A method and apparatus for fast random addressing of scanning fluorescence correlation spectroscopy

Abstract

The application provides a fast random addressing scanning fluorescence correlation spectroscopy method and device thereof, comprising the following steps: S1, using a surface array camera to collect a series of wide field images under light sheet illumination, and obtaining a global low time resolution image fluorescence correlation spectroscopy diffusion image by calculation; S2, selecting an interested region according to the wide field fluorescence correlation spectroscopy image and setting a scanning array generation condition; S3, automatically generating a scanning array according to the selected arbitrary interested region and the set condition.The fast random addressing scanning fluorescence correlation spectroscopy method and device thereof provided by the application can solve the problem that the traditional scanning FCS cannot detect fast kinetics due to slow scanning speed, and can also solve the problem that the traditional scanning FCS cannot simultaneously monitor multiple regions due to the mechanical scanning of the scanning galvanometer.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy, and more particularly to a fast random addressing scanning fluorescence correlation spectroscopy method and apparatus thereof. Background Technology

[0002] Fluorescence correlation spectroscopy (FCS) is a powerful tool for measuring local concentrations, molecular weights, translational and rotational diffusion coefficients, chemical rate constants, binding and dissociation constants, and photodynamics in vitro and in vivo. However, compared to FCS in solution, the diffusion of membrane proteins is more complex and rapid, posing additional challenges to the measurement of fluorophores diffused in membranes. To collect a sufficient number of samples to satisfy the sampling theorem and obtain good experimental results, very long continuous measurement times (at least 10 times longer than the diffusion time) are required. To avoid photobleaching, very low excitation power must be used, ultimately leading to a reduction in the signal-to-noise ratio.

[0003] The basic idea behind scanning FCS is to move the detection volume relative to the sample, thereby reducing the residence time of fluorophores and improving statistical accuracy. However, traditional scanning FCS is limited by the physical speed of the scanning galvanometer, and can only scan continuously at a relatively slow speed. Therefore, it is impossible to achieve rapid FCS scanning or cross-region FCS scanning.

[0004] Therefore, it is necessary to provide a fast random addressing scanning fluorescence correlation spectroscopy method and apparatus to solve the above-mentioned technical problems. Summary of the Invention

[0005] This invention provides a fast random addressing scanning fluorescence correlation spectroscopy method and apparatus, which solves the problems mentioned above.

[0006] To solve the above-mentioned technical problems, the present invention provides a fast random addressing scanning fluorescence correlation spectroscopy method, characterized by comprising the following steps:

[0007] S1. A series of wide-field images under light sheet illumination are acquired using an area array camera, and the global low-temporal resolution fluorescence-related spectral diffusion image is obtained by calculation.

[0008] S2. Select the region of interest and set the scanning array generation conditions based on the wide-field fluorescence correlation spectrum image;

[0009] S3. Automatically generate a scanning array according to the selected region of interest and the conditions set above;

[0010] S4. The excitation light is controlled by the first and second acousto-optic deflectors to irradiate the scanning array in a sequential and cyclic manner, and the fluorescence signal sequence of the corresponding array is synchronously acquired by the signal acquisition module on the acquisition card.

[0011] S5. Perform scanning fluorescence correlation spectroscopy calculations on the fluorescence signals generated at the scanning array to obtain ultra-high temporal resolution dynamic information of the region of interest.

[0012] Further, step S1 includes:

[0013] Control the movable mirror to switch to wide-field imaging mode;

[0014] Controlling the wide-field excitation of samples by continuous laser;

[0015] Acquire and store the wide-field fluorescence image sequence of the sample;

[0016] Correlation calculations were performed on the wide-field image sequence, and fluorescence correlation spectral diffusion images of the images were plotted.

[0017] Furthermore, the method for acquiring and storing the wide-field fluorescence image sequence of the sample is as follows:

[0018] Wide-field fluorescence image sequences were acquired at equal time intervals, denoted as . The sequence length is Acquire the collection time of each wide-field fluorescence image. Let the pixels of the scanned pixel plane be denoted as The fluorescence signal intensity of each fluorescence image is ,in The sequence number of the acquired wide-field fluorescence image. and These are the x and y coordinates of the pixel;

[0019] The formula for calculating the correlation of a wide-field image sequence is as follows:

[0020]

[0021] in To normalize the correlation function, These are the pixel coordinates. The value is , , Until the image collection is complete, for The fluorescence intensity of each pixel in a wide-field fluorescence image recorded at all times;

[0022] pass The diffusion rate at each point is calculated by fitting the diffusion model, and the fluorescence-related spectral diffusion image is plotted.

