Large-depth two-photon three-dimensional super-resolution imaging system and method

Abstract

The application provides a large-depth two-photon three-dimensional super-resolution imaging system and method, and the system comprises: a first beam splitter which divides laser emitted by a femtosecond pulse laser into two parts, transmits light, and converts the light into a three-dimensional cage light beam with zero central intensity after twice non-coherent modulation of a spatial light modulator; an angle reflector which adjusts the pulse interval of the Gaussian light beam and the cage light beam to 50%-100% of the fluorescent lifetime of a used fluorescent dye; a second beam splitter which combines the Gaussian light beam and the cage light beam, and transmits the combined light beam to a sample on a three-dimensional moving object platform through a galvanometer and a microscope; two-photon fluorescence generated by the sample returns to a dichroic mirror in the original path, is reflected to a photomultiplier for amplification and processing, and is subjected to imaging processing by a time-correlated single-photon counter and an imaging processing terminal. The application has the beneficial effect of significantly improving the three-dimensional imaging resolution under a large depth.

Description

Technical Field

[0001] This invention relates to the technical field of optical microscope imaging, and in particular to a deep two-photon three-dimensional super-resolution imaging system and method. Background Technology

[0002] Optical microscopes, with their non-invasive nature and high resolution, are ideally suited for in vivo biological imaging. However, much important structural and functional information within organisms resides deep within tissues. Furthermore, light undergoes diffraction during propagation, with intense scattering occurring particularly within biological tissues. This causes excitation and emission signals to rapidly attenuate with increasing imaging depth, significantly hindering our exploration of the internal world of living organisms. Therefore, achieving high-quality imaging of deep biological tissues and obtaining effective structural and functional information remains a hot topic and a significant challenge in current biomedical photonics research.

[0003] Two-photon microscopy (TPM) utilizes a nonlinear optical effect, where a ground-state fluorescent molecule absorbs two photons simultaneously, transitions to an excited state, and then returns to the ground state, releasing a fluorescence signal. TPM employs long-wavelength excitation, offering advantages such as high signal-to-noise ratio and large penetration depth (hundreds of micrometers), making it a crucial technology in modern biomedical research. However, the use of long wavelengths and the presence of strong scattering in tissues significantly reduce imaging resolution. Furthermore, traditional TPM remains limited by the optical diffraction limit. To overcome this limit, super-resolution imaging techniques have emerged. In recent years, two-photon imaging technology, combined with super-resolution imaging methods based on different principles, including stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM), has improved the resolution of two-photon imaging from hundreds of nanometers to tens of nanometers, enabling the resolution of finer biological tissues and structures. However, these techniques are all based on the principle of fluorescence "on / off" states, which not only requires fluorescent probes with special photophysical and chemical properties, but also introduces a series of new problems when pursuing the limits of super-resolution, such as high laser power, low imaging speed, complex imaging systems, and high experimental costs. Furthermore, these techniques have not yet achieved super-resolution imaging at depths greater than 100 micrometers. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a deep two-photon three-dimensional super-resolution imaging system, which aims to improve imaging resolution.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a deep two-photon three-dimensional super-resolution imaging system, comprising: a femtosecond pulsed laser, a first beam splitter, a spatial light modulator, a corner reflector, a second beam splitter, a dichroic mirror, a photomultiplier tube, a time-correlated single-photon counter, an imaging processing terminal, a galvanometer, a microscope, and a three-dimensional moving platform.

[0006] The first beam splitter is positioned on the output optical path of the femtosecond pulsed laser to split the Gaussian beam emitted by the femtosecond pulsed laser into two, forming a transmission optical path and a reflection optical path. The spatial light modulator is positioned on the transmission optical path to convert the transmission laser beam into a three-dimensional cage beam with zero central intensity after two incoherent modulations. The corner reflector is positioned on the reflection optical path to adjust the pulse interval between the Gaussian beam and the three-dimensional cage beam, with the pulse interval adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used. The second beam splitter is used to combine the three-dimensional cage beam with the Gaussian beam from the corner reflector.

