CN117783070A - Fluorescent signal coding super-resolution microscopic imaging method - Google Patents

Abstract

The invention discloses a fluorescent signal coding super-resolution microscopic imaging method, which is applied to the technical field of optical microscope imaging.

Description

A fluorescence signal encoding super-resolution microscopy imaging method

Technical Field

The invention belongs to the technical field of optical microscope imaging, and in particular relates to a fluorescence signal encoding super-resolution microscopic imaging method.

Background technique

The visualization of intracellular macromolecular complexes is a basic requirement of modern cell biology. Observing the dynamic interactions between organelles and in-depth analysis of the rules of action are of great significance for revealing the mechanisms behind physiological and pathological process phenomena. Due to the Abbe diffraction limit However, the resolution of traditional optical microscopes is limited. In the visible light band, the lateral resolution of traditional optical microscopes is only 200-300nm, and the axial resolution is only 500-800nm. Due to the structure and morphological characteristics of organelles and the interactions between organelles, The dynamic changes are often smaller than the diffraction limit, so imaging technology that breaks through the optical diffraction limit has become a key technology for studying cell organelles.

In the past few decades, scientists represented by Eric Betzig, Stefan W. Hell and W.E. Moerner have proposed super-resolution microscopy imaging technology based on fluorescence switching effect and single-molecule localization, which has improved the lateral resolution and axial resolution by at least 10 times and 6 times. Currently, Stimulated Emission Depletion (STED), Single-molecule Localization Microscopy (SMLM) and Structured Light Illumination Microscopy (SIM) are the three mainstream far-field super-resolution microscopes. Imaging technology. In recent years, super-resolution optical imaging technology has developed rapidly, providing new means for studying the interaction processes and rules of organelles in living cells, making the connection between optical microscopy and biomedicine and other fields closer.

However, there are still some shortcomings in existing research, such as the dynamic imaging of samples is limited by time resolution, high-power excitation light irradiation causes damage to biological samples, and photobleaching of fluorescent dyes, which limits the choice of dyes. Therefore, developing a far-field super-resolution microscopy technology that is easy to implement and does not rely on laser energy can broaden the selection of dyes and provide technical support for the study of more active organelle interactions.

Contents of the invention

The purpose of the present invention is to propose a fluorescence signal encoding super-resolution microscopic imaging method, which has the advantage of secondary excitation of the sample by Gaussian pulse laser and ring pulse laser within a nanosecond scale, encoding the fluorescence signal and then utilizing The time-correlated single photon counter module collects the spatiotemporal information of fluorescent molecules, and then uses the photon extraction method based on frequency domain phasor analysis to decode the fluorescence signal, thereby achieving super-resolution imaging in a traditional confocal microscope system.

The above technical objectives of the present invention are achieved through the following technical solutions: a fluorescence signal encoding super-resolution microscopy imaging method, including a laser scanning confocal imaging system based on time-resolved detection, a laser scanning confocal imaging system based on time-resolved detection The system includes:

Laser, a pulsed laser produced by a picosecond laser;

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

Polarizer, used for laser beam splitting, can be used with half-wave plate to control the energy ratio of the outgoing laser;

Reflector, used to change the transmission direction of laser;

Vortex glass slide, used for wavefront modulation of laser light and converting Gaussian light into ring light;

The corner reflector is used to adjust the pulse interval between the Gaussian laser and the ring laser, that is, moving the position of the corner reflector to extend or shorten the optical path of the ring excitation spot, and to control the time when the Gaussian laser and the ring laser reach the sample;

Beam splitter, used for laser beam combining;

Dichroic mirror, used to transmit Gaussian excitation light and ring excitation light, and reflect fluorescence signals;

Scanning galvanometer is used to synchronously scan two beams of excitation light to achieve area array confocal imaging of the sample;

The scanning lens is placed after the scanning galvanometer and is used to collect the laser beam for area array scanning;

Tube lenses, combined with objective lenses, form a microscope system;

Objective lens, used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample;

Stage, used to place and fix the sample and control the three-dimensional movement of the sample;

Lenses, used to focus the beam;

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

Detectors, using photomultiplier tubes or avalanche photodiodes, are used to collect signals and amplify fluorescent signals;

High-speed photodiode detector, used to detect the laser reflected by the first polarizer as a reference signal for fluorescence time-resolved detection;

Time-correlated single photon counter, used for fluorescence time-resolved detection, recording the spatiotemporal information of the encoded fluorescence signal;

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

The invention is further configured as follows: after the laser emits laser light, it is divided into two beams through the first polarizer, in which the reflected light is detected by a high-speed photodiode detector and used as a reference signal in fluorescence time-resolved detection, and the other beam is transmitted again Beam split by the second polarizer.