[0023] Furthermore, the aforementioned through The specific model for calculating the diffusion time at each point under the illumination field of the light sheet by fitting the diffusion model is as follows:

[0024]

[0025] in

[0026]

[0027] in In pixels To observe the number of particles within the volume, The diffusion coefficient is... for Point spread function in direction, The thickness of the sheet.

[0028] Furthermore, through The fitting methods for calculating the diffusion time and diffusion coefficient at each point using the diffusion model are: Levenberg-Marquardt and trust region reflection methods, which calculate the feature diffusion coefficient for each pixel. .

[0029] Furthermore, the steps in S2 include:

[0030] Obtain the X and Y coordinates of the polygon vertices in the region of interest.

[0031] Input threshold As a condition for generating the scan array.

[0032] Furthermore, the method of S3 is as follows:

[0033] Determine the feature diffusion coefficient of each pixel. Is it greater than the threshold? If the diffusion coefficient Greater than the threshold And the pixel If the point is located inside the polygon within the region of interest, it is recorded and added to the scan array, denoted as . ,in The pixel number is the number of the pixel in the scan array, and the scan array length is... .

[0034] Furthermore, the steps in S4 include:

[0035] Control the movable reflector to switch to point scan mode;

[0036] The control acquisition card generates digital signals and acquires analog signals. The digital signals are used to control the first and second acousto-optic deflectors to control the excitation light to irradiate the scanning array in sequence. The acquired analog signals are converted into digital signals by the analog-to-digital converter in the acquisition card 13 and stored.

[0037] Furthermore, the method for using the digital signal to control the first and second acousto-optic deflectors is as follows:

[0038] According to the scan array coordinates The corresponding sound wave frequency is calculated, and the corresponding analog signal is calculated based on the sound wave frequency, which is then used by the acquisition card to generate the corresponding digital signal.

[0039] Furthermore, the method for sequentially irradiating the scanning array with excitation light is as follows:

[0040] According to the scan array coordinates ,according to From smallest to largest, the data acquisition cards are arranged at equal time intervals. Output the corresponding digital signal when scanning reaches the length of the scan array. Then, the scanning starts again from the first pixel coordinate of the scan array, and this process is repeated until the loop completes. Second-rate.

[0041] Furthermore, the steps involved in converting the acquired analog signals into digital signals using an analog-to-digital converter in the acquisition card and storing them include:

[0042] After the acquisition card outputs the corresponding digital signal, it synchronously acquires the current analog signal and records it. ,in The number of loops. The pixel number in the scan array is the time at which this data is recorded. .

[0043] The calculation formula for S5, which calculates the scanning fluorescence correlation spectrum of the fluorescence signal generated at the scanning array, is as follows:

[0044]

[0045] in To normalize the correlation function, This represents the number of iterations. The value is , , Until the scanning array fluorescence collection is complete, for The fluorescence intensity of each pixel in the scanned fluorescence image is recorded at any time, where This represents the pixel number in the scan array.

[0046] pass The diffusion rate at each point was calculated by fitting the diffusion model, and a scanning fluorescence-correlated spectral diffusion image was plotted.

[0047]

[0048] Furthermore, the aforementioned through The specific model for calculating the diffusion time at each point by fitting the diffusion model is as follows:

[0049]

[0050] in The characteristic diffusion time is denoted as . and These are the optical waist radius and optical axis radius of the laser focal point, respectively.

[0051] The specific model for the diffusion coefficient is as follows:

[0052]

[0053] Furthermore, through The fitting method for calculating the diffusion time and diffusion coefficient at each point using the diffusion model is the Levenberg-Marquardt method and the trust region reflection method, which calculates the feature diffusion coefficient for each pixel. .

[0054] A fast random addressing scanning fluorescence correlation spectroscopy device includes a first acousto-optic deflector and a cylindrical lens. A second acousto-optic deflector is provided at the output end of the first acousto-optic deflector, and a high-reflection, low-transmission dichroic mirror is provided at the output end of the second acousto-optic deflector. An objective lens is provided between the cylindrical lens and the high-reflection, low-transmission dichroic mirror, and a sample is provided on the objective lens.