[0007] The dichroic mirror, galvanometer, microscope, and three-dimensional moving platform are sequentially positioned in the output light path of the combined beam from the second beam splitter. The dichroic mirror forms a 45° angle with the combined beam. The combined beam passes sequentially through the galvanometer and microscope to the three-dimensional moving platform, illuminating the sample placed on the platform. The two-photon fluorescence generated by the sample returns to the dichroic mirror along the original path, and the dichroic mirror reflects the two-photon fluorescence to a photomultiplier tube for photoelectric signal conversion and electrical signal amplification.

[0008] The photomultiplier tube, time-correlated single-photon counter, and imaging processing terminal are connected by electrical signals in sequence. The time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal after fluorescence amplification acquired by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal. The imaging processing terminal is used to process the spatiotemporal information of the photon fluorescence signal to form a sample image.

[0009] Furthermore, a half-glass slide and a first reflecting mirror are provided behind the first beam splitter. The half-glass slide is used to adjust the polarization of the transmitted laser beam. The first reflecting mirror is used to direct the adjusted transmitted laser beam onto the spatial light modulator with a 45° polarization direction.

[0010] Furthermore, the transmitted light path is also provided with a quarter-glass slide, a first lens, and a second reflector; the liquid crystal surface of the spatial light modulator is divided into two equal regions, and a vortex phase is loaded in the left region of the liquid crystal surface of the spatial light modulator to modulate the polarization p component of the incident beam. The reflected light passes through the first lens and the quarter-glass slide in sequence, is reflected by the second reflector, and then passes through the first lens and the quarter-glass slide to reach the right region of the liquid crystal surface of the spatial light modulator. A 0 / π phase is loaded in the right region of the liquid crystal surface of the spatial light modulator to modulate the other vertical component of the incident beam. The quarter-glass slide rotates the polarization vector of the beam by 90°, allowing the vertical component of the incident light, i.e., the s component, to be modulated through the right side of the spatial light modulator; the second reflector and the light modulator are respectively located at the front and rear focal points of the first lens to maintain the conjugate modulation of the laser wavefront.

[0011] Furthermore, a third and a fourth reflecting mirror are provided in the transmission light path. The third reflecting mirror reflects the outgoing light beam from the right side region of the liquid crystal surface of the spatial light modulator to the fourth reflecting mirror; the fourth reflecting mirror reflects the outgoing light beam reflected by the third reflecting mirror to the second beam splitter.

[0012] Furthermore, a fifth and a sixth reflecting mirror are provided on the reflected light path. The fifth reflecting mirror reflects the reflected light from the first beam splitter onto the corner reflector, and the sixth reflecting mirror reflects the Gaussian beam from the corner reflector onto the second beam splitter.

[0013] Furthermore, a second lens and a filter are provided between the dichroic mirror and the photomultiplier tube.

[0014] Furthermore, the microscope comprises, in sequence, a scanning lens, a tube lens, and an objective lens.

[0015] This invention also provides a deep-depth two-photon three-dimensional super-resolution imaging method, applied to the aforementioned deep-depth two-photon three-dimensional super-resolution imaging system, comprising:

[0016] The Gaussian beam emitted by the femtosecond pulsed laser is split into two by the first beam splitter to form a transmission optical path and a reflection optical path.

[0017] The Gaussian beam in the transmission optical path is incoherently modulated twice by the spatial light modulator to produce a three-dimensional cage beam with zero central intensity.

[0018] The Gaussian beam in the reflected light path is adjusted by a corner reflector to change the pulse interval between the Gaussian beam and the three-dimensional cage beam. The pulse interval is adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used.

[0019] The three-dimensional cage beam modulated by the spatial light modulator and the Gaussian beam adjusted by the corner reflector are combined through the second beam splitter.

[0020] The combined beam is sequentially transmitted through a dichroic mirror, reflected by a galvanometer, transmitted through a scanning lens, and focused by a microscope before illuminating the sample.

