t can be expressed as follows:
δF(t)＝F(t)-<F(t)>
wherein F (t) represents the total fluorescence intensity of the system at the moment t, and the symbol represents the average value of the solving function in a certain time; correlating the fluorescence rise and fall values δf (t) within the detection microcell with the delay time, the normalized time autocorrelation function G (τ) can be expressed as:
wherein the delay time τ represents the residence time of fluorescent molecules in the detection volume, F (t+τ) represents the fluorescence intensity after the delay time τ at any time t, G (τ) represents the degree of change of the molecular motion state in the detection volume after the delay time τ, and the data acquired by the single photon counter 15 are subjected to partition processing by using a time autocorrelation function to obtain an FCS curve, so as to explore information such as molecular dynamics in living cell biology.
The invention extracts molecular dynamics information from a raster scan image by utilizing raster image correlation spectrum analysis, which comprises the following two steps:
firstly, removing stationary or slowly moving objects by background subtraction, and subtracting an average value of a group of continuous images from the obtained images needing to be analyzed by average background subtraction;
in the second step, a hidden time structure exists between any two pixels under raster scanning, if a certain correlation exists between fluorescence intensities between the two pixels, the correlation can be displayed by a spatial autocorrelation function, and the spatial autocorrelation function can be expressed as:
wherein, I (x, y) represents the fluorescence intensity on each pixel point, xi and psi respectively represent the variation in the x direction and y direction space on the raster scanning image, the symbol represents the average value, the acquired raster image is analyzed by using the space autocorrelation function to obtain the raster image correlation spectrum curve, and the information such as molecular dynamics in living cell biology is explored.
The fluorescent probe on the photodetector 14 of the present invention may be any one of a quantum dot, an organic dye, or a rare earth up-conversion nanoparticle which can be used with a NOBF 4 And (3) reacting to remove oleic acid ligand on the particle surface, so that the rare earth up-conversion nano particles are dissolved in water for application.
A fluorescence correlation spectrum intelligent acquisition method based on accurate addressing comprises the following two acquisition modes:
s1, a point scanning FCS acquisition mode based on accurate addressing is carried out, before FCS data are acquired, first addressing raster scanning imaging is carried out, according to raster scanning imaging results, an FPGA development board 16 finds out a target area with fluorescence, the FPGA development board 16 records coordinates of all the target areas, and controls deflection angles of a scanning galvanometer to acquire a Time-Trace curve long enough until all the target areas are acquired, as shown in FIG. 2, FIG. 2 shows the point scanning FCS acquisition mode based on accurate addressing in the invention.
S2, a grating image related spectrum acquisition mode based on accurate addressing. Before the related spectrum data of the grating image are collected, first addressing grating scanning imaging is carried out, according to the scanning imaging result, an FPGA development board is used for accurately addressing to find a target area with fluorescence, and the FPGA development board is used for controlling a scanning galvanometer to carry out grating scanning in the target area, as shown in fig. 3, and fig. 3 shows a related spectrum collection mode of the grating image based on accurate addressing in the invention.
Example 1
The example shows the data collected by the FCS based on the accurate addressing point scanning based on the accurate addressing fluorescence correlation spectrum intelligent collection method in the specific embodiment;
excitation of NaYF using a continuous beam of near infrared excitation light 4 Yb/Tm (18/2%) nano-particles, in the example, the wavelength is 980nm, a group of Time-Trace tracks collected by a single photon counter 15 are shown in figure 4 under the excitation of 980nm near infrared laser with certain power, the collection Time of 60s is used for collecting fluorescent signals of all target areas, and the data of 6 target areas are required to be divided and then are subjected to data analysis;
the embodiment carries out accurate addressing to find out 6 fluorescent target areas for data acquisition, the FPGA development board 16 controls the deflection angle of the vibration describing mirror 6 to carry out single-point FCS data acquisition on 8 target areas, signals are transmitted to an external computer, and the external computer analyzes the acquired data through a time autocorrelation function to obtain 6 FCS curves;
as shown in FIG. 5, FIG. 5 shows a trace of 8 FCS curves, the average diffusion time of the molecules obtained by this analysis was 7.687ms, and the diffusion coefficient was 3.9467. Mu.m 2 ·s -1 。
The embodiment provides a precisely addressed fluorescence correlation spectrum intelligent acquisition device, the structure of which is shown in fig. 1, and the device comprises an excitation light generation module, a micro scanning module, a photoelectric detection module and an FPGA development board 16.