[0055] The output end of the high-reflection, low-transmission dichroic mirror is equipped with a movable reflector via a filter. The output end of the movable reflector is equipped with a lens and a filter. The output end of the lens is equipped with an area array camera. The output end of the filter is equipped with a fluorescence collection device. The output ends of the area array camera and the fluorescence collection device are both equipped with acquisition cards. The acquisition cards are equipped with computers.

[0056] Compared with related technologies, the fast random addressing scanning fluorescence correlation spectroscopy method and apparatus provided by the present invention have the following advantages:

[0057] This invention provides a rapid random addressing scanning fluorescence correlation spectroscopy method and apparatus. It uses an acousto-optic deflector to control the excitation light to irradiate the scanning array sequentially and cyclically, and a signal acquisition module to synchronously acquire the fluorescence signal sequence of the corresponding array. This solves the problem that traditional scanning FCS cannot detect rapid dynamic processes due to slow scanning speed. It also solves the problem that traditional scanning FCS cannot simultaneously monitor the dynamics of multiple regions because the scanning galvanometer is mechanical and cannot be randomly addressed. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the process of the present invention;

[0059] Figure 2 This is a schematic diagram of the structure of the device of the present invention;

[0060] Figure 3 This is a schematic diagram of the image diffusion coefficient acquisition steps in this invention;

[0061] Figure 4 This is a schematic diagram of the diffusion coefficient image thresholding process in this invention;

[0062] Figure 5 This is a schematic diagram of the scanning correlation spectral image acquisition steps in this invention.

[0063] The following are the labels in the diagram: 1. First acousto-optic deflector; 2. Second acousto-optic deflector; 3. High-reflection, low-transmission dichroic mirror; 4. Objective lens; 5. Sample; 6. Cylindrical lens; 7. Filter; 8. Movable mirror; 9. Lens; 10. Area array camera; 11. Filter; 12. Fluorescence collection device; 13. Data acquisition card; 14. Computer. Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0065] Please refer to the following: Figure 1-5 As shown, a fast random addressing scanning fluorescence correlation spectroscopy device includes a first acousto-optic deflector 1 and a cylindrical lens 6. A second acousto-optic deflector 2 is provided at the output end of the first acousto-optic deflector 1, and a high-reflection, low-transmission dichroic mirror 3 is provided at the output end of the second acousto-optic deflector 2. An objective lens 4 is provided between the cylindrical lens 6 and the high-reflection, low-transmission dichroic mirror 3, and a sample 5 is provided on the objective lens 4.

[0066] The output end of the high-reflection, low-transmission dichroic mirror 3 is equipped with a movable reflector 8 via a filter 7. The output end of the movable reflector 8 is equipped with a lens 9 and a filter 11. The output end of the lens 9 is equipped with a field array camera 10. The output end of the filter 11 is equipped with a fluorescence collection device 12. The output ends of both the field array camera 10 and the fluorescence collection device 12 are equipped with a data acquisition card 13. A computer 14 is installed on the data acquisition card 13.

[0067] A fast random-addressing scanning fluorescence correlation spectroscopy method includes the following steps:

[0068] S1. A series of wide-field images under light sheet illumination are acquired using area array camera 10, and the global low temporal resolution image fluorescence correlation spectral diffusion image is obtained by calculation.

[0069] S2. Select the region of interest and set the scanning array generation conditions based on the wide-field fluorescence correlation spectrum image;

[0070] S3. Automatically generate a scanning array based on the selected region of interest and the conditions set above.

[0071] S4. The excitation light is controlled by the first acousto-optic deflector 1 and the second acousto-optic deflector 2 to irradiate the scanning array in a sequential cycle, and the fluorescence signal sequence of the corresponding array is synchronously acquired by the signal acquisition module on the acquisition card 13.

[0072] S5. Perform scanning fluorescence correlation spectroscopy calculations on the fluorescence signals generated at the scanning array to obtain ultra-high temporal resolution dynamic information of the region of interest;

[0073] The steps in S1 include:

[0074] Control the movable reflector 8 to switch to wide-field imaging mode;

[0075] Controlled continuous laser wide-field excitation of sample 5;

[0076] Acquire and store the wide-field fluorescence image sequence of sample 5;

[0077] Correlation calculations were performed on the wide-field image sequence, and fluorescence correlation spectral diffusion images of the images were plotted.

