A fluorescence modulation multicolor super-resolution microscopy imaging method with single wavelength excitation

Abstract

The present invention provides a single-wavelength excited fluorescence modulation multicolor super-resolution microscopy imaging method, comprising the following steps: S1, first, according to the laser scanning confocal imaging and fluorescence lifetime imaging principles, a fluorescence modulation microscopy imaging system is constructed; S2, a sample is marked with two dyes with similar spectra but different fluorescence lifetimes, and fluorescence lifetime imaging is performed on the prepared sample. At this time, two laser spots separated by a light source simultaneously illuminate the sample, collect fluorescence signals and save fluorescence lifetime data , The present invention realizes super-resolution imaging in the spatial dimension by using a method that requires the Gaussian laser spot and the annular laser spot to be precisely overlapped in the focal plane. A gold nanoparticle sample can be used to adjust the positions of the two laser spots in real time through scattering imaging so that they can be precisely overlapped in space.

Description

Technical Field

[0001] The present invention belongs to the field of optical microscope imaging, and more specifically, relates to a nanometer microscope technology for realizing multi-color super-resolution imaging under a single-wavelength pulse laser and a fluorescence detector. Background Art

[0002] Optical microscopy for living cell research has the advantages of non-contact, non-destructive and specific. It can conduct in-situ, real-time and dynamic research on cells, and deeply understand the interactions and physiological processes between organelles, proteins and molecules, which has greatly promoted the progress and development of life sciences. However, the resolution of optical microscopes is limited by optical diffraction, and biological structures with a size below 200nm cannot be clearly distinguished. In order to study and reveal the interactions and action rules of subcellular structures and related molecules, it is urgent to break through the optical diffraction limit of imaging solutions, so super-resolution microscopy technology came into being. In the past three decades, super-resolution imaging methods based on different principles have been proposed one after another, making optical microscopes gradually enter the era of nano-imaging. In 2014, the Nobel Prize in Chemistry was awarded to three scientists, Eric Betzig, Stefan W. Hell and WE Moerner, in recognition of their great contributions in the field of super-resolution optical microscopy.

[0003] In recent years, the continuous development of super-resolution optical imaging technology has made the connection between optical microscopy and biomedicine and other fields closer. However, almost all super-resolution imaging modes will produce new problems when pursuing the super-resolution limit, such as high laser power, low imaging speed, special fluorescent dyes, complex imaging systems and expensive experimental costs. Among them, the stimulated emission depletion (STED) technology consumes extremely high laser power, resulting in serious sample damage and dye photobleaching, so it is not suitable for long-term super-resolution imaging of living samples such as cells; single-molecule localization microscopy (SMLM) technology requires image reconstruction, so the temporal resolution is low and the imaging depth is limited, which limits its application in dynamic imaging of biological samples. Most importantly, multi-color super-resolution imaging basically adopts spectral separation technology, which requires multiple lasers with different wavelengths as light sources and multiple detectors to collect fluorescence with different emission spectra. The multi-color imaging system based on spectral separation technology not only has a complex optical path and poor imaging stability, but also has a high cost, which hinders the promotion and application of super-resolution technology. To this end, a single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging method is proposed. Summary of the invention

[0004] In view of this, the main purpose of the present invention is to propose a nano-microscopy imaging technology that breaks through the optical diffraction limit and realizes multi-color super-resolution imaging in a simple optical imaging system based on a single-wavelength pulsed laser and a detector, reduce the complexity and cost of the multi-color super-resolution microscopy imaging system, improve the stability of multi-color super-resolution imaging, and provide technical support for low-cost super-resolution imaging of living cells and other living organisms and the study of the dynamic processes of subcellular structure interactions.

[0005] The technical solution of the embodiment of the present invention is implemented as follows: a single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging method comprises the following steps:

[0006] S1. First, build a fluorescence modulation microscopy system based on the principles of laser scanning confocal imaging and fluorescence lifetime imaging;

[0007] S2, labeling the sample with two dyes with similar spectra but different fluorescence lifetimes, and performing fluorescence lifetime imaging on the prepared sample. At this time, two laser spots separated by the light source irradiate the sample simultaneously, collect fluorescence signals and save fluorescence lifetime data;

[0008] S3, separating the time dimension of the fluorescence lifetime data, and dividing it into two time series graph stacks based on the time channel where the ring laser pulse is located;

