A pixel-relocation-based super-resolution fluorescence lifetime microscopic imaging method and system

Abstract

The application relates to a kind of super-resolution fluorescence lifetime microscopic imaging method and system based on pixel relocation, comprising the following steps: S1, using acoustooptic deflector to carry out multi-focus parallel scanning to sample;S2, by surface array detector and point detector synchronous acquisition fluorescence intensity image and fluorescence lifetime data;S3, based on pixel relocation algorithm, fluorescence intensity image is super-resolution reconstructed, and fluorescence lifetime data is mapped to reconstructed image, and super-resolution fluorescence lifetime microscopic image is generated, the application adopts AOD to replace scanning galvanometer in traditional TCSPC-FLIM imaging system as light beam scanner, in combination with carefully designed multi-path synchronous signal, the addressing scanning of AOD, image acquisition of EMCCD and the lifetime data acquisition of PMT and TCSPC card are closely linked, and through the data processing method of later period, fluorescence lifetime data is distributed to each point array, so that the organic combination of super-resolution and FLIM can be realized without complex equipment, and the SR-FLIM image of sample is obtained.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy, specifically to a super-resolution fluorescence lifetime microscopy method and system based on pixel relocation. Background Technology

[0002] Optical microscopy, with its advantages of high sensitivity, high spatiotemporal resolution, and non-invasiveness, is one of the main tools in biomedical research. However, due to diffraction effects, the resolution of traditional optical microscopy is limited, typically to 250-300 nm. Laser scanning confocal microscopy (LSCM) achieves higher image contrast and optical tomography by adding a confocal pinhole to filter out light signals outside the focal plane of the sample. Furthermore, when the pinhole size is small (<1.0 AU, where AU is the Airy unit, and a 1.0 AU pinhole means the pinhole diameter is equal to the Airy disk diameter of the system's point spread function), the resolution can be further improved; theoretically, the smaller the pinhole, the higher the resolution. However, as the pinhole size decreases, the light flux also decreases, leading to a lower image signal-to-noise ratio. Therefore, using this method for super-resolution imaging is not very practical. Image scanning microscopy (ISM) uses area array detectors instead of the single-point detectors in LSCM. Since the equivalent pixel size of the area array detector on the sample surface is similar to or even smaller than the confocal pinhole size in LSCM, each pixel essentially functions as a single-point detector with its own pinhole. This allows the spatial resolution of the scanned image to be comparable to that of LSCM. Furthermore, the simultaneous acquisition of signals by multiple single-point detectors significantly improves energy efficiency, thus enabling better super-resolution imaging. However, the ISM approach of using single-point scanning combined with area array detectors results in a slow imaging speed. Multifocal structured illumination microscopy (MSIM) addresses this by introducing parallel excitation modes, which greatly improves the imaging speed and has become one of the more commonly used super-resolution microscopy imaging methods.

[0003] On the other hand, in most optical microscopy imaging techniques, the intensity or color of the detected light signal is usually used as image contrast. However, in reality, each fluorescent molecule, in addition to having a unique emission spectrum, also has a specific lifetime, which reflects the time the fluorophore is in an excited state before emitting a photon. This lifetime can sensitively reflect changes in the microenvironment in which the fluorophore is located or the energy transfer that occurs. Therefore, fluorescence lifetime imaging microscopy (FLIM), which forms image contrast by detecting fluorescence lifetime, can provide more information for biomedical optical imaging and is widely used in biological microenvironment monitoring, protein interaction research, and other fields. Currently, the commonly used method for detecting fluorescence lifetime in FLIM is time-correlated single photon counting (TCSPC). This method requires using a point detector with single photon counting capability to record the arrival time of each photon. After accumulating a sufficient number of photons, lifetime curve fitting or calculation is performed. It can usually be well combined with LSCM. However, since traditional area array detectors do not have single-photon counting capabilities, it is difficult to combine the super-resolution principles of ISM and MSIM to achieve super-resolution fluorescence lifetime imaging microscopy (SR-FLIM).

