A fluorescence signal encoding super-resolution microscopy imaging method

Abstract

The present invention discloses a fluorescence signal encoding super-resolution microscopic imaging method, which is applied in the field of optical microscope imaging technology. The present invention performs secondary excitation on a sample within a nanosecond time scale by using a Gaussian pulse laser and a ring pulse laser, and after encoding the fluorescence signal, uses a time-correlated single-photon counter module to collect the spatiotemporal information of the fluorescent molecules, and then uses a photon extraction method based on frequency domain phasor analysis to decode the fluorescence signal, thereby realizing super-resolution imaging in a traditional confocal microscope system.

Description

Technical Field

[0001] 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 Art

[0002] Visualization of intracellular macromolecular complexes is a basic requirement of modern cell biology. Observing the dynamic interactions between organelles and deeply analyzing the laws of action are of great significance for revealing the mechanisms behind physiological and pathological phenomena. Due to the existence of the Abbe diffraction limit, 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. Since the structure and morphological characteristics of organelles and the dynamic changes of interactions between organelles are often smaller than the diffraction limit, imaging technology that breaks through the optical diffraction limit has become a key technology for studying organelles.

[0003] In the past few decades, scientists represented by Eric Betzig, Stefan W. Hell and WE Moerner have proposed super-resolution microscopy technology based on fluorescence switching effect and single-molecule localization, which has increased the lateral resolution and axial resolution by at least 10 times and 6 times, respectively. At present, stimulated emission depletion (STED), single-molecule localization microscopy (SMLM) and structured illumination microscopy (SIM) are the three mainstream far-field super-resolution microscopy technologies. In recent years, super-resolution optical imaging technology has developed rapidly, providing a new means for studying the interaction processes and laws of organelles in living cells, and making the connection between optical microscopy and fields such as biomedicine closer.

[0004] However, there are still some shortcomings in the existing research, such as the dynamic imaging of samples is limited by the time resolution, the high-power excitation light irradiation causes damage to biological samples, and the photobleaching of fluorescent dyes, which in turn limits the choice of dyes. Therefore, the development of an easy-to-implement, laser-independent far-field super-resolution microscopy technology can broaden the range of dye selection and provide technical support for more active organelle interaction research. Summary of the invention

[0005] The purpose of the present invention is to propose a fluorescence signal encoding super-resolution microscopy imaging method, which has the advantage of performing secondary excitation on the sample within nanosecond time by Gaussian pulse laser and ring pulse laser, encoding the fluorescence signal, collecting the spatiotemporal information of the fluorescent molecules by using a time-correlated single photon counter module, and then decoding the fluorescence signal by using a photon extraction method based on frequency domain phasor analysis, thereby realizing super-resolution imaging in a traditional confocal microscope system.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A fluorescence signal encoding super-resolution microscopic imaging method, including a laser scanning confocal imaging system based on time-resolved detection, and the laser scanning confocal imaging system based on time-resolved detection includes:

[0007] Laser, which generates pulsed laser using picosecond laser;

[0008] Half-wave plate, used to adjust the polarization direction of the laser;

[0009] Polarizer, used for laser beam splitting, can be used with half-wave plate to control the energy ratio of the outgoing laser;

[0010] A reflector, used to change the transmission direction of the laser;

[0011] Vortex glass slide, used to modulate the laser wavefront and convert Gaussian light into ring light;

[0012] 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;

[0013] Beam splitters, used for laser beam combining;

[0014] A dichroic mirror, used to transmit Gaussian excitation light and annular excitation light and reflect fluorescence signals;

[0015] Scanning galvanometer, used to synchronously scan two beams of excitation light to achieve area array confocal imaging of the sample;

[0016] The scanning lens is placed behind the scanning galvanometer and is used to collect the laser beam for area array scanning;

[0017] The tube lens, together with the objective lens, forms a microscope system;

[0018] The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample;

[0019] The stage is used to place and fix the sample and to control the three-dimensional movement of the sample;

[0020] A lens, used to focus the light beam;

[0021] Filters are used to remove stray light other than fluorescence and improve the image signal-to-noise ratio;

[0022] Detectors, using photomultiplier tubes or avalanche photodiodes, are used to collect signals and amplify the fluorescence signal;

[0023] 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;

[0024] Time-correlated single-photon counter, used for fluorescence time-resolved detection, recording the temporal and spatial information of the encoded fluorescence signal;

[0025] Computer, used to control the software to acquire images, store data and process image data, etc.