The present invention is further configured such that the light transmitted by the second polarizer is still Gaussian laser, and the reflected light beam is modulated by a 0-2π vortex glass plate or vortex phase plate to generate a ring laser.

The invention is further configured as follows: two laser beams meet at the spectroscope and overlap accurately in space. After the sample is excited, it emits a fluorescence signal. The fluorescence signal is collected by the objective lens and then returns to the original path. After being reflected by the dichroic mirror, it passes through the lens and filter in sequence. The chip reaches the detector, which transmits the fluorescence signal collected by the detector to a time-correlated single photon counter, and saves the data to the computer.

The invention is further configured as follows: after the laser is emitted, it passes through two pairs of half-wave plates and polarizers in sequence, and forms three optical paths, one of which is collected by a high-speed photodiode detector and used as a reference signal during fluorescence time-resolved detection;

The other two light beams are used to excite the sample. Their wavefronts are Gaussian and annular respectively. The two light beams are combined after passing through the beam splitter, and then pass through the dichroic mirror, scanning galvanometer, scanning lens, and tube lens in sequence, and are focused by the objective lens to illuminate the sample. After the sample is excited, fluorescence is generated. The fluorescence is collected by the same objective lens and returns along the original path. After being reflected by the dichroic mirror, it is focused by the lens and filtered before reaching the detector. The time-correlated single-photon counter simultaneously receives the reference signal and fluorescence signal collected by the two detectors, and transmits the data to the computer for storage and processing.

The invention is further configured as follows: the arrangement of the laser pulse sequence encodes the spontaneous emission transition process of fluorescence photons, uses fluorescence time-resolved detection technology to record nanosecond-scale time information and nano-scale spatial information during the photon emission process, and finally uses the frequency domain Fluorescence signal decoding using phasor analysis technology.

The invention is further configured as follows: the two laser beams accurately overlap in space, the center intensity of the Gaussian beam is the largest, and the center intensity of the annular beam is zero, fluorescence lifetime imaging is performed on the sample labeled with the fluorescent dye, and the space-time of the fluorescence photons is collected and analyzed. Information, according to the distribution of photons in the time channel, the spatial coordinates involved in the two light spots can be divided into five areas. Area A, such as coordinate (33), at this time the sample is only excited by the Gaussian laser and is not affected by the ring laser. , thus producing a single-exponential fluorescence decay curve;

Area B, such as coordinates (23, 32, 34, 43), at this time the sample is excited by two laser beams, but the Gaussian laser intensity in the corresponding area is greater than the ring laser intensity, so a double-peak fluorescence attenuation curve is generated, and the first peak The number of photons is greater than the second peak;

Area C, such as coordinates (22, 24, 42, 44), when the sample is excited by two laser beams, the Gaussian laser intensity in the corresponding area is the same as the ring laser intensity, so the peak photon number of the bimodal fluorescence decay curve is the same;

Area D, such as coordinates (13, 31, 35, 53), at this time the sample is excited by two laser beams, but the Gaussian laser intensity in the corresponding area is less than the ring laser intensity, so a double-peak fluorescence attenuation curve is generated, and the first peak The number of photons is smaller than the second peak;

In region E, such as coordinates (12, 21, 14, 41, 25, 52, 45, 54), the sample is only excited by the ring laser and is not affected by the Gaussian laser, thus generating a single exponential fluorescence decay curve with a certain time delay.