[0004] In 2019, Marco Castello et al. proposed an ISM technique with a single-photon avalanche diode (SPAD) array detector. This technique uses an array of 5×5 SPAD detectors instead of a traditional area array detector and proposes an adaptive pixel relocation algorithm to compensate for the error between the actual and ideal offset vectors, reducing the size of the system's point spread function (PSF) and improving imaging resolution. Simultaneously, due to the single-photon counting capability of the SPAD array, this method successfully combines ISM with FLIM, thus achieving SR-FLIM. In 2025, Cuifang Kuang et al. proposed a fluorescence emission difference (FED) technique using a detector array composed of seven avalanche photodiodes (APDs) and a TCSPC module. This technique improves quantum efficiency through parallel detection of the APD array, effectively suppresses the stacking effect of TCSPC, and reduces the system PSF size by combining FED and pixel relocation methods, achieving PDFED-FLIM with a 1.6-fold improvement in spatial resolution. However, both of these methods either require the additional purchase of expensive and immature SPAD array detectors, or necessitate the self-combination of multiple point detectors and multiple sets of TCSPC modules, which is not only costly but also places very high demands on the user. Furthermore, because both types of array detectors have very few pixels, their imaging method, like ISM, is based on single-point scanning and area array detector detection, resulting in slow imaging speeds. Therefore, how to conveniently and effectively implement SR-FLIM using area array detectors such as charge-coupled devices (CCDs) and complementary metal-oxide-semiconductors (CMOS), which are more commonly used in optical microscopy, and point detectors such as photomultiplier tubes (PMTs), commonly used in traditional FLIM imaging, is a problem that urgently needs to be solved for the development and widespread application of SR-FLIM technology. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art, adapt to practical needs, and provide a super-resolution fluorescence lifetime microscopy imaging method and system based on pixel relocation. This solves the current technical problem of needing to purchase expensive and immature SPAD array detectors, or needing to combine multiple point detectors and multiple sets of TCSPC modules, which is not only costly but also places very high demands on users. In addition, since the number of pixels in the above two types of array detectors is very small, the imaging method is based on single-point scanning and area array detector detection, just like ISM, resulting in a slow imaging speed.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows: A super-resolution fluorescence lifetime microscopy imaging method based on pixel relocation is designed, comprising the following steps:

[0007] S1. Use an acousto-optic deflector (AOD) to perform multi-focal parallel scanning of the sample;

[0008] S2. Simultaneously acquire fluorescence intensity images and fluorescence lifetime data using area array detectors and point detectors;

[0009] S3. The fluorescence intensity image is super-resolution reconstructed based on the pixel relocation algorithm, and the fluorescence lifetime data is mapped to the reconstructed image to generate a super-resolution fluorescence lifetime microscopy (SR-FLIM) image.

[0010] Preferably, the area array detector is an electron multiplier charge-coupled device (EMCCD) or a scientific-grade complementary metal-oxide-semiconductor (sCMOS) camera; the point detector is a photomultiplier tube (PMT) or an avalanche photodiode (APD).

[0011] Preferably, the scanning control of the AOD is achieved through digital signals, which include acoustic frequency codes in the X and Y directions, and the scanning timing is strictly synchronized with the exposure signal of the area array detector and the single photon counting signal of the point detector.

[0012] Preferably, the pixel relocation algorithm includes the following steps:

[0013] Adjust the focal spot size or spacing according to the ratio of excitation wavelength to fluorescence emission wavelength;

[0014] Multifocal scan image sequences are spatially superimposed, and the resolution is further improved through deconvolution processing.

[0015] Preferably, the fluorescence lifetime data is acquired by a time-correlated single-photon counting (TCSPC) module, and the photon count-time histogram of each scan point is assigned with the relative intensity value of the corresponding pixel of the area array detector as the weight.