[0026] The present invention is further configured as follows: after the laser emits laser light, it is split into two beams through the first polarizer, wherein the reflected light is detected by a high-speed photodiode detector and used as a reference signal for fluorescence time-resolved detection, and the other beam is transmitted and split again through the second polarizer.

[0027] 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 or a vortex phase plate to generate a ring laser.

[0028] The present invention is further configured as follows: two laser beams meet at a spectroscope and overlap precisely in space; the sample is excited to emit a fluorescence signal; the fluorescence signal is collected by an objective lens and then returns along the original path; after being reflected by a dichroic mirror, it passes through a lens and a filter in sequence to reach a detector; the fluorescence signal collected by the detector is transmitted to a time-correlated single-photon counter, and the data is saved in a computer.

[0029] The present invention is further configured as follows: after the laser is emitted, it passes through two pairs of half-wave plates and polarizers in sequence to form three light paths, one of which is collected by a high-speed photodiode detector and used as a reference signal for fluorescence time-resolved detection;

[0030] 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.

[0031] The present invention is further configured as follows: the arrangement of the laser pulse sequence encodes the spontaneous radiation transition process of the fluorescent photons, the fluorescence time-resolved detection technology is used to record the nanosecond scale time information and nanometer scale space information during the photon emission process, and finally the frequency domain phasor analysis technology is used to decode the fluorescent signal.

[0032] The present invention is further configured as follows: the two laser beams are precisely overlapped in space, the central intensity of the Gaussian beam is the maximum, and the central intensity of the annular beam is zero, the fluorescence lifetime imaging is performed on the sample labeled with the fluorescent dye, and the spatiotemporal information of the fluorescence photons is collected and analyzed. According to the distribution of the 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;

[0033] Region 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 decay curve is generated, and the number of photons in the first peak is greater than that in the second peak;

[0034] Region C, such as coordinates (22, 24, 42, 44), at this time the sample is excited by two laser beams, and the Gaussian laser intensity in the corresponding area is the same as the ring laser intensity, so the peak photon number of the double-peak fluorescence decay curve is the same;

[0035] Region 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 decay curve is generated, and the number of photons in the first peak is less than that in the second peak;

[0036] 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.

[0037] The present invention is further configured as follows: the phasor space includes a semicircular trajectory with coordinates (0.5, 0) as the center and a radius of 0.5, wherein the coordinates (0, 0) represent that the fluorescence lifetime is infinite, and the coordinates (1, 0) represent that 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 to phasor coordinates (g, s), and the collection of all phasor points forms a phasor diagram. The fluorescence lifetime value of a single-component sample must fall on this semicircular curve, and different positions (coordinates) on the semicircle represent different fluorescence lifetimes. Therefore, when only a single lifetime component exists in the sample, the phasor center coordinates must be located on the semicircular curve. If the fluorescence emission contains two One or more components, the phasor center coordinates will no longer be on the semicircular curve. Relative to the reference pulse signal, the ring laser pulse has a larger time delay than the Gaussian laser pulse. Therefore, in the phasor space, the photon phasor points in the area that is more affected by the ring laser have a larger phase delay, that is, a larger φ value, relative to the photon phasor points in the central area of ​​the Gaussian laser. The change in the φ value is the result of encoding the fluorescence signal. Pixels with similar phase delay (φ) and amplitude modulation (m) are clustered in 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 are therefore clustered in the area closer to the coordinates (1, 0).

[0038] 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;

[0039] 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 coordinates of the photon in the phasor space move to position E.