The present invention is further configured as follows: the phasor space contains a semicircular trajectory with coordinates (0.5, 0) as the center and a radius of 0.5, where the coordinates (0,0) represent that the fluorescence lifetime is infinite, and the coordinates (1,0) represent The fluorescence lifetime is zero. After frequency domain conversion, each pixel in the spatial domain image corresponds to a phasor point in the phasor space, that is, the spatial coordinates (x, y) are converted into phasor coordinates (g, s), and all phases The collection of measuring points forms a phasor diagram. The fluorescence lifetime value of a single-component sample must fall on this semicircle curve, and different positions (coordinates) on the semicircle represent different fluorescence lifetimes. Therefore, when there is only a single component in the sample, When the lifetime component exists, the phasor center coordinate must be located on the semicircle curve. If the fluorescence emission contains two or more components, the phasor center coordinate will no longer be located on the semicircle curve. Relative to the reference pulse signal, the ring laser pulse is consistent with the Gaussian Laser pulses have a greater time delay than laser pulses. Therefore, in phasor space, the photon phasor points in the larger area affected by the ring laser have a larger phase delay relative to the photon phasor points in the central area of the Gaussian laser, that is, Larger φ values, changes in φ values are the result of encoding the fluorescence signal, and pixels with similar phase delays (φ) and amplitude modulation degrees (m) are clustered on the phasor diagram, where, g=m×cos (φ), s=m×sin(φ), in the phasor diagram, the pixels in the area excited only by Gaussian light have the smallest phase delay, and therefore are gathered in the area closer to the coordinate (1, 0).

The present invention is further configured as follows: five positions A, B, C, D and E are calibrated in the phasor space, corresponding to different relative intensities of Gaussian and annular spots in space, wherein A is closer to the short-life region on the semicircular curve, and E is closer to the long-life region on the semicircular curve. The encoded fluorescence emission presents a two-component fluorescence attenuation characteristic, so the phasor point coordinates are located on the line connecting the positions A and E, and the specific position on the line is related to the ratio of the number of photons of the two components, that is, the contribution value of the photon under Gaussian light excitation is a1=k1/(k1+k2), and the contribution value of the photon under annular light excitation is a2=k2/(k1+k2), wherein k1 and k2 represent the distances between the center coordinates of the overall phasor diagram and A and E, respectively. Since the high-frequency super-resolution signal is located at the center of the laser spot and is greatly affected by the Gaussian spot, the coordinates of the photon in the phasor space are close to position A;

The low-frequency non-super-resolution signal is located at the edge of the light spot and is affected by both the Gaussian spot and the annular spot, so the coordinates of the photon in the phasor space move toward position E.

The present invention is further configured such that when photons in all regions (I) of the phase space are extracted, the image formed is still limited by optical diffraction and the resolution is not improved;

When photons close to the short-lived region (II) in the phasor space are extracted, the resolution of the image formed is improved;

When photons in the region (III) corresponding to the fluorescence signal generated only under Gaussian laser excitation are further extracted, the resolution of the image is further improved.

To sum up, the present invention has the following beneficial effects:

The sample is excited twice in the nanosecond scale by Gaussian pulse laser and ring pulse laser. After encoding the fluorescence signal, the time-correlated single photon counter module is used to collect the spatiotemporal information of the fluorescent molecules, and then based on frequency domain phasor analysis The photon extraction method is used to decode the fluorescence signal, thereby achieving super-resolution imaging in a traditional confocal microscope system.

Description of drawings

Figure 1 is a schematic diagram of a super-resolution optical microscopy imaging system for encoding and decoding fluorescence information according to the present invention;

Figure 2 is a schematic diagram of the present invention for realizing fluorescence information encoding and decoding super-resolution optical microscopy imaging.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings.

Referring to FIG1-2, a fluorescence signal encoding super-resolution microscopic imaging method includes a laser scanning confocal imaging system based on time-resolved detection, and the laser scanning confocal imaging system based on time-resolved detection includes:

Laser, a pulsed laser produced by a picosecond laser;

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

Polarizer, used for laser beam splitting, can be used with half-wave plate to control the energy ratio of the outgoing laser;

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

Vortex glass slide, used to modulate the laser wavefront and convert Gaussian light into ring light;

Corner reflector, used to adjust the pulse interval between Gaussian laser and ring laser, that is, to move the position of the corner reflector to extend or shorten the optical path of the ring-shaped excitation spot, and to control the time when Gaussian laser and ring laser reach the sample;

Beam splitter, used for laser beam combining;