[0016] A super-resolution fluorescence lifetime microscopy system based on pixel relocation, comprising:

[0017] The wide-field illumination module, including light-emitting diodes, a condenser lens, and a dichroic mirror, is used to illuminate the sample;

[0018] The multifocal excitation module includes a femtosecond pulsed laser, a half-wave plate, a polarizing beam splitter prism, a dispersion compensation prism, a two-dimensional acousto-optic deflector, a dichroic mirror, two beam expander lens groups, and a pair of mirrors, which are used to control the laser beam to perform multifocal scanning in the region of interest to excite the sample to generate a fluorescence signal.

[0019] The image detection module includes an emission filter, a tube mirror, and an EMCCD camera, used to acquire fluorescence dot matrix images generated by the multifocal scanning;

[0020] The lifetime acquisition module, including a beam splitter, PMT, and TCSPC card, is used to acquire fluorescence lifetime data for each scan point.

[0021] The control module includes a data acquisition card, a computer terminal, and a control program running on it, which is used to output a synchronization signal to realize the synchronous control of the multifocal scanning, fluorescence array image acquisition, and fluorescence lifetime data acquisition.

[0022] Preferably, the wide-field illumination module uses a light-emitting diode as the light source. The light emitted by the light source is focused at the entrance pupil of the objective lens by a condenser lens and a dichroic mirror. After passing through the objective lens, the light is illuminated on the sample in a parallel light illumination manner, thereby achieving wide-field illumination and excitation.

[0023] Preferably, the image detection module and the lifetime acquisition module are precisely synchronized through a synchronization signal, which includes digital signals, analog signals, and three analog pulse signals for pixels, lines, and frames, to ensure that the image detection module and the lifetime acquisition module can acquire data simultaneously and accurately during the scanning process of the acousto-optic deflector.

[0024] Preferably, it also includes a dispersion compensation prism or grating pair for correcting spatial and temporal dispersion generated by AOD scanning.

[0025] Preferably, the multi-focus excitation module supports a multi-focus parallel scanning mode, and the scanning area can be dynamically adjusted according to the region of interest selected in the wide-field fluorescence image to reduce invalid scans and reduce phototoxicity.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention achieves SR-FLIM imaging by using only a single PMT and a single TCSPC module, which are already present in traditional TCSPC-FLIM, as well as an EMCCD or sCMOS camera commonly used in super-resolution fluorescence microscopy, thus significantly reducing system costs. At the same time, since the detection area of ​​EMCCD and sCMOS is larger than that of existing SPAD array detectors or multiple APD combinations, it is no longer limited to the single-point scanning ISM super-resolution principle, and can greatly improve the imaging speed by utilizing multi-focal parallel scanning MSIM super-resolution.

[0028] 2. This invention introduces a wide-field illumination optical path, facilitating the user's search for structures of interest. Combined with the system's AOD (Alignment On Demand) technology, it enables addressable scanning imaging of the region of interest. Imaging speed can be further improved by reducing the scanning area. Furthermore, by combining the pixel relocation method with TCSPC-FLIM, the resolution of the processed fluorescence lifetime image is significantly improved compared to traditional TCSPC-FLIM images. It can distinguish microstructures or microenvironments in cells that cannot be resolved using traditional TCSPC-FLIM, and the spatial resolution of the obtained SR-FLIM image is comparable to that of the super-resolution fluorescence intensity image obtained by directly relocating the pixels of the dot matrix fluorescence intensity image. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the system of the present invention;

[0030] Figure 2 The flowchart of AOD scanning and data processing of the present invention is shown in ((a) AOD scanning sequence diagram (b) Synchronization signal diagram (c) TCSPC data storage sequence diagram (d) Coordinate mapping diagram).

[0031] Figure 3 Two methods for pixel repositioning in this invention are (a) reducing the focal spot size and (b) increasing the focal spot spacing.

[0032] Figure 4 This is a schematic diagram of the photon number histogram allocation of the present invention ((a) photon number histogram of a certain scanning point (b) focal spot captured by EMCCD when AOD scans the point (c) focal spot after pixel repositioning (d) photon number histogram allocated with the relative intensity value of each pixel as weight).