[0078] Furthermore, the method for obtaining and storing the wide-field fluorescence image sequence of sample 5 is as follows:

[0079] Wide-field fluorescence image sequences were acquired at equal time intervals, denoted as . The sequence length is Acquire the collection time of each wide-field fluorescence image. Let the pixels of the scanned pixel plane be denoted as The fluorescence signal intensity of each fluorescence image is ,in The sequence number of the acquired wide-field fluorescence image. and These are the x and y coordinates of the pixel;

[0080] The formula for calculating the correlation of a wide-field image sequence is as follows:

[0081]

[0082] in To normalize the correlation function, These are the pixel coordinates. The value is , , Until the image collection is complete, for The fluorescence intensity of each pixel in a wide-field fluorescence image recorded at all times;

[0083] pass The diffusion rate at each point is calculated by fitting the diffusion model, and the fluorescence-related spectral diffusion image is plotted.

[0084] This invention is achieved through The specific model for calculating the diffusion time at each point under the illumination field of the light sheet by fitting the diffusion model is as follows:

[0085]

[0086] in

[0087]

[0088] in In pixels To observe the number of particles within the volume, The diffusion coefficient is... for Point spread function in direction, The thickness of the sheet.

[0089] This invention is achieved through The fitting methods for calculating the diffusion time and diffusion coefficient at each point using the diffusion model are: Levenberg-Marquardt and trust region reflection methods, which calculate the feature diffusion coefficient for each pixel. .

[0090] Step S2 of the present invention includes:

[0091] Obtain the X and Y coordinates of the polygon vertices in the region of interest.

[0092] Input threshold As a condition for generating the scan array.

[0093] The method of S3 in this invention is as follows:

[0094] Determine the feature diffusion coefficient of each pixel. Is it greater than the threshold? If the diffusion coefficient Greater than the threshold And the pixel If the point is located inside the polygon within the region of interest, it is recorded and added to the scan array, denoted as . ,in The pixel number is the number of the pixel in the scan array, and the scan array length is... .

[0095] Step S4 of the present invention includes:

[0096] Control the movable reflector 8 to switch to point scan mode;

[0097] The present invention controls the acquisition card 13 to generate digital signals and acquire analog signals. The digital signals are used to control the first acousto-optic deflector 1 and the second acousto-optic deflector 2 to control the excitation light to irradiate the scanning array in sequence. The acquired analog signals are converted into digital signals by the analog-to-digital converter in the acquisition card 13 and stored.

[0098] The method of using digital signals to control the first acousto-optic deflector 1 and the second acousto-optic deflector 2 according to the present invention is as follows:

[0099] Based on the scan array coordinates The corresponding sound wave frequency is calculated, and the corresponding analog signal is calculated based on the sound wave frequency. The corresponding digital signal is then generated by the acquisition card 13.

[0100] The method of sequentially irradiating the scanning array with excitation light according to the present invention is as follows:

[0101] Based on the scan array coordinates ,according to In ascending order, data is collected from data acquisition card 13 at equal time intervals. Output the corresponding digital signal when scanning reaches the length of the scan array. Then, the scanning starts again from the first pixel coordinate of the scan array, and this process is repeated until the loop completes. Second-rate.

[0102] The steps of converting the analog signal acquired by this invention into a digital signal via the analog-to-digital converter in the acquisition card 13 and storing it include:

[0103] After the acquisition card 13 outputs the corresponding digital signal, it synchronously acquires the current analog signal and records it. ,in The number of loops. The pixel number in the scan array is the time at which this data is recorded. .

[0104] The formula for calculating the scanning fluorescence correlation spectrum of the fluorescence signal generated at the scanning array in S5 is as follows:

[0105]

[0106] in To normalize the correlation function, This represents the number of iterations. The value is , , Until the scanning array fluorescence collection is complete, for The fluorescence intensity of each pixel in the scanned fluorescence image is recorded at any time, where This represents the pixel number in the scan array.

[0107] pass The diffusion rate at each point was calculated by fitting the diffusion model, and a scanning fluorescence-correlated spectral diffusion image was plotted.