Dichroic mirror, used to transmit Gaussian excitation light and ring excitation light, and reflect fluorescence signals;

Scanning galvanometer is used to synchronously scan two beams of excitation light to achieve area array confocal imaging of the sample;

The scanning lens is placed after the scanning galvanometer and is used to collect the laser beam for area array scanning;

Tube lenses, combined with objective lenses, form a microscope system;

Objective lens, used to focus the laser onto the sample and collect the fluorescence signal reflected back from the sample;

Stage, used to place and fix the sample and control the three-dimensional movement of the sample;

Lenses, used to focus the beam;

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

Detectors, using photomultiplier tubes or avalanche photodiodes, are used to collect signals and amplify fluorescent signals;

A high-speed photodiode detector is used to detect the laser reflected by the first polarizer as a reference signal for time-resolved fluorescence detection;

Time-correlated single photon counter, used for fluorescence time-resolved detection, recording the spatiotemporal information of the encoded fluorescence signal;

Computers are used to control software to collect images, store data, and process image data. They use Gaussian pulse lasers and ring pulse lasers to excite the sample twice within a nanosecond scale. The fluorescence signal is encoded and then time-correlated single photons are used. The counter module collects the spatiotemporal information of fluorescent molecules, and then uses a photon extraction method based on frequency domain phasor analysis to decode the fluorescence signal, thereby achieving super-resolution imaging in a traditional confocal microscope system.

As shown in Figure 1, after the laser is emitted, it passes through two pairs of half-wave plates and polarizers in sequence, and forms three optical paths. One of the light paths is collected by a high-speed photodiode detector and used as a reference signal for fluorescence time-resolved detection; the other two paths Light is used to excite the sample, and their wavefronts are Gaussian and annular respectively. The two lights are combined after passing through the spectroscope, and then pass through the dichroic mirror, scanning galvanometer, scanning lens, and tube lens in sequence, and then focus and illuminate the sample through the objective lens. , the sample is excited and produces fluorescence. The fluorescence is collected by the same objective lens and then returns along the original path. It is reflected by the dichromatic mirror and then reaches the detector after being focused by the lens and filter. The time-correlated single photon counter simultaneously receives the reference signals collected by the two detectors. and fluorescence signals, and transmit the data to a computer for storage and processing.

As shown in Figure 2, the spontaneous emission transition process of fluorescence photons is encoded through the arrangement of laser pulse sequences, and fluorescence time-resolved detection technology is used to record nanosecond-scale time information and nano-scale spatial information during the photon emission process. Finally, frequency Domain phasor analysis technology is used to decode fluorescence signals;

Figure 2(a) is a schematic diagram of the wavefront image of Gaussian and ring-shaped laser spots, the spatial overlap image of the two beam spots, and the intensity distribution of their point spread function. Under the same imaging system and all excitation spots fill the entrance pupil of the objective lens, the ring-shaped The spatial size of the light spot is larger than the Gaussian light spot;

Figure 2(b) is a schematic diagram of the laser pulse sequence of the reference signal, Gaussian beam and ring beam, as well as the fluorescence decay curve generated under the excitation of the above laser pulse sequence, the pulse interval between the Gaussian laser and the ring laser and the fluorescence lifetime of the fluorescent dye Relevantly, the specific value of the pulse interval is related to the fluorescence lifetime of the dye used for imaging, and can be adjusted through the corner reflector in the optical system;

Figure 2(c) shows the spatial and temporal distribution of the encoded fluorescence information. The two laser beams overlap precisely in space, the central intensity of the Gaussian beam is the largest, and the central intensity of the annular beam is zero. Fluorescence lifetime imaging is performed on the sample labeled with fluorescent dyes to collect and obtain the spatiotemporal information of fluorescence photons. According to the distribution of photons in the time channel, the spatial coordinates involved in the two light spots can be divided into five regions. In region A, such as coordinate (33), the sample is only excited by the Gaussian laser and is not affected by the annular laser, so a single-exponential fluorescence decay curve is generated; in region B, such as coordinates (23, 32, 34, 43), the sample is excited by two laser beams, but the Gaussian laser intensity in the corresponding area is greater than the annular laser intensity, so a double-peak fluorescence decay curve is generated. And the photon number of the first peak is greater than that of the second peak; in region C, such as the coordinates (22, 24, 42, 44), the sample is excited by two laser beams, and the Gaussian laser intensity in the corresponding region is the same as the ring laser intensity, so the peak photon number of the double-peak fluorescence decay curve is the same; in region D, such as the coordinates (13, 31, 35, 53), the sample is excited by two laser beams, but the Gaussian laser intensity in the corresponding region is less than the ring laser intensity, so a double-peak fluorescence decay curve is generated, and the photon number of the first peak is less than that of the second peak; in region E, such as the coordinates (12, 21, 14, 41, 25, 52, 45, 54), the sample is only excited by the ring laser and is not affected by the Gaussian laser, so a single exponential fluorescence decay curve with a certain time delay is generated;