[0033] Figure 5 This is a schematic diagram of the selection area operation interface of the control program of the present invention;

[0034] Figure 6 This is a schematic diagram of the data acquisition operation interface of the control program of the present invention;

[0035] Figure 7The following are images showing the implementation results of the present invention: (a) conventional fluorescence intensity image, (b) super-resolution fluorescence intensity image, (c) conventional fluorescence lifetime image, and (d) super-resolution fluorescence lifetime image.

[0036] In the picture: Figure 1 The English labels in this document are defined as follows: Laser: Laser device; HWP: Half-wave plate; PBS: Polarizing beam splitter prism; M: Mirror; BE1: First beam expander and shaping lens pair; Prism: Dispersion compensation prism; AOD: Acousto-optic deflector; BE2: Second beam expander and shaping lens pair; DM: Dichroic mirror; Objective: Objective lens; Sample: Sample (located on the stage); LED: Light-emitting diode light source; BS: Beam splitter; PMT: Photomultiplier tube; TCSPC card: Time-correlated single-photon counter card; EMCCD: Electron multiplier charge-coupled device; DAQ card: Data acquisition card; frame: Frame signal; line: Line signal; pixel: Pixel signal; digital signal: Digital signal; analog signal: Analog signal; PC: Computer terminal. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0038] A super-resolution fluorescence lifetime microscopy method based on pixel relocation, see [link to relevant documentation]. Figures 1 to 7 The specific steps are as follows:

[0039] S1. Use LED light source to illuminate the sample to obtain a wide-field fluorescence image. Select the region of interest in the control software written in LabVIEW, and the software will automatically calculate the pixel coordinate information of the MSIM scan array corresponding to the region of interest.

[0040] S2. Switch to laser illumination mode to acquire super-resolution fluorescence lifetime microscopic imaging data. The control software controls the data acquisition card to output digital signals, analog signals and three synchronous signals according to the coordinate information of the selected region of interest. The digital signals control the AOD to scan the selected region of interest, the analog signals control the EMCCD to synchronously acquire fluorescence images of each scan array, and the three synchronous signals control the TCSPC to store fluorescence lifetime data, saving the fluorescence image sequence and fluorescence lifetime data file respectively.

[0041] S3. Offline data processing was performed using data processing software written in Matlab. First, the collected dot matrix fluorescence image sequence was relocated to pixels. Then, the fluorescence lifetime data was distributed to each pixel to obtain multiple dot matrix images containing fluorescence lifetime information, which were then superimposed. Finally, fluorescence lifetime analysis was performed on the superimposed image containing fluorescence lifetime information to obtain a super-resolution fluorescence lifetime image corresponding to the region of interest.

[0042] Specifically, in S1, an LED light source is used to excite the fluorescence of the sample, and an EMCCD is used for imaging. Focusing is then used to obtain a clear image of the sample structure. When using LED illumination to select the region of interest, care must be taken to block the optical path of the PMT (Programmable Module) or to turn off the TCSPC card to prevent irreversible damage to the PMT caused by strong light while it is powered on. The captured images can be saved in any file path for use in data processing software. The region of interest to be scanned can be manually selected by the experimenter on the selection interface of the control software. The region shape is generally set to rectangle, such as... Figure 5 As shown. After the selection is completed, the control software will calculate and save the pixel coordinate information of the multifocal array scanning points corresponding to the selected region of interest. The saved pixel coordinate file will be used in S2.

[0043] Furthermore, in S2, the control software utilizes the linear correspondence between pixel coordinates and acoustic frequencies to calculate the acoustic frequency corresponding to each pixel position and convert it into a 32-bit digital signal required by the data acquisition card. This signal loads the acoustic frequency onto the AOD, enabling it to scan specific pixel positions of the sample. The AOD is controlled to perform addressing and scanning according to the saved scan point pixel position coordinates. Simultaneously, based on the scan coordinates of the dot matrix, the control program... Figure 2 (b) The relationship simultaneously outputs the corresponding analog signal and three synchronization signals. The analog signal controls the EMCCD to achieve synchronization between exposure triggering and dot matrix scanning. That is, after each dot matrix scan by the AOD, the camera performs one exposure, finally capturing a series of dot matrix fluorescence images. The three synchronization signals are also analog signals used to control the location of the lifetime data stored in the TCSPC. The pixel signal is output as the AOD scan occurs, the line signal is output when the AOD scans the first point of each dot matrix image, and the frame signal is output when the AOD scans the first point of the entire area. Before the synchronization signal is generated, it is first... Figure 6 In the control program's data acquisition interface, the PMT and EMCCD are instructed to enter an external trigger waiting state in advance, and then... Figure 5 By confirming the output synchronization signal in the selected area operation interface, synchronization of AOD scanning, TCSPC storage, and EMCCD and PMT acquisition can be achieved.