[0040] 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;

[0041] When photons in the phase space close to the short-life region (Ⅱ) are extracted, the resolution of the formed image is improved;

[0042] When the photons in the area (III) corresponding to the fluorescence signal generated only under Gaussian laser excitation are further extracted, the resolution of the image is further improved.

[0043] In summary, the present invention has the following beneficial effects:

[0044] The sample is re-excited by Gaussian pulse laser and ring pulse laser in nanosecond time. The fluorescence signal is encoded and the spatiotemporal information of the fluorescent molecules is collected using a time-correlated single-photon counter module. The fluorescence signal is then decoded using a photon extraction method based on frequency domain phasor analysis, thereby achieving super-resolution imaging in a traditional confocal microscope system. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of a super-resolution optical microscopic imaging system for implementing fluorescence information encoding and decoding of the present invention;

[0046] Figure 2 It is a schematic diagram of the principle of realizing fluorescence information encoding and decoding super-resolution optical microscopic imaging of the present invention. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0048] refer to Figure 1-2 , a fluorescence signal encoding super-resolution microscopic imaging method, including a laser scanning confocal imaging system based on time-resolved detection, the laser scanning confocal imaging system based on time-resolved detection includes:

[0049] Laser, which generates pulsed laser using picosecond laser;

[0050] Half-wave plate, used to adjust the polarization direction of the laser;

[0051] Polarizer, used for laser beam splitting, can be used with half-wave plate to control the energy ratio of the outgoing laser;

[0052] A reflector, used to change the transmission direction of the laser;

[0053] Vortex glass slide, used to modulate the laser wavefront and convert Gaussian light into ring light;

[0054] 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;

[0055] Beam splitters, used for laser beam combining;

[0056] A dichroic mirror, used to transmit Gaussian excitation light and annular excitation light and reflect fluorescence signals;

[0057] Scanning galvanometer, used to synchronously scan two beams of excitation light to achieve area array confocal imaging of the sample;

[0058] The scanning lens is placed behind the scanning galvanometer and is used to collect the laser beam for area array scanning;

[0059] The tube lens, together with the objective lens, forms a microscope system;

[0060] The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample;

[0061] The stage is used to place and fix the sample and to control the three-dimensional movement of the sample;

[0062] A lens, used to focus the light beam;

[0063] Filters are used to remove stray light other than fluorescence and improve the image signal-to-noise ratio;

[0064] Detectors, using photomultiplier tubes or avalanche photodiodes, are used to collect signals and amplify the fluorescence signal;

[0065] 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;

[0066] Time-correlated single-photon counter, used for fluorescence time-resolved detection, recording the temporal and spatial information of the encoded fluorescence signal;

[0067] The computer is used to control the software to collect images, store data and process image data. The sample is re-excited by Gaussian pulse laser and ring pulse laser in nanosecond time. After encoding the fluorescence signal, the time-correlated single photon counter module is used to collect the spatiotemporal information of the fluorescent molecules. The fluorescence signal is then decoded using a photon extraction method based on frequency domain phasor analysis, thereby achieving super-resolution imaging in a traditional confocal microscope system.

[0068] like Figure 1As shown, after the laser is emitted, it passes through two pairs of half-wave plates and polarizers in sequence, and forms three light 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 light paths are used to excite the sample, and their wavefronts are Gaussian and annular, respectively. The two light paths are combined after passing through a beam splitter, and then pass through a dichroic mirror, a scanning galvanometer, a scanning lens, and a tube lens in sequence, and are focused by an objective lens to irradiate the sample. The sample is excited to produce fluorescence, which is collected by the same objective lens and returns to the original path. After being reflected by the dichroic mirror, the fluorescence 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 a computer for storage and processing.

[0069] like Figure 2 As shown, the spontaneous radiation transition process of fluorescent photons is encoded by arranging laser pulse sequences, and the fluorescence time-resolved detection technology is used to record the nanosecond-scale time information and nanometer-scale spatial information in the photon emission process. Finally, the frequency-domain phasor analysis technology is used to decode the fluorescence signal.