[0009] S4, respectively converting the two time series graph stacks into phasor space by sine and cosine transforms to form phasor diagrams of the two graph stacks;

[0010] S5. Using the phasor diagram analysis method to separate and extract target photons with different labeling structures, four fluorescence images of the two dyes produced under Gaussian and ring laser excitation were obtained;

[0011] S6, then spatially modulating the intensity value of each pixel of the image, subtracting the fluorescence signal photons of the annular image from the Gaussian signal photons, and compressing the Gaussian excitation point spread function to achieve super-resolution imaging;

[0012] S7. Finally, the two super-resolution images are applied with different pseudo colors and then superimposed to obtain a two-color super-resolution image. Multiple dyes with similar spectra but different fluorescence lifetimes are selected and multi-target structures of the sample are marked. The above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging.

[0013] Preferably, in step S1, the light source of the optical imaging system is a single-wavelength picosecond pulse laser.

[0014] Preferably, in step S3, the two time series image stacks are fluorescence signals excited by Gaussian light and annular light respectively.

[0015] Preferably, in step S2, the fluorescence lifetime data includes three dimensions: x, y and t.

[0016] Preferably: in step S5, the phasor diagram analysis method is to obtain fluorescence lifetime information by converting the fitting in the time domain into mathematical calculation in the frequency domain, and the two-dimensional view of the distribution of fluorescence lifetime information enables the observer to quickly distinguish different lifetime populations in a fluorescence lifetime image.

[0017] Preferably, in S5, the time difference between the Gaussian image and the annular image is in the order of nanoseconds, and the spatial position information of the Gaussian spot and the annular spot is recorded in real time.

[0018] Preferably, in S6, a weight factor is introduced to enhance the intensity of the annular image, and the suppression effect on the luminous area around the Gaussian spot is adjusted through a spatial modulation process to remove the diffraction-limited low-frequency signal and background noise.

[0019] A single-wavelength excited fluorescence modulation multi-color super-resolution microscopic imaging system, comprising

[0020] Pulse laser, using picosecond laser to generate 40MHz pulsed excitation light;

[0021] Half-wave plate, used to adjust the polarization direction of the laser;

[0022] Glan laser prism, used to separate lasers with different polarization directions;

[0023] A lens that converges the parallel transmitted laser beam;

[0024] Beam splitters, used for laser beam splitting and combining;

[0025] A spiral phase plate is used to transform the laser wavefront from a Gaussian shape to a ring shape;

[0026] A reflector, used to change the transmission direction of the laser;

[0027] A corner reflector, the position of which is adjusted to change the optical path of the annular excitation light spot, and is used to control the pulse interval between the Gaussian laser and the annular laser in time;

[0028] A quarter glass slide converts linearly polarized laser light into right-handed circularly polarized light, improving the quality of the annular spot.

[0029] A dichroic mirror, used to transmit the excitation light and reflect the fluorescence signal;

[0030] The galvanometer is used to synchronously scan the excitation light to achieve area array imaging of the sample;

[0031] The scanning lens is placed behind the galvanometer and is used to collect the laser beam for area array scanning;

[0032] The tube lens, together with the objective lens, forms a microscope system;

[0033] The objective lens is used to focus the laser light to the focal plane and collect the fluorescence signal reflected from the sample;

[0034] A stage, used to place and fix the sample and to move the sample in three dimensions;

[0035] Filters are used to remove stray light other than fluorescence and improve the image signal-to-noise ratio;

[0036] Multimode optical fiber, used to transmit the fluorescence signal to the photomultiplier tube detector, and its core is used as the detection aperture of the confocal system;

[0037] Photomultiplier tube, used to collect fluorescent photons, amplify fluorescent signals, and output electrical signals to a computer;

[0038] High-speed photodiode detector, used to detect the laser beam after the Glan laser prism splits it, and serves as a reference signal for measuring the fluorescence lifetime;

[0039] Time-correlated single-photon counter, used to measure and record the spatiotemporal information of fluorescence photons;

[0040] Computer, used to control the software to acquire images, store data and process image data, etc.

[0041] Preferably, the position of the corner reflector is adjusted to lengthen or shorten the optical path of the annular excitation light spot.

[0042] The embodiment of the present invention has the following advantages due to the adoption of the above technical solution:

[0043] 1. The present invention realizes super-resolution imaging in the spatial dimension by using this method, which requires the Gaussian laser spot and the annular laser spot to be precisely overlapped in the focal plane. Gold nanoparticle samples can be used to adjust the positions of the two laser spots in real time through scattering imaging so that they can be precisely overlapped in space.