[0044] It is worth noting that in S3, the fluorescence dot matrix image sequence acquired by the EMCCD in S2 is first processed for pixel relocation and saved. Simultaneously, each scan point in each dot matrix image is marked according to the scanning sequence. Since AOD, EMCCD, and TCSPC are all controlled by synchronization signals output from the data acquisition card, the correspondence between each scan point in each dot matrix image and the photon count-time histogram stored in the PMT and TCSPC can be confirmed based on the generation method of the synchronization signal. Then, by reassigning the photon count-time histogram of a focal spot recorded by the PMT and TCSPC cards using the relative intensity values ​​of the corresponding pixels on the EMCCD image as weights, multiple sub-histograms are generated for each pixel within the corresponding focal spot area, i.e., a series of dot matrix images containing fluorescence lifetime information. Subsequent processing is similar to traditional pixel relocation algorithms; simply slicing and summing the series of dot matrix images yields a complete super-resolution image. Fluorescence lifetime analysis of the photon count-time histograms contained in each pixel in the image provides a super-resolution fluorescence lifetime image.

[0045] It is worth noting that among the aforementioned components, the EMCCD can be replaced with an sCMOS camera or other highly sensitive area array detectors, the PMT can be replaced with an APD (Aspect Ratio Detector), and the prism can be replaced with a prism pair or a grating pair. In the illumination path, the LED lamp can be replaced with a mercury lamp or a xenon lamp. A zoom lens can be added to the optical path to achieve three-dimensional scanning imaging.

[0046] Example 1

[0047] Synchronous scanning control and multifocal excitation method based on AOD

[0048] S1. The sample is scanned in parallel with multiple focal points using an acousto-optic deflector (AOD). The scanning of the AOD is controlled by a 32-bit digital signal output from the data acquisition card. The lower 16 bits encode the acoustic frequency in the X direction, and the higher 16 bits encode the acoustic frequency in the Y direction. Each pixel position corresponds to a specific digital signal to achieve rapid beam deflection.

[0049] S2. Within one exposure cycle of the EMCCD, control the AOD to scan a preset 3×3 dot matrix (or other dot matrix size), and move the dot matrix position sequentially in subsequent exposure cycles to cover the entire field of view or select the region of interest (ROI).

[0050] S3. Generate an analog signal synchronized with the scanning dot matrix to control the exposure time of the EMCCD and ensure that the dot matrix scanning cycle is strictly matched with the camera exposure.

[0051] S4. Implement parallel scanning through a multi-focus excitation module (including a femtosecond pulsed laser and a dispersion compensation prism), and preselect the ROI in combination with a wide-field illumination module (LED or mercury lamp) to reduce the ineffective scanning area.

[0052] Example 2

[0053] Pixel repositioning algorithm and super-resolution reconstruction method

[0054] S1. After collecting the fluorescence dot matrix image sequence of multi-focus scanning, perform super-resolution reconstruction using the pixel repositioning algorithm:

[0055] Method 1: Reduce the image size of each focus to 1 / k 2 / (k 2 +n) times of the original image and then stack them;

[0056] Method 2: Keep the focal spot size unchanged, increase its spacing to (k 2 +n) / k 2 times of the original spacing and then stack them;

[0057] As Figure 3 shown, where the constant k represents the ratio of the excitation light wavelength to the fluorescence emission wavelength, and the constant n takes 1 in the case of single-photon excitation and 2 in the case of two-photon excitation;