[0070] Figure 2 (a) is a schematic diagram of the wavefront images of Gaussian and annular laser spots, the spatial overlap image of the two spots, and the intensity distribution of the point spread function. Under the same imaging system and when all the excitation spots fill the objective entrance pupil, the spatial size of the annular spot is larger than that of the Gaussian spot.

[0071] Figure 2 (b) is a schematic diagram of a laser pulse sequence of a reference signal, a Gaussian beam and a ring beam, and a fluorescence decay curve generated under the excitation of the above laser pulse sequence. The pulse interval between the Gaussian laser and the ring laser is related to the fluorescence lifetime of the fluorescent dye. The specific value of the pulse interval is related to the fluorescence lifetime of the dye used for imaging and can be adjusted by a corner reflector in the optical system;

[0072] Figure 2(c) is 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. Region A, such as coordinate (33), is where the sample is only excited by the Gaussian laser and is not affected by the annular laser, thus generating a single exponential fluorescence decay curve; region B, such as coordinates (23, 32, 34, 43), is where the sample is excited by two laser beams, but the Gaussian laser intensity in the corresponding region is greater than the annular laser intensity, thus generating a double-peak fluorescence decay curve, 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;

[0073] Figure 2(d) is the phasor diagram generated by frequency domain transformation of the encoded fluorescence information into the phasor space. The phasor space contains a semicircular trajectory with coordinates (0.5, 0) as the center and a radius of 0.5, where coordinates (0, 0) represent infinite fluorescence lifetimes and coordinates (1, 0) represent zero fluorescence lifetimes. After frequency domain transformation, 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 to phasor coordinates (g, s). The collection of all phasor points forms a phasor diagram. The fluorescence lifetime value of a single-component sample must fall on this semicircular curve, and different positions (coordinates) on the semicircle represent different fluorescence lifetimes. Therefore, when there is only a single lifetime component in the sample, the phasor center coordinates must be on the semicircular curve. If the fluorescence emission contains two or more components, the phasor center coordinates will no longer be on the semicircular curve. Relative to the reference pulse signal, the ring laser pulse has a larger time delay than the Gaussian laser pulse. Therefore, in the phasor space, the photon phasor points in the area affected by the ring laser have a larger phase delay, that is, a larger φ value, relative to the photon phasor points in the central area of ​​the Gaussian laser. The change in the φ value is the result of the encoding of the fluorescence signal by this method. Pixels with similar phase delay (φ) and amplitude modulation (m) are clustered in 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, so they are clustered in the area closer to the coordinates (1, 0). Figure 2 (d) In the phasor space, five positions A, B, C, D and E are marked, corresponding to the different relative intensities of Gaussian and annular spots in space, where A is closer to the short-life region on the semicircular curve, while E is closer to the long-life region on the semicircular curve. The encoded fluorescence emission shows a two-component fluorescence attenuation characteristic, so the phasor point 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 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), where k1 and k2 represent the distances from the center coordinate of the overall phasor diagram to 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 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;

[0074] Figure 2(e) is a schematic diagram of the fluorescence decoding method based on frequency domain phasor analysis for photon extraction. When photons in all regions of the phasor space (I) 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 corresponding to the fluorescence signal generated only under Gaussian laser excitation (III) are further extracted, the resolution of the image is further improved.