[0044] Second, the present invention selects multiple dyes with similar spectra but different fluorescence lifetimes and performs multi-target structural labeling on the sample. The above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging, thereby revealing the dynamic process of the interaction between organelles such as mitochondria, lysosomes and microtubules in living cells;

[0045] 3. The method proposed in the present invention can achieve multi-color super-resolution imaging under the excitation of a single-wavelength pulsed laser and a fluorescence detector, as well as microwatt-level laser power, providing technical support for the study of the dynamic processes and interactions of subcellular structures in living cells.

[0046] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 This is a light path diagram of a fluorescence modulation multi-color super-resolution microscopic imaging system based on single-wavelength excitation provided by an embodiment of the present invention;

[0049] Figure 2a-2b A schematic diagram of the effect of the excitation light in the time and space dimensions in the fluorescence modulation multi-color super-resolution microscopy imaging method based on single-wavelength excitation provided by an embodiment of the present invention;

[0050] Figure 3a to 3e It is a schematic diagram of the process of a fluorescence modulation multi-color super-resolution microscopic imaging method based on single-wavelength excitation provided by an embodiment of the present invention;

[0051] Figures 4a to 4g This is the imaging result of a fluorescent bead sample based on single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0052] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0053] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0054] Example

[0055] The present invention provides a fluorescence modulation multi-color super-resolution microscopic imaging method based on single-wavelength excitation, which is a new method that breaks through the optical diffraction limit on the basis of a laser scanning confocal imaging system, reduces the complexity and cost of the multi-color super-resolution imaging system, and improves the stability of the imaging system.

[0056] A single-wavelength excited fluorescence modulation multi-color super-resolution microscopic imaging method comprises the following steps:

[0057] S1. First, build a fluorescence modulation microscopy system based on the principles of laser scanning confocal imaging and fluorescence lifetime imaging;

[0058] S2, labeling the sample with two dyes with similar spectra but different fluorescence lifetimes, and performing fluorescence lifetime imaging on the prepared sample. At this time, two laser spots separated by the light source irradiate the sample simultaneously, collect fluorescence signals and save fluorescence lifetime data;

[0059] S3, separating the time dimension of the fluorescence lifetime data, and dividing it into two time series graph stacks based on the time channel where the ring laser pulse is located;

[0060] S4, respectively converting the two time series graph stacks into phasor space by sine and cosine transforms to form phasor diagrams of the two graph stacks;

[0061] S5. Using the phasor diagram analysis method to separate and extract target photons with different labeling structures, four fluorescence images of the two dyes produced under Gaussian and ring laser excitation were obtained;

[0062] S6, then spatially modulating the intensity value of each pixel of the image, subtracting the fluorescence signal photons of the annular image from the Gaussian signal photons, and compressing the Gaussian excitation point spread function to achieve super-resolution imaging;

[0063] S7. Finally, the two super-resolution images are applied with different pseudo colors and then superimposed to obtain a two-color super-resolution image. Multiple dyes with similar spectra but different fluorescence lifetimes are selected and multi-target structures of the sample are marked. The above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging.

[0064] The embodiment of the present invention is implemented as follows: a laser scanning confocal fluorescence lifetime imaging system, comprising:

[0065] Pulsed laser: a picosecond laser generates 40MHz pulsed excitation light;

[0066] Half-wave plate (HWP), used to adjust the polarization direction of the laser;

[0067] Glan-laser polarizer (GLP), used to separate lasers with different polarization directions;

[0068] Lens (Lens, L), converges the parallel transmitted laser beam;

[0069] Beam splitter (BS), used for laser beam splitting and combining;

[0070] Vortex phase plate (VPP), used to convert the laser wavefront from Gaussian to annular;

[0071] Mirror (M), used to change the transmission direction of the laser;

[0072] Corner reflector (RR), whose position is adjusted to change (lengthen or shorten) the optical path of the annular excitation spot, and is used to control the pulse interval between the Gaussian laser and the annular laser in time;

[0073] Quarter-wave plate (QWP), which converts linearly polarized laser light into right-handed circularly polarized light, improving the quality of the annular spot;

[0074] Dichroic mirror (DM), used to transmit excitation light and reflect fluorescence signals;

[0075] Galvanometer mirror (GS), used to synchronously scan the excitation light to achieve area array imaging of the sample;