Figure 2(d) is a phasor diagram generated by frequency domain transformation of the encoded fluorescence information into phasor space. The phasor space contains a semicircular trajectory centered on coordinates (0.5, 0) and with a radius of 0.5, where , the coordinate (0, 0) represents the fluorescence lifetime is infinite, the coordinate (1, 0) represents the fluorescence lifetime is zero, after frequency domain conversion, each pixel in the spatial domain image corresponds to a phasor point in the phasor space, That is, the spatial coordinates (x, y) are converted into phasor coordinates (g, s). The set of all phasor points forms a phasor diagram. The fluorescence lifetime value of the single-component sample must fall on this semicircle curve, and the semicircle Different positions (coordinates) on represent different fluorescence lifetimes. Therefore, when only a single lifetime component exists in the sample, the phasor center coordinate must be located on the semicircle curve. If the fluorescence emission contains two or more components, the phasor The center coordinate will no longer be located on the semicircle curve. Relative to the reference pulse signal, the ring laser pulse has a greater time delay than the Gaussian laser pulse, so it appears in the phase space as the photon phase of a larger area affected by the ring laser. Compared with the photon phasor point in the central area of the Gaussian laser, the quantum point has a larger phase delay, that is, a larger φ value. The change in φ value is the result of encoding the fluorescence signal by this method, with similar phase delay (φ) and amplitude. Pixels with modulation degree (m) are gathered into groups on the phasor diagram, where, g=m×cos(φ), s=m×sin(φ). In the phasor diagram, the area pixels excited only by Gaussian light It has the smallest phase delay, so it gathers in the area closer to the coordinate (1, 0). The five positions A, B, C, D and E are calibrated in the phasor space of Figure 2(d), corresponding to the Gaussian sum in the space. Different relative intensities of the annular light spot, where A is closer to the short-lifetime region on the semicircle curve, and E is closer to the long-lifetime region on the semicircle curve. The encoded fluorescence emission exhibits a two-component fluorescence attenuation characteristic, so the phasor point The coordinates are located on the line connecting positions A and E, and the specific position on the line is related to the ratio of the number of photons of these two components, that is, the contribution value of photons under Gaussian light excitation a1 = k1/(k1 +k2), the contribution value of photons under ring light excitation a2=k2/(k1+k2), where k1 and k2 represent the distance from the center coordinate of the overall phasor diagram to A and E respectively. Due to the high-frequency super-resolution The signal is located at the center of the laser spot and is greatly affected by the Gaussian spot, so the coordinates of the photon in the phasor space are close to position A; the low-frequency non-super-resolution signal is located at the edge of the spot and is affected by both the Gaussian spot and the annular spot, so the photon is in the phasor space. The coordinates of space move toward position E;

Figure 2(e) is a schematic diagram of the fluorescence decoding method for photon extraction based on frequency domain phasor analysis. When photons are extracted from all regions (I) of the phasor space, the image formed is still limited by optical diffraction, and the resolution is not improved; When the photons in the phasor space close to the short-lived region (II) are extracted, the resolution of the image formed is improved; when the photons in the region (III) corresponding to the fluorescence signal generated only under Gaussian laser excitation are further extracted, the image resolution is improved. The resolution is further improved.