The excitation light generation module comprises a near infrared continuous laser 1, a light filter 2, a collimation beam expander 3 (comprising a pinhole filter), a half wave plate 4, a polaroid 5, a near infrared laser 1, a light filter 2, a collimation beam expander 3, a rotatable mounting seat, a linear polarizer 5 and a laser beam adjusting device, wherein the light filter 2 filters stray light of other wave bands in laser, the collimation beam expander 3 enlarges the excitation light spot size, improves the utilization rate of the power of the excitation light, and meanwhile, the pinhole filter is placed at a focus to filter high-frequency stray light, and the half wave plate 4 is arranged on the rotatable mounting seat and matched with the linear polarizer 5 to adjust the power of the laser beam.
The multi-photon microscopic scanning module comprises a scanning galvanometer 6, a scanning lens 8, a tube lens 9, a high-reflection low-transmission dichroic mirror 12 and an objective lens 11, wherein the scanning galvanometer 6 controls the light path deflection of a laser beam to realize two-dimensional scanning of a sample, the high-reflection low-transmission dichroic mirror 12 reflects the laser beam, the scanning lens 8 and the tube lens 9 focus and collimate the emergent light beam of the scanning galvanometer 6, so that the laser beam still matches the entrance pupil size of the microscope objective lens in the scanning process, and finally the objective lens 11 focuses the laser beam to the reflecting mirror sample.
The photoelectric detection module comprises a focusing lens 13, a photoelectric detector 14 and a single photon counter 15, wherein the focusing lens 13 and the photoelectric detector 14 are arranged in the advancing direction of fluorescence collected along the objective lens 11, the photoelectric detector is connected with an FPGA development board 16, in a grating image correlation spectrum collection mode, after the photoelectric detector 14 receives detection signals once, the photoelectric detector sends signals to the FPGA development board 16, then the FPGA development board 16 controls the scanning galvanometer 6 to rotate, a sample point-by-point scanning mode of a focusing light spot is utilized, all signals are transmitted to an external computer for processing, finally, a two-dimensional laser scanning super-resolution image is obtained for carrying out space autocorrelation analysis, in an FCS collection mode, the single photon counter 15 collects a group of data, the FPGA development board 16 controls the deflection angle of the scanning galvanometer 6 until all interested areas are collected, and the signals are transmitted to the external computer for carrying out Time-Trace Time autocorrelation analysis.
The FPGA development board 16 performs accurate addressing according to the results of the one-time addressing raster scan imaging image, and according to two different acquisition modes: FCS and grating image related spectrum, controlling the photoelectric detection module to collect data; the FPGA in FCS mode controls the deflection angle of the scanning galvanometer 6, and the FPGA development board 16 in raster image correlation spectrum mode controls the scanning galvanometer 6 to perform raster scanning.
Example 2
Based on the intelligent fluorescence correlation spectrum acquisition method based on accurate addressing in the specific embodiment, the example shows the data acquired based on the grating image correlation spectrum based on accurate addressing.
NaYF synthesized by excitation of a continuous beam of near infrared excitation light 4 Yb/Tm (20/10%) nanoparticles, in this exampleThe wavelength was chosen to be 980nm. Under the excitation of 980nm near-infrared laser with certain power, the photoelectric detector 14 collects the signal of each raster scanning pixel and transmits the signal to an external computer, and the external computer sorts the signal to obtain a pair of two-dimensional laser scanning super-resolution fluorescent images;
as shown in fig. 6, fig. 6 shows a two-dimensional laser scanning fluorescence image obtained in the grating image correlation spectrum acquisition mode, and the image can be put into a computer for spatial autocorrelation analysis.
The example obtains a two-dimensional laser scanning fluorescence image, and puts the image into a computer for space autocorrelation analysis to obtain a three-dimensional track diagram of a grating image correlation spectrum, as shown in fig. 7;
FIG. 7 shows a three-dimensional trace obtained by correlation spectroscopy of a grating image, the average diffusion coefficient of the molecules obtained by the analysis being 1.2034 μm 2 ·s -1 。
The foregoing is only illustrative of the present invention and is not to be construed as limiting the scope of the invention, and all equivalent structures or equivalent flow modifications which may be made by the teachings of the present invention and the accompanying drawings or which may be directly or indirectly employed in other related art are within the scope of the invention.
Claims (7)
1. The intelligent fluorescence correlation spectrum acquisition method with accurate addressing is characterized by comprising the following specific steps:
step one, a microscope scanning module performs one-time addressing raster scanning imaging;
accurately addressing a target area with fluorescent signals by using an intelligent acquisition module, recording coordinates of the target area, and inputting the coordinates of the target area into a microscope scanning module to realize accurate acquisition of data of the target area;
and thirdly, the intelligent acquisition module synchronously controls the microscope detection module to transmit the acquired fluorescence signals to a computer, and analyzes the data according to a time or space autocorrelation function to obtain a Fluorescence Correlation Spectrum (FCS) curve or a grating image correlation spectrum curve.