[0076] Scan lens (SL), placed behind the galvanometer, is used to collect the laser beam for area scanning;

[0077] Tube lens (TL), together with the objective lens, constitutes a microscope system;

[0078] Objective (Obj), used to focus the laser to the focal plane and collect the fluorescence signal reflected by the sample;

[0079] The stage (3D Stage) is used to place and fix the sample and move the sample in three dimensions;

[0080] Filter: used to remove stray light other than fluorescence and improve the image signal-to-noise ratio;

[0081] Multimode fiber (MMF) is used to transmit the fluorescence signal to the photomultiplier tube detector, and its core is used as the detection aperture of the confocal system;

[0082] Photomultiplier tube (PMT), used to collect fluorescent photons, amplify fluorescent signals, and output electrical signals to a computer;

[0083] High speed photodiode detector (HSPD), used to detect the laser beam after Glan laser prism splitting, as a reference signal for measuring fluorescence lifetime;

[0084] Time-correlated single photon counting (TSCPC), used to measure and record the temporal and spatial information of fluorescence photons;

[0085] Personal computer (PC), used to control the software to collect images, store data and process image data;

[0086] Figure 1 The red solid line in the middle represents a Gaussian laser. After the laser is emitted, it passes through a half-wave plate and is split into two by a Glan laser prism. One beam is reflected and focused on a high-speed photodiode detector by a lens as a reference signal for fluorescence lifetime imaging. The other beam is transmitted and split again by a beam splitter (BS). The light transmitted by the beam splitter is still a Gaussian laser (red solid line). The reflected beam is modulated by a 0-2π spiral phase plate (VPP) to form a ring laser (red hollow line). The above two laser beams overlap at the second beam splitter. The green solid line represents the fluorescence signal reflected back by the sample. The signal collected by the photomultiplier tube is transmitted to the TCSPC and the data is saved to the computer. When working, turn on the laser, adjust the pulse frequency to 40MHz, and adjust the pulse interval between the Gaussian laser and the ring laser by moving the corner reflector (RR).

[0087] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not limited to the present invention.

[0088] The implementation of the present invention is described in detail below in conjunction with embodiments.

[0089] Figure 1The optical path diagram of the fluorescence modulation multicolor super-resolution microscopy imaging system based on single-wavelength excitation provided by the embodiment of the present invention. As can be seen from the figure, this system only uses a pulsed picosecond laser as the light source, and the detection end also uses only a photomultiplier tube detector to collect fluorescence. After the laser is emitted, it passes through a half-wave plate, and after being split by a Glan laser prism and a beam splitter, three optical paths are formed. One of the light paths is collected by a high-speed photodiode detector as a reference signal for fluorescence lifetime imaging; the other two light paths are used to excite the sample, and the wavefront of one of the light paths is not modulated and is still Gaussian; the wavefront of the other light path is a hollow ring distribution after being modulated by a spiral phase plate. The optical path of the ring laser is changed by adjusting the corner reflector, so that it produces a certain time delay relative to the Gaussian laser pulse, and the delay time is greater than the fluorescence lifetime of the dye being measured and less than half of the laser light source pulse period. The above two excitation lights are combined after passing through the second beam splitter, and after passing through the λ / 4 glass slide, dichroic mirror, galvanometer, scanning lens and tube lens in sequence, they are focused on the sample through the objective lens. The sample marked with dye is excited by laser to produce fluorescence, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, it is focused on the core of the multimode optical fiber through the lens and filter. After being transmitted in the multimode optical fiber, the fluorescence finally reaches the photomultiplier tube detector. The time-correlated single photon counter simultaneously receives the reference signal and fluorescence signal collected by the high-speed photodiode detector and the photomultiplier tube, and transmits the data to the computer for storage and processing.