[0021] The two-photon fluorescence generated by the sample is returned to the dichroic mirror, which reflects the two-photon fluorescence to the photomultiplier tube for photoelectric signal conversion and electrical signal amplification.

[0022] The time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal after fluorescence amplification acquired by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal;

[0023] The imaging processing terminal processes the spatiotemporal information of the two-photon fluorescence signal to form a sample image.

[0024] Furthermore, the time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal amplified by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal, specifically including:

[0025] Fluorescence lifetime data were obtained by acquiring two-photon fluorescence generated after two laser pulses using a time-correlated single-photon counter.

[0026] Using the time channel where the intensity abrupt change in the fluorescence decay curve is located as the boundary, the signal before the channel is extracted to form the light cage image, and the signal after the channel is extracted to form the mixed image. At this time, the mixed image contains some fluorescence signals excited by the light cage beam. The mixing point spread function of the mixed image includes the Gaussian point spread function and the light cage point spread function.

[0027] After obtaining the blended image and the light cage image, intensity difference is performed on the blended image and the intensity-enhanced light cage image, i.e., I 超分辨图像 (x,y)=I 混合图像 (x,y)-K×I 光笼图像 (x,y), where K>1;

[0028] The sample was subjected to three-dimensional imaging and fluorescence lifetime data was collected. After processing all the data, a series of two-dimensional super-resolution images were obtained. The final three-dimensional super-resolution image was obtained by superimposing all the two-dimensional super-resolution images.

[0029] Furthermore, the deep-depth two-photon three-dimensional super-resolution imaging method also includes: adjusting the scanning lens and the microscope according to the real-time image of the sample to adjust the spot size so that the focal planes of the three-dimensional cage beam and the Gaussian beam of the combined beam coincide in space.

[0030] The beneficial effects of this invention are as follows: by combining two-photon imaging, signal timing control and light field modulation technology to improve the depth of optical microscopy and the resolution of three-dimensional imaging, and by extracting the high-frequency signal at the center of the beam by intensity difference of photons on the periphery of the excitation beam, a lateral resolution of better than 100 nm and an axial resolution of 120 nm can be achieved within a depth of 500 micrometers. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0032] Figure 1 This is an optical path diagram of the deep-depth two-photon three-dimensional super-resolution imaging system according to an embodiment of the present invention;

[0033] Figure 2 This is a schematic diagram illustrating the principle of deep-depth two-photon three-dimensional super-resolution imaging according to an embodiment of the present invention.

[0034] Figure 3 This is a flowchart of the deep two-photon three-dimensional super-resolution imaging method according to an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] It should be noted that the descriptions involving "first," "second," etc., in this invention are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Furthermore, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0037] like Figure 1As shown, an embodiment of the present invention is: a deep-depth two-photon three-dimensional super-resolution imaging system, comprising: a femtosecond pulse laser 101, a first beam splitter 102, a spatial light modulator 105, a corner reflector 112, a second beam splitter 114, a dichroic mirror 115, a photomultiplier tube 125, a time-correlated single-photon counter 126, an imaging processing terminal 127, a galvanometer 116, a microscope 117, and a three-dimensional moving platform 122;

[0038] The first beam splitter 102 is disposed on the output optical path of the femtosecond pulsed laser 101 to split the Gaussian beam emitted by the femtosecond pulsed laser 101 into two, forming a transmission optical path and a reflection optical path; the spatial light modulator 105 is disposed on the transmission optical path to convert the transmission laser beam into a three-dimensional cage beam with zero central intensity after two incoherent modulations; the corner reflector 112 is disposed on the reflection optical path to adjust the pulse interval between the Gaussian beam and the three-dimensional cage beam, with the pulse interval adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used; the second beam splitter 114 is used to combine the three-dimensional cage beam and the Gaussian beam from the corner reflector 112.