[0058] S2. For the case of single-photon excitation, generally the fluorescence emission wavelength is only slightly longer than the excitation light wavelength, at this time k≈1. After processing according to the above method, the adjacent focal spot spacing is about 2 times the original. Stacking the processed dot matrix can increase the image resolution by about times; while for the case of two-photon excitation, 1<k<2. Taking 800nm excitation and 550nm emission as an example, it can be calculated that k≈1.45. After processing according to the above method, the adjacent focal spot spacing is about 1.95 times the original, and the corresponding image resolution can be increased by about 1.4 times;

[0059] S3. Further optimize the reconstructed image in combination with the deconvolution algorithm to increase the resolution to 2 times.

[0060] Example 3

[0061] Fluorescence lifetime data synchronous acquisition and mapping scheme

[0062] S1. Use a PMT and a TCSPC module to collect the fluorescence lifetime data of each focus, and control the data storage through pixel, row, and frame three-way synchronous signals:

[0063] Pixel signal: Switch the storage location after scanning each focus;

[0064] Row signal: Switch to the starting position of the next row after completing one dot matrix scan;

[0065] Frame signal: Reset the storage location after completing the full field of view scan;

[0066] S2. Remap the lifetime data (photon number-time histogram) recorded by TCSPC according to the AOD scan coordinates so that the lifetime data of each focal spot corresponds to the actual spatial location.

[0067] S3. Set a merging factor (e.g., bin=1) to weight and merge lifetime data of adjacent pixels to improve lifetime fitting accuracy in low photon number regions.

[0068] Example 4

[0069] Super-resolution fluorescence lifetime image fusion method

[0070] S1. Perform pixel relocation processing on the fluorescence dot matrix image acquired by EMCCD to generate a super-resolution fluorescence intensity image;

[0071] S2. Distribute the lifetime data recorded by the PMT and TCSPC modules to the super-resolution image as follows:

[0072] Using the relative intensity of each pixel in the EMCCD image as the weight, the lifetime histogram of a single focal spot is split into multiple sub-histograms.

[0073] The sub-histograms are assigned to the corresponding pixels, and the fluorescence lifetime value of each pixel is obtained by fitting.

[0074] S3. The lifetime data is fused with the super-resolution intensity image to generate a three-dimensional array of SR-FLIM images, which include spatial coordinates and fluorescence decay information.

[0075] Example 5

[0076] System integration and experimental verification

[0077] S1. Build the imaging system, including a wide field illumination module, a multifocal excitation module (AOD + femtosecond laser), an EMCCD / sCMOS camera, a PMT + TCSPC module, and a synchronization control module.

[0078] S2. An experiment was conducted using lily of the valley rhizome samples as an example:

[0079] (1) Select an appropriate wavelength for LED illumination, set the EMCCD for continuous imaging, adjust the objective lens position up and down to focus the imaging plane onto the sample surface, adjust the stage position forward, backward, left and right to find the cell region of interest, acquire a wide-field fluorescence image of the sample, load the acquired wide-field fluorescence image under "File Path", select the region of interest, and input the two key parameters: the interval D of the scanning array and the size d of the scanning step size. The other parameters are left as default. The program will automatically calculate the coordinate values ​​of each point in the scanning array based on the size of the selected area and the settings of D and d.

[0080] (2) Turn on the pulsed laser and adjust the wavelength to 800nm. The laser power can be adjusted by rotating the half-wave plate HWP1. Turn on the controller power for the LED, DAQ card, EMCCD, TCSPC card, PMT, and AOD. Open the control software on the computer. Figure 6 In the control program, the number of frames acquired by the synchronous camera is (D / d)*(D / d), and the exposure time is N*(1 / v), where N is the number of points scanned in a dot matrix image, and v is the AOD scanning frequency. Change the shooting mode of the EMCCD to the external trigger mode, click "StartAcquisition" to put the camera into a trigger-ready state, and click "Start" to put the PMT into a trigger-ready state as well. Click "Start Scan" to make the data acquisition card output three synchronous signals, digital signals and analog signals at the same time. At this time, the AOD starts scanning, the EMCCD starts exposure imaging, and the PMT records the fluorescence signal and sends it to the TCSPC card for single photon counting. After the acquisition is completed, the image results will be displayed in real time in the program. After the data acquisition is completed, save the lifetime data file obtained by the TCSPC, save the fluorescence dot matrix image captured by the EMCCD and the scanning coordinates of the AOD for later image reconstruction.