[0090] FIG2 is a schematic diagram showing the effects of the excitation light in the time and space dimensions in the fluorescence modulation multi-color super-resolution microscopy imaging method based on single-wavelength excitation provided by an embodiment of the present invention. Figure 2a The first row in the figure is the laser pulse sequence collected by the high-speed photodiode detector, which serves as the reference signal (starting point or end point) for fluorescence lifetime imaging; Figure 2a The second row in the figure shows the Gaussian laser pulse sequence and the fluorescence decay curve (solid black line) generated under Gaussian laser excitation; Figure 2a The third row is a ring laser pulse sequence and a fluorescence decay curve (black dashed line) generated under ring laser excitation; there is a certain time interval between the two laser pulse sequences, and the specific value of the time interval is between the fluorescence lifetime (τ) of the measured dye and the laser light source pulse period (T), where τ <T; Figure 2a The last line in the figure is the attenuation curve of the fluorescence emitted by the sample when two laser beams irradiate the sample at the same time. In the present invention, relative to the reference signal pulse, the Gaussian laser pulse can be in front and the ring laser pulse can be in the back; or the ring laser pulse can be in front and the Gaussian laser pulse can be in the back. In the spatial dimension, this method requires the Gaussian laser spot and the ring laser spot to be precisely overlapped in the focal plane to achieve super-resolution imaging. Gold nanoparticle samples can be used to adjust the positions of the two laser spots in real time through scattering imaging so that they can be precisely overlapped in space, such as Figure 2b .

[0091] FIG3 is a schematic diagram of a flow chart of a method for fluorescence modulation multi-color super-resolution microscopy imaging based on single wavelength excitation provided by an embodiment of the present invention. Figure 3a First, according to the principles of laser scanning confocal imaging and fluorescence lifetime imaging, a fluorescence modulation microscopy system is built. The light source of this optical imaging system is a single-wavelength picosecond pulse laser, and the detection end uses only a photomultiplier tube detector to collect fluorescence signals. Taking two-color imaging as an example, the sample is marked with two dyes with similar spectra but different fluorescence lifetimes, and the prepared sample is imaged for fluorescence lifetime. At this time, the two laser spots (Gaussian and annular wavefronts) separated by the light source simultaneously illuminate the sample, collect fluorescence signals and save data. The data contains x, y and t Three dimensions, such as Figure 3b . The time dimension of the fluorescence lifetime data ( t ) is separated and divided into two time series stacks based on the time channel where the ring laser pulse is located. They contain the fluorescence signals excited by Gaussian light and ring light respectively, such as Figure 3c . The two time series stacks are converted to phasor space respectively by sine and cosine transforms to form phasor diagrams of the two stacks. Phasor plot analysis is a commonly used fluorescence lifetime data analysis method. It obtains fluorescence lifetime information by converting the fitting in the time domain into mathematical calculations in the frequency domain, so no fitting process is required. The fluorescence lifetime data processing method based on phasor plot analysis is simple and direct. It provides a two-dimensional view of the lifetime distribution, allowing the observer to quickly distinguish different lifetime populations in a fluorescence lifetime image. According to the fluorescence lifetimes of the two dyes, the phasor plot analysis method is used to separate and extract target photons with different labeling structures, and four fluorescence images of the two dyes produced under Gaussian and ring laser excitation are obtained ( I g1 , I g2 , I d1 and I d2 ),like Figure 3d Since the laser pulse period is 40MHz, the Gaussian image and the ring image ( I g and I d ) is in the nanosecond range (~10ns), which is equivalent to recording the spatial position information of the Gaussian spot and the annular spot in real time. Then the intensity value of each pixel in the image is spatially modulated, and the fluorescence signal photons of the annular image are subtracted from the Gaussian signal photons ( I g - Id ), compressing the Gaussian excitation point spread function to achieve super-resolution imaging. Introducing the weight factor ( β ) enhances the intensity of the annular image, adjusts the suppression effect of the luminous area around the Gaussian spot through the spatial modulation process, removes the diffraction-limited low-frequency signal and background noise, and further improves the resolution, that is, I s = I g - β × I d Finally, the two super-resolution images ( I s1 and I s2 ) and then superimposed with different pseudo colors, a two-color super-resolution image is obtained ( I S ),like Figure 3e By selecting multiple dyes with similar spectra but different fluorescence lifetimes and performing multi-target structural labeling on the sample, the above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging, thereby revealing the dynamic process of the interaction between organelles such as mitochondria, lysosomes and microtubules in living cells.