[0039] The dichroic mirror 115, galvanometer 116, microscope 117, and three-dimensional moving platform 122 are sequentially arranged in the output light path of the combined beam of the second beam splitter 114. The dichroic mirror 115 forms a 45° angle with the combined beam. The combined beam passes sequentially through the galvanometer 116 and microscope 117 to reach the three-dimensional moving platform 122, illuminating the sample 121 placed on the three-dimensional moving platform 122. The two-photon fluorescence generated by the sample 121 returns to the dichroic mirror 115 along the original path, and the dichroic mirror 115 reflects the two-photon fluorescence to the photomultiplier tube 125 for photoelectric signal conversion and electrical signal amplification.

[0040] The photomultiplier tube 125, the time-correlated single-photon counter 126, and the imaging processing terminal 127 are connected by electrical signals in sequence. The time-correlated single-photon counter 126 is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulse laser 101 and the two-photon fluorescence signal after fluorescence amplification collected by the photomultiplier tube 125, and to measure and record the spatiotemporal information of the two-photon fluorescence signal. The imaging processing terminal 127 is used to process the spatiotemporal information of the photon fluorescence signal to form a sample image.

[0041] The first beam splitter 102 is provided with a half glass plate 103 and a first reflector 104. The half glass plate 103 is used to adjust the polarization of the transmitted laser beam. The first reflector 104 is used to direct the adjusted transmitted laser beam onto the spatial light modulator 105 with a polarization direction of 45°.

[0042] The transmission light path also includes a quarter-glass slide 106, a first lens 107, and a second reflector 108. The liquid crystal surface of the spatial light modulator 105 is divided into two equal regions. A vortex phase is loaded on the left region of the liquid crystal surface of the spatial light modulator 105 to modulate the polarization p component of the incident beam. The reflected light passes sequentially through the first lens 107 and the quarter-glass slide 106, is reflected by the second reflector, and then passes through the first lens 107 and the quarter-glass slide 106 again to reach the right region of the liquid crystal surface of the spatial light modulator 105. A 0 / π phase is loaded on the right region of the liquid crystal surface of the spatial light modulator 105 to modulate the other vertical component of the incident beam. The quarter-glass slide 106 rotates the polarization vector of the beam by 90°, allowing the vertical component, i.e., the s component, of the incident light to be modulated through the right side of the spatial light modulator 105. The second reflector 108 and the light modulator 105 are respectively located at the front and rear focal points of the first lens 107 to maintain the conjugate modulation of the laser wavefront.

[0043] The transmission light path is also provided with a third reflecting mirror 109 and a fourth reflecting mirror 110. The third reflecting mirror 109 reflects the emitted light beam from the right side region of the liquid crystal surface of the spatial light modulator 105 to the fourth reflecting mirror 110. The fourth reflecting mirror 110 reflects the emitted light beam reflected by the third reflecting mirror 109 to the second beam splitter 114.

[0044] The reflected light path is provided with a fifth reflecting mirror 111 and a sixth reflecting mirror 113. The fifth reflecting mirror 111 reflects the reflected light from the first beam splitter 102 onto the corner reflector 112, and the sixth reflecting mirror 113 reflects the Gaussian beam from the corner reflector 112 onto the second beam splitter 114.

[0045] A second lens 123 and a filter 124 are provided between the dichroic mirror 115 and the photomultiplier tube 125.

[0046] The microscope 117 includes, in sequence, a scanning lens 118, a tube lens 119, and an objective lens 120.

[0047] In this deep-depth two-photon three-dimensional super-resolution imaging system, there is only one femtosecond pulse laser source with a tunable wavelength (680-1300nm) to better meet the actual excitation wavelength requirements.

[0048] This embodiment combines two-photon imaging, signal timing control, and light field manipulation techniques to improve the depth and three-dimensional imaging resolution of optical microscopy. By performing intensity difference analysis on the photons around the excitation beam, the high-frequency signal at the center of the beam is extracted. Within a depth of 500 micrometers, a lateral resolution better than 100 nm and an axial resolution of 120 nm can be achieved.

[0049] like Figure 3As shown, another embodiment of the present invention is: a deep-depth two-photon three-dimensional super-resolution imaging method, applied to the above-mentioned deep-depth two-photon three-dimensional super-resolution imaging system, comprising:

[0050] S10. The Gaussian beam emitted by the