[0081] (3) Offline data processing was performed using data processing software written in Matlab. First, the fluorescence dot matrix image of the lily of the valley rhizome sample was processed and saved using the pixel relocation algorithm. Then, the processed dot matrix image, AOD scan coordinates, and lifetime data file were processed. The corresponding super-resolution fluorescence lifetime image can be obtained by processing according to the method described above. The results are as follows. Figure 7 As shown, Figure 7 (a) A conventional fluorescence intensity image obtained by EMCCD. Figure 7 (b) is the super-resolution fluorescence intensity image obtained after pixel relocation. Figure 7 (c) A conventional fluorescence lifetime image obtained by directly performing pixel mapping on fluorescence lifetime data acquired by PMT and TCSPC cards. Figure 7 (d) is a super-resolution fluorescence lifetime image obtained using the above method. Different colors in the lifetime image represent different fluorescence lifetime values, as shown by the color scale in the figure.

[0082] S3. Compare conventional FLIM and SR-FLIM images (e.g.) Figure 7 This study aimed to verify the resolution improvement effect and the ability to capture micro-environment information.

[0083] Example 6

[0084] Hardware alternatives

[0085] S1 and EMCCD can be replaced with sCMOS camera, PMT can be replaced with APD detector, and dispersion compensation prism can be replaced with grating pair;

[0086] S2. The wide-field illumination source can be a xenon lamp or a laser wide-field illumination module to suit different sample excitation requirements.

[0087] In addition, all components designed in this invention are general standard parts or components known to those skilled in the art. Their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods. They can be fully implemented by those skilled in the art, so there is no need to elaborate. The content protected by this invention does not involve improvements to the internal structure and methods.

[0088] The embodiments disclosed in this invention are preferred embodiments, but are not limited thereto. Those skilled in the art can easily understand the spirit of this invention based on the above embodiments and make different extensions and variations, but as long as they do not depart from the spirit of this invention, they are all within the protection scope of this invention.

Claims

1. A super-resolution fluorescence lifetime microscopy imaging method based on pixel relocation, characterized in that, Includes the following steps: S1. Use an acousto-optic deflector to perform multi-focal parallel scanning of the sample; S2. Simultaneously acquire fluorescence intensity images and fluorescence lifetime data using area array detectors and point detectors; S3. Based on the pixel relocation algorithm, the fluorescence intensity image is super-resolution reconstructed. The focal spot size or spacing is adjusted according to the ratio of the excitation wavelength to the fluorescence emission wavelength. The multi-focal scanning image sequence is spatially superimposed and the resolution is further improved by deconvolution processing. The fluorescence lifetime data is mapped to the reconstructed image to generate a super-resolution fluorescence lifetime microscopic image.

2. The super-resolution fluorescence lifetime microscopy method based on pixel relocation as described in claim 1, characterized in that, The area array detector is an electron multiplier charge-coupled device or a scientific-grade complementary metal-oxide-semiconductor camera; the point detector is a photomultiplier tube or an avalanche photodiode.

3. The super-resolution fluorescence lifetime microscopy method based on pixel relocation as described in claim 1, characterized in that, The scanning control of the acousto-optic deflector is achieved through digital signals, which include acoustic frequency codes in the X and Y directions, and the scanning timing is strictly synchronized with the exposure signal of the area array detector and the single-photon counting signal of the point detector.

4. The super-resolution fluorescence lifetime microscopy method based on pixel relocation as described in claim 1, characterized in that, The fluorescence lifetime data is acquired through a time-correlated single-photon counting module, and the photon count-time histogram for each scanning point is weighted by the relative intensity value of the corresponding pixel of the area array detector.

Images