2. The intelligent acquisition method of the precisely addressed fluorescence correlation spectrum according to claim 1, wherein the intelligent acquisition module is controlled in real time by a Field Programmable Gate Array (FPGA) development board (16), the intelligent acquisition module firstly carries out data acquisition on coordinates obtained by precisely addressing, and the two acquisition modes sequentially carry out FCS analysis of single-point scanning of the coordinates or grating image correlation spectrum analysis of grating scanning of one area, and control the X axis and the Y axis of a scanning galvanometer to realize data acquisition of all target areas.
3. The intelligent acquisition method of the precisely addressed fluorescence correlation spectrum according to claim 2, wherein after the microscope scanning system finishes addressing raster scanning, the following scanning modes are adopted for single-point scanning and raster scanning:
the first and recorded coordinates are sequentially arranged in space, an FPGA development board (16) transmits the coordinates of the first pixel to a microscope scanning system, the deflection angles of an X axis and a Y axis of a scanning galvanometer are controlled, the scanning time is fixed, a single photon counter (15) detects data, after scanning is completed, the FPGA development board (16) transmits the second coordinates to the microscope scanning system, the process is repeated until all the accurately positioned coordinates on the whole two-dimensional pixel surface are acquired, and the acquired data are transmitted to a computer for time autocorrelation analysis;
the second, recorded coordinates are arranged in sequence in space, the FPGA development board (16) finds 256 pixels with the largest addressing coordinates, scans the first row from the beginning until the 256 pixels of the first row are scanned, then transfers to the second row for scanning, repeats the above process until the 256 rows are scanned, repeats the raster scanning from the first pixel, scans at least 10 complete images with 256 pixels, detects fluorescent signals by the photoelectric detector (14), and sends the signals to the computer for space autocorrelation analysis.
4. The intelligent fluorescence correlation spectrum acquisition method based on accurate addressing according to claim 3, wherein the fluctuation value δF (t) caused by the change of the intensity of the fluorescence signal in the detection micro-area at any time t can be expressed as follows:
δF(t)＝F(t)-<F(t)>
wherein F (t) represents the total fluorescence intensity of the system at the moment t, and the symbol represents the average value of the solving function in a certain time; correlating the fluorescence rise and fall values δf (t) within the detection microcell with the delay time, the normalized time autocorrelation function G (τ) can be expressed as:
wherein the delay time τ represents the time that the fluorescent molecule stays in the detection volume;
f (t+τ) represents the fluorescence intensity after the delay time τ at any time t, G (τ) represents the degree of change of the molecular motion state in the detection volume after the delay time τ, and the data acquired by the single photon counter (15) are subjected to partition processing by using a time autocorrelation function to obtain an FCS curve, so that information such as molecular dynamics in living cell biology is explored.
5. The intelligent acquisition method of fluorescence correlation spectroscopy based on accurate addressing according to claim 4, wherein molecular dynamics information is extracted from a raster scan image by using raster image correlation spectroscopy, and the raster image correlation spectroscopy comprises the following two steps:
firstly, removing stationary or slowly moving objects by background subtraction, and subtracting an average value of a group of continuous images from the obtained images needing to be analyzed by average background subtraction;
in the second step, a hidden time structure exists between any two pixels under raster scanning, if a certain correlation exists between fluorescence intensities between the two pixels, the correlation can be displayed by a spatial autocorrelation function, and the spatial autocorrelation function can be expressed as:
wherein, I (x, y) represents the fluorescence intensity on each pixel point, xi and psi respectively represent the variation in the x direction and y direction space on the raster scanning image, the symbol represents the average value, the acquired raster image is analyzed by using the space autocorrelation function to obtain the raster image correlation spectrum curve, and the information such as molecular dynamics in living cell biology is explored.
6. The intelligent fluorescence correlation spectrum acquisition device based on accurate addressing according to claim 5, wherein the fluorescent probe can be any one of quantum dots, organic dyes or rare earth up-conversion nano particles, and the rare earth up-conversion nano particles can be prepared by combining with NOBF 4 And (3) reacting to remove oleic acid ligand on the particle surface, so that the rare earth up-conversion nano particles are dissolved in water for application.
7. The precisely addressed fluorescence correlation spectrum intelligent acquisition device according to claim 6, which is characterized by comprising an excitation light generation module, a microscanning module, an FPGA development board (16) and a photoelectric detection module;
the excitation light generation module comprises an infrared continuous laser (1), an optical filter (2), a collimation beam expander (3), a half wave plate (4) and a polaroid (5);
the microscopic scanning module comprises a scanning galvanometer (6), a scanning lens (8), a tube mirror (9), a high-reflection low-transmission dichroic mirror (12) and an objective lens (11);
the photoelectric detection module comprises a focusing lens (13), a photoelectric detector (14) and a single photon counter (15).
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