[0092] FIG. 4 is an imaging result of a fluorescent bead sample by fluorescence modulation multi-color super-resolution microscopy imaging based on single-wavelength excitation provided by an embodiment of the present invention. Figure 4a The confocal intensity image of two fluorescent beads (both with a diameter of 23 nm) labeled with dyes STAR 635P and Alexa 647 respectively. The two fluorescent particles cannot be distinguished or identified from the image. Fluorescence lifetime imaging was performed under the simultaneous irradiation of two laser spots (both with power in the microwatt range) to obtain the fluorescence decay curve, as shown in the figure below. Figure 4b . The first complete fluorescence decay process is the autofluorescence signal generated under Gaussian light excitation, and the second complete fluorescence decay process is the autofluorescence signal generated under annular light excitation. After separating the data into time channels, two time series stacks are obtained, and two phasor diagrams are obtained after sine and cosine transformation, as shown in Figure 4c . Two photon extractions are performed in two phase diagrams (STAR ​​635P is the blue solid line and dashed line area, Alexa 647 is the red solid line and dashed line area) to form four images, as shown in Figure 2. Figure 4d . The intensity value of each pixel of the image is spatially modulated. β = 1, we get two super-resolution images ( I s1 = I g1 - I d1 and Is2 = I g2 - I d2 ),like Figure 4e The images of the two dye-labeled fluorescent beads are superimposed with different pseudo colors to obtain a two-color super-resolution image, such as Figure 4f The intensity distribution of the two fluorescences in the dual-color super-resolution image was Gaussian fitted to obtain super-resolution point spread functions of 87 nm and 92 nm, respectively, as shown in Figure 4g .contrast Figure 4a and Figure 4f It can be seen that Figure 4f Not only is the resolution higher, but it can also distinguish two fluorescent beads labeled with different dyes, achieving dual-color super-resolution imaging. Therefore, the method proposed in the present invention can achieve multi-color super-resolution imaging under the excitation of a single-wavelength pulsed laser and a fluorescence detector, as well as microwatt-level laser power, providing technical support for the study of the dynamic process and interaction of subcellular structures in living cells.

[0093] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging method, characterized in that: The following steps are involved: S1. First, build a fluorescence modulation microscopy system based on the principles of laser scanning confocal imaging and fluorescence lifetime imaging; S2, labeling the sample with two dyes with similar spectra but different fluorescence lifetimes, and performing fluorescence lifetime imaging on the prepared sample. At this time, two laser spots separated by the light source irradiate the sample simultaneously, collect fluorescence signals and save fluorescence lifetime data; S3, separating the time dimension of the fluorescence lifetime data, dividing it into two time series stacks based on the time channel where the ring laser pulse is located, wherein the two time series stacks are fluorescence signals excited by Gaussian light and ring light respectively; S4, respectively converting the two time series graph stacks into phasor space by sine and cosine transforms to form phasor diagrams of the two graph stacks; S5. Using the phasor diagram analysis method to separate and extract target photons with different labeling structures, four fluorescence images of the two dyes produced under Gaussian and ring laser excitation were obtained; S6, then spatially modulating the intensity value of each pixel of the image, subtracting the fluorescence signal photons of the annular image from the Gaussian signal photons, and compressing the Gaussian excitation point spread function to achieve super-resolution imaging; S7. Finally, the two super-resolution images are applied with different pseudo colors and then superimposed to obtain a two-color super-resolution image. Multiple dyes with similar spectra but different fluorescence lifetimes are selected and multi-target structures of the sample are marked. The above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging.

2. The method for fluorescence modulation multi-color super-resolution microscopy with single wavelength excitation according to claim 1, characterized in that: In the step S1, the light source of the fluorescence modulation microscopy system is a single-wavelength picosecond pulse laser.

3. The method for fluorescence modulation multi-color super-resolution microscopy imaging with single wavelength excitation according to claim 1, characterized in that: In step S2, the fluorescence lifetime data includes three dimensions: x, y and t.

4. The method for fluorescence modulation multi-color super-resolution microscopy imaging with single wavelength excitation according to claim 1, characterized in that: In step S5, the phasor diagram analysis method obtains fluorescence lifetime information by converting the fitting in the time domain into mathematical calculation in the frequency domain. The two-dimensional view of the distribution of fluorescence lifetime information enables the observer to quickly distinguish different lifetime populations in a fluorescence lifetime image.

5. The single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging method according to claim 1, characterized in that: In S5, the time difference between the Gaussian image and the annular image is in the order of nanoseconds, and the spatial position information of the Gaussian spot and the annular spot is recorded in real time.

6. The method for fluorescence modulation multi-color super-resolution microscopy imaging with single wavelength excitation according to claim 1, characterized in that: The S6 also includes introducing a weight factor to enhance the intensity of the annular image, and adjusting the suppression effect of the luminous area around the Gaussian spot through a spatial modulation process to remove the diffraction-limited low-frequency signal and background noise.

Images