[0108] This invention is achieved through The specific model for calculating the diffusion time at each point by fitting the diffusion model is as follows:

[0109]

[0110] in The characteristic diffusion time is denoted as . and These are the optical waist radius and optical axis radius of the laser focal point, respectively.

[0111] The specific model for the diffusion coefficient is as follows:

[0112]

[0113] This invention is achieved through The fitting method for calculating the diffusion time and diffusion coefficient at each point using the diffusion model is the Levenberg-Marquardt method and the trust region reflection method, which calculates the feature diffusion coefficient for each pixel. .

[0114] Example

[0115] like Figure 2 As shown, the solid line represents the laser propagation path, and the dashed line represents the fluorescence or signal propagation path. The path direction is indicated by the arrow. The laser generation includes, but is not limited to, the use of gas lasers, solid-state lasers, and semiconductor lasers. The area array camera 10 includes, but is not limited to, the use of CCD photosensitive elements or CMOS photosensitive elements.

[0116] like Figure 2 As shown, the first acousto-optic deflector 1 and the second acousto-optic deflector 2 are used to change the deflection angle of the laser in the X and Y directions, respectively.

[0117] The first acousto-optic deflector 1 and the second acousto-optic deflector 2 of the present invention are scanned under the digital signal control of the acquisition card 13;

[0118] The cylindrical lens 6 of this invention is used to generate an illumination light field.

[0119] The fluorescence signal acquired by this invention is transmitted to the computer 14 via the acquisition card 13 for data processing;

[0120] In this embodiment, a complete scanning fluorescence correlation spectroscopy acquisition cycle is as follows:

[0121] First, turn off the point scanning laser, that is, the laser at the positions of the first acousto-optic deflector 1 and the second acousto-optic deflector 2, and turn on the single-plane illumination light, that is, the laser at the position of the cylindrical lens 6.

[0122] The present invention controls the mirror to switch to wide-field imaging mode, that is, the movable mirror 8 is placed in the optical path, and the single-plane illumination light irradiates the sample 5 to generate fluorescence. The fluorescence passes through the objective lens 4, the high-reflection low-transmission dichroic mirror 3, the filter 7, the movable mirror 8, and the lens 9 in sequence to reach the area array camera 10. After continuous synchronous acquisition, the fluorescence image sequence signal is obtained.

[0123] like Figure 3 As shown, the obtained Five fluorescence images were generated, and the fluorescence signal intensity of each image was denoted as . ,in The sequence number of the acquired wide-field fluorescence image. and These are the x and y coordinates of the pixel;

[0124] The calculation formula for correlation calculation of the wide-field image sequence of the present invention is as follows:

[0125]

[0126] in To normalize the correlation function, These are the pixel coordinates. The value is , , Until the image collection is complete, for The fluorescence intensity of each pixel in a wide-field fluorescence image recorded at all times.

[0127] This invention uses the Levenberg-Marquardt and trust region reflection methods to fit the relevant model and obtain the corresponding diffusion coefficient D. The specific model is as follows:

[0128] in

[0129]

[0130] in In pixels To observe the number of particles within the volume, Where is the diffusion coefficient. for Point spread function in direction, The thickness of the sheet;

[0131] This invention selects a polygonal region of interest in a computer, records the X and Y coordinates of the polygon's vertices, and inputs a threshold. As a condition for generating the scanning array;

[0132] like Figure 4 As shown, this invention determines the feature diffusion coefficient of each pixel. Is it greater than the threshold? If the diffusion coefficient Greater than the threshold And the pixel If the point is located inside the polygon within the region of interest, it is recorded and added to the scan array, denoted as . ,in The pixel number is the number of the pixel in the scan array, and the scan array length is... The image on the right is The generated mask;

[0133] This invention controls the reflector to switch to point scanning mode, turns on the point scanning laser, that is, the laser at the position of the first acousto-optic deflector 1 and the second acousto-optic deflector 2, and turns off the single-plane illumination light, that is, the laser at the position of the cylindrical lens 6.

[0134] In this invention, the reflector is switched to point scanning mode, i.e., the reflector 8 is moved outside the optical path. After the single-plane illumination light shines on the sample, fluorescence is generated. The fluorescence passes through the objective lens 4, the high-reflection low-transmission dichroic mirror 3, the filter 7, the movable reflector 8, the filter 11, and the fluorescence collection device 12 to acquire the fluorescence image sequence signal.