This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims (10)

1. A fluorescence signal coding super-resolution microscopic imaging method comprises a laser scanning confocal imaging system based on time-resolved detection, and is characterized in that: the laser scanning confocal imaging system based on time resolution detection comprises:

a laser for generating pulse laser by a picosecond laser;

the half wave plate is used for adjusting the polarization direction of the laser;

the polarizer is used for splitting laser beams and can control the energy proportion of emergent laser when being matched with the half-wave plate;

a reflecting mirror for changing the transmission direction of the laser;

the vortex slide is used for carrying out wave front modulation on laser and converting Gaussian light into annular light;

the angle reflector is used for adjusting the pulse interval between the Gaussian laser and the annular laser, namely, the position of the angle reflector is moved to prolong or shorten the optical path of the annular excitation light spot, and the time for the Gaussian laser and the annular laser to reach the sample is controlled in time;

a beam splitter for laser beam combining;

a dichroic mirror for transmitting Gaussian excitation light and annular excitation light, reflecting fluorescent signals;

the scanning galvanometer is used for synchronously scanning the two beams of excitation light to realize the area array confocal imaging of the sample;

the scanning lens is arranged behind the scanning galvanometer and is used for collecting laser beams scanned by the area array;

a tube lens matched with the objective lens to form a microscope system;

the objective lens is used for focusing the laser to the sample and collecting fluorescent signals reflected by the sample;

the objective table is used for placing and fixing the sample and carrying out three-dimensional movement control on the sample;

a lens for focusing the light beam;

the optical filter is used for removing stray light except fluorescence and improving the signal-to-noise ratio of the image;

a detector using a photomultiplier tube or an avalanche photodiode for collecting the signal and amplifying the fluorescent signal;

the high-speed photodiode detector is used for detecting the laser reflected by the first polarizer and used as a reference signal for fluorescence time resolution detection;

the time-dependent single photon counter is used for fluorescence time resolution detection and recording the time-space information of the coded fluorescence signal;

and the computer is used for controlling software to collect images, store data, process image data and the like.

2. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 1, wherein: the laser emits laser light and then is divided into two beams by the first polarizer, wherein the reflected light is detected by the high-speed photodiode detector and used as a reference signal during fluorescence time resolution detection, and the other beam is transmitted and then is divided into two beams by the second polarizer.

3. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 2, wherein: the light transmitted by the second polarizer is still Gaussian laser, and the reflected light beam is modulated by a 0-2 pi vortex slide or a vortex phase plate to generate annular laser.

4. A fluorescent signal encoding super resolution microscopic imaging method according to claim 3, wherein: the two laser beams meet at the spectroscope, and are precisely overlapped in space, the sample is excited and then emits a fluorescent signal, the fluorescent signal is collected by the objective lens and returned in the original path, the fluorescent signal is reflected by the dichroscope and then sequentially reaches the detector through the lens and the optical filter, the fluorescent signal collected by the detector is transmitted to a time-related single photon counter, and the data are stored in a computer.

5. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 1, wherein: after laser is emitted, the laser sequentially passes through two pairs of half wave plates and a polarizer, and three light paths are formed, wherein one light path is collected by a high-speed photodiode detector and then used as a reference signal during fluorescence time resolution detection;

the other two paths of light are used for exciting the sample, the wave fronts of the two paths of light are respectively Gaussian and annular, the two paths of light are combined after passing through a spectroscope, then sequentially pass through a dichroic mirror, a scanning galvanometer, a scanning lens and a tube mirror, the sample is focused and irradiated through an objective lens, fluorescence is generated after the sample is excited, the fluorescence returns from the original path after being collected by the same objective lens, the fluorescence is reflected by the dichroic mirror, reaches a detector after passing through the lens for focusing and an optical filter, and a time-related single photon counter receives reference signals and fluorescence signals collected by the two detectors at the same time and transmits data to a computer for storage and processing.

6. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 1, wherein: the arrangement of the laser pulse sequences encodes the spontaneous radiation transition process of fluorescent photons, the fluorescence time resolution detection technology is used for recording nanosecond-scale time information and nanometer-scale space information in the photon emission process, and finally the frequency domain phasor analysis technology is used for fluorescent signal decoding.

7. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 6, wherein: the two laser beams are precisely overlapped in space, the center intensity of the Gaussian beam is the maximum, the center intensity of the annular beam is zero, fluorescent life imaging is carried out on a sample marked by fluorescent dye, the space-time information of fluorescent photons is collected and analyzed, the space coordinates related to the two light spots can be divided into five areas according to the distribution condition of the photons in a time channel, and the areas A are like the coordinates (33), at the moment, the sample is only excited by Gaussian laser and is not influenced by the annular laser, so that a single-index fluorescent attenuation curve is generated;

region B, such as coordinates (23, 32, 34, 43), where the sample is excited by two lasers, but the gaussian laser intensity of the corresponding region is greater than the ring laser intensity, thus producing a bimodal fluorescence decay curve with a first peak having a greater number of photons than the second peak;

region C, such as coordinates (22, 24, 42, 44), where the sample is excited by two lasers, the gaussian laser intensity of the corresponding region is the same as the ring laser intensity, and thus the number of peak photons producing a bimodal fluorescence decay curve is the same;

region D, such as coordinates (13, 31, 35, 53), where the sample is excited by two lasers, but the gaussian laser intensity of the corresponding region is less than the ring laser intensity, thus producing a bimodal fluorescence decay curve with a first peak having a smaller number of photons than the second peak;

region E, such as coordinates (12, 21, 14, 41, 25, 52, 45, 54), where the sample is excited only by the ring laser and is not affected by the gaussian laser, thus producing a single exponential fluorescence decay curve with a certain time delay.

8. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 7, wherein: the phasor space comprises a semicircular track centered on a coordinate (0.5, 0) with a radius of 0.5, wherein the coordinate (0, 0) represents a fluorescence lifetime of infinity, the coordinate (1, 0) represents a phasor point in the corresponding phasor space for each pixel in the spatial domain image after frequency domain conversion, i.e. the spatial coordinates (x, y) are converted into phasor coordinates (g, s), the set of all phasor points forming a phasor diagram, the fluorescence lifetime value of a single component sample necessarily falls on this semicircular curve, and the different positions (coordinates) on the semicircle represent different fluorescence lifetimes, whereby, when only a single lifetime component is present in the sample, the phasor center coordinates must be located on the semicircular curve, if the fluorescence emission comprises two or more components, the phasor center coordinates will no longer be located on the semicircular curve, compared to the reference pulse signal, the ring laser pulse has a larger time delay compared to the laser pulse, and therefore represents a photon with a larger area in the phasor space affected by the ring phase, and the photon peak value has a larger value compared to the laser peak value in the phase position x, i.e. the phase value of the laser signal is more delayed compared to the phase value m, the phase value is more than the excitation signal is plotted in the phase value m, and the phase value is more m is the same, and the value is the peak value is plotted in the phase value is compared to the phase value is compared with the phase value m, 0) Is a region of (a) in the above-mentioned region(s).

9. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 8, wherein: the method comprises the steps that A, B, C, D and E five positions are calibrated in a phasor space, corresponding to different relative intensities of Gaussian and annular light spots in the space, wherein A is closer to a short-life area on a semicircular curve, E is closer to a long-life area on the semicircular curve, coded fluorescence emission shows the fluorescence attenuation characteristics of two components, so that phasor point coordinates are located on a connecting line of the positions A and E, a specific position on the connecting line is related to the ratio of photon numbers of the two components, namely a contribution value a1=k1/(k1+k2) of photons under Gaussian light excitation, a contribution value a2=k2/(k1+k2) of photons under annular light excitation, wherein k1 and k2 respectively represent the distance from the center coordinates of the whole phasor diagram to the positions A and E, and the coordinates of the photons in the phasor space are close to the position A because a high-frequency super-resolution signal is located at the center of a laser spot and is greatly influenced by the Gaussian light spot;

the low frequency non-super-resolution signal is located at the edge of the spot and is affected by the gaussian spot and the annular spot, so that the photon moves to the position E at the coordinates of the phasor space.

10. The fluorescence signal encoding super-resolution microscopic imaging method according to claim 1, wherein: when extracting photons in all areas (I) of the phasor space, the formed image is still limited by optical diffraction, and the resolution is not improved;

when photons of which the phasor space is close to the short-life region (II) are extracted, the resolution of the formed image is improved;

when photons of the region (III) corresponding to the fluorescence signal generated only under Gaussian laser excitation are further extracted, the resolution of the image is further improved.