[0075] 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, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A fluorescence signal encoding super-resolution microscopic imaging method, comprising a laser scanning confocal imaging system based on time-resolved detection, characterized in that: The laser scanning confocal imaging system based on time-resolved detection includes: Laser, which generates pulsed laser using 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 splitters, used for laser beam combining; A dichroic mirror, used to transmit Gaussian excitation light and annular excitation light and reflect fluorescence signals; Scanning galvanometer, used to synchronously scan two beams of excitation light to achieve area array confocal imaging of the sample; The scanning lens is placed behind the scanning galvanometer and is used to collect the laser beam for area array scanning; The tube lens, which is paired with the objective lens to form a microscope system; The objective lens is used to focus the laser onto the sample and collect the fluorescence signal reflected by the sample; The stage is used to place and fix the sample and to control the three-dimensional movement of the sample; A lens, used to focus the light 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 the fluorescence signal; 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 temporal and spatial information of the encoded fluorescence signal; Computer, used to control the software to acquire images, store data and process image data; After the laser is emitted, it passes through two pairs of half-wave plates and polarizers in sequence, and forms three light 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 beams of light are used to excite the sample. Their wavefronts are Gaussian and annular respectively. The two beams are combined after passing through a beam splitter, and then sequentially pass through a dichroic mirror, a scanning galvanometer, a scanning lens, and a tube lens, and then are focused by an objective lens to illuminate the sample. The sample is excited to produce fluorescence, which is collected by the same objective lens and then returned along the original path. After being reflected by the dichroic mirror, the fluorescence is focused by a lens and filtered before reaching the detector. A time-correlated single-photon counter simultaneously receives the reference signal and fluorescence signal collected by the two detectors, and transmits the data to a computer for storage and processing; The spontaneous radiation transition process of fluorescent photons is encoded by arranging laser pulse sequences, and the fluorescence time-resolved detection technology is used to record the nanosecond time information and nanometer spatial information of the photon emission process. Finally, the frequency domain phasor analysis technology is used to decode the fluorescence signal. Under the same imaging system and when all the excitation spots fill the objective entrance pupil, the spatial size of the annular spot is larger than that of the Gaussian spot. The two laser beams overlap precisely in space, the central intensity of the Gaussian beam is the largest, while the central intensity of the annular beam is zero. Fluorescence lifetime imaging is performed on the sample labeled with fluorescent dyes, and the spatiotemporal information of fluorescent photons is collected and analyzed. According to the distribution of photons in the time channel, the spatial coordinates involved in the two beams can be divided into five areas. In area A, 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; Region B: 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 decay curve is generated, and the number of photons in the first peak is greater than that in the second peak; Region C, at this time, the sample is excited by two laser beams, and the Gaussian laser intensity in the corresponding area is the same as the ring laser intensity, so the peak photon number of the double-peak fluorescence decay curve is the same; Region D: 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 decay curve is generated, and the number of photons in the first peak is less than that in the second peak; In region E, 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 phasor space contains a semicircular trajectory with the coordinate (0.5, 0) as the center and a radius of 0.5, where the coordinate (0, 0) represents an infinite fluorescence lifetime and the coordinate (1, 0) represents a fluorescence lifetime of 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 coordinate (x, y) is converted to the phasor coordinate (g, s). The collection of all phasor points forms a phasor diagram. The fluorescence lifetime value of a single-component sample must fall on this semicircular curve, and different position coordinates on the semicircle represent different fluorescence lifetimes. Therefore, when there is only a single lifetime component in the sample, the phasor center coordinate must be on the semicircular curve. If the fluorescence emission contains two or more Components, the phasor center coordinates will no longer be located on the semicircular curve. Relative to the reference pulse signal, the ring laser pulse has a larger time delay than the Gaussian laser pulse. Therefore, in the phasor space, the photon phasor points in the 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, a larger φ value. The change in the φ value is the result of encoding the fluorescence signal. Pixels with similar phase delay φ and amplitude modulation index m are clustered in 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, so they are gathered in the area closer to the coordinate (1, 0); Five positions A, B, C, D and E are calibrated in the phasor space, corresponding to the different relative intensities of Gaussian and annular spots in space, where 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 shows a two-component fluorescence attenuation characteristic, so the phasor point 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 the two components, that is, the contribution value of photons under Gaussian light excitation is a1=k1 / (k1+k2), and the contribution value of photons under annular light excitation is a2=k2 / (k1+k2), where k1 and k2 represent the distances from the center coordinates of the overall phasor diagram to 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 photons 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 light spot and the annular light spot, so the coordinates of the photon in the phasor space move toward position E; When extracting photons from all regions I of the phase space, the image formed is still limited by optical diffraction, and the resolution is not improved; When extracting photons close to the short-lifetime region II in phase space, the resolution of the formed image is improved; When the photons in region III corresponding to the fluorescence signal generated only under Gaussian laser excitation are further extracted, the resolution of the image is further improved.

Images