[0135] It should be noted that the fluorescence collection device 12 includes, but is not limited to, the use of a photomultiplier tube (PMT), an avalanche photodiode (APD), and a transimpedance amplifier.

[0136] In this invention, the data acquisition card 13 generates digital signals and acquires analog signals. The digital signals are used to control the first acousto-optic deflector 1 and the second acousto-optic deflector 2 to control the excitation light to irradiate the scanning array in sequence. The acquired analog signals are converted into digital signals by the analog-to-digital converter in the acquisition card 13 and stored.

[0137] This invention, based on the coordinates of the scanning array The corresponding sound wave frequency is calculated, and the corresponding analog signal is calculated based on the sound wave frequency. The corresponding digital signal is then generated by the acquisition card 13.

[0138] like Figure 5 As shown, in this invention, based on the coordinates of the scanning array... ,according to From smallest to largest, the data acquisition cards are arranged at equal time intervals. Output the corresponding digital signal when scanning reaches the length of the scan array. Then, the scanning starts again from the first pixel coordinate of the scan array, and this process is repeated until the loop completes. Second-rate;

[0139] After the acquisition card 13 of this invention outputs the corresponding digital signal, it synchronously acquires the current analog signal and records it. ,in The number of loops. The pixel number in the scan array is the time at which this data is recorded. ;

[0140] This invention performs scanning fluorescence correlation spectroscopy calculations on the fluorescence signals generated at the scanning array. The calculation formula is as follows:

[0141]

[0142] in To normalize the correlation function, The number of loops. The value is , , Until the scanning array fluorescence collection is complete, for The fluorescence intensity of each pixel in the scanned fluorescence image is recorded at any time, where This represents the pixel number in the scan array.

[0143] The Levenberg-Marquardt and trust region reflection methods were used to fit the relevant model, and the feature diffusion coefficient of each pixel was calculated. The specific model is as follows:

[0144]

[0145] in

[0146]

[0147] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A fast random addressing scanning fluorescence correlation spectroscopy method, characterized in that, Includes the following steps: S1. Control the movable mirror (8) to switch to wide field imaging mode, use the area array camera (10) to acquire a series of wide field images under light sheet illumination, calculate the global low temporal resolution fluorescence correlation spectral diffusion image, and calculate the characteristic diffusion coefficient of each pixel. S2. Select the region of interest and set the scanning array generation conditions based on the fluorescence correlation spectral diffusion image; S3. Automatically generate a scanning array based on the selected region of interest and the conditions set above. S4. The excitation light is controlled by the first acousto-optic deflector (1) and the second acousto-optic deflector (2) to irradiate the scanning array in sequence, and the fluorescence signal sequence of the corresponding array is synchronously acquired by the signal acquisition module on the acquisition card (13). S5. Perform scanning fluorescence correlation spectroscopy calculations on the fluorescence signals generated at the scanning array to obtain ultra-high temporal resolution dynamic information of the region of interest; The steps in S2 include: Obtain the X and Y coordinates of the vertices of the polygon in the region of interest. Input threshold As a condition for generating the scanning array; The method for S3 is as follows: Determine the feature diffusion coefficient of each pixel. Is it greater than the threshold? If the diffusion coefficient Greater than the threshold And the pixel Within the polygon of the region of interest, the pixel is recorded and added to the scan array, denoted as . ,in The pixel number is the number of the pixel in the scan array, and the scan array length is... ; The steps in S4 include: Control the movable reflector (8) to switch to point scan mode; The control acquisition card (13) generates digital signals and acquires analog signals. The digital signals are used to control the first acoustic-optic deflector (1) and the second acoustic-optic deflector (2) to control the excitation light to irradiate the scanning array in sequence. The acquired analog signals are converted into digital signals by the analog-to-digital converter in the acquisition card (13) and stored. The method by which the digital signal controls the first acoustic-optical deflector (1) and the second acoustic-optical deflector (2) is as follows: Based on the scan array coordinates The corresponding sound wave frequency is calculated, and the corresponding analog signal is calculated based on the sound wave frequency. The corresponding digital signal is generated by the acquisition card (13). The method of sequentially irradiating the scanning array with excitation light is as follows: According to the scan array coordinates ,according to In ascending order, the data is collected by the acquisition card (13) at equal time intervals. Output the corresponding digital signal when scanning reaches the length of the scan array. Then, the scanning starts again from the first pixel coordinate of the scan array, and this process is repeated until the loop completes. Second-rate.

2. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 1, characterized in that, The steps in S1 include: Controlling the wide-field excitation of the sample with continuous laser (5); Acquire and store the wide-field fluorescence image sequence of sample (5); Correlation calculations were performed on the wide-field image sequence, and fluorescence correlation spectral diffusion images were plotted.

3. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 2, characterized in that, The method for obtaining and storing the wide-field fluorescence image sequence of sample (5) is as follows: Wide-field fluorescence image sequences were acquired at equal time intervals, denoted as . The sequence length is Acquire the collection time of each wide-field fluorescence image. Let the pixels of the scanned pixel plane be denoted as The fluorescence signal intensity of each fluorescence image is ,in The sequence number of the acquired wide-field fluorescence image. and These are the x and y coordinates of the pixel; The formula for calculating the correlation of a wide-field image sequence is as follows: in To normalize the correlation function, The value is , , Until the image collection is complete, for The fluorescence intensity of each pixel in a wide-field fluorescence image recorded at all times; pass The diffusion coefficient at each point is calculated by fitting the diffusion model, and the fluorescence-related spectral diffusion image is plotted.

4. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 3, characterized in that, The passage The specific model for calculating the diffusion coefficient at each point using a diffusion model under light sheet illumination is as follows: in in In pixels To observe the number of particles within the volume, The diffusion coefficient is... for Point spread function in direction, The thickness of the sheet.

5. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 4, characterized in that, pass The fitting methods for calculating the diffusion coefficient at each point using the diffusion model are: Levenberg-Marquardt and trust region reflection methods, which calculate the feature diffusion coefficient for each pixel. .

6. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 5, characterized in that, The steps for converting the acquired analog signal into a digital signal and storing it via the analog-to-digital converter in the acquisition card (13) include: After the acquisition card (13) outputs the corresponding digital signal, it synchronously acquires the current analog signal and records it. ,in The number of loops. The pixel number in the scan array is used to record the time of the analog signal. , The calculation formula for S5, which calculates the scanning fluorescence correlation spectrum of the fluorescence signal generated at the scanning array, is as follows: in To normalize the correlation function, The number of loops. The value is , , Until the scanning array fluorescence collection is complete, for The fluorescence intensity of each pixel in the scanned fluorescence image is recorded at any time, where The pixel number in the scan array. pass The diffusion coefficient at each point is calculated by fitting the diffusion model, and a scanning fluorescence-correlated spectral diffusion image is plotted.

7. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 6, characterized in that, The passage The specific model for calculating the diffusion time at each point by fitting the diffusion model is as follows: in Characteristic diffusion time, and These are the optical waist radius and optical axis radius of the laser focal point, respectively. The specific model for the diffusion coefficient is as follows: 。 8. The fast random addressing scanning fluorescence correlation spectroscopy method according to claim 7, characterized in that, pass The fitting method for calculating the diffusion time and diffusion coefficient at each point using the diffusion model is the Levenberg-Marquardt method and the trust region reflection method, which calculates the feature diffusion coefficient for each pixel. .

9. A fast random addressing scanning fluorescence correlation spectroscopy method according to any one of claims 1-8, characterized in that, The method is based on a fast random addressing scanning fluorescence correlation spectroscopy device, which includes a first acousto-optic deflector (1) and a cylindrical lens (6). A second acousto-optic deflector (2) is provided at the output end of the first acousto-optic deflector (1), and a high-reflection, low-transmission dichroic mirror (3) is provided at the output end of the second acousto-optic deflector (2). An objective lens (4) is provided between the cylindrical lens (6) and the high-reflection, low-transmission dichroic mirror (3), and a sample (5) is provided on the objective lens (4). The output end of the high-reflection, low-transmission dichroic mirror (3) is provided with a movable reflector (8) through a first filter (7). The output end of the movable reflector (8) is provided with a lens (9) and a second filter (11). The output end of the lens (9) is provided with an area array camera (10). The output end of the second filter (11) is provided with a fluorescence collection device (12). The output ends of the area array camera (10) and the fluorescence collection device (12) are both provided with a data acquisition card (13). The data acquisition card (13) is communicatively connected to a computer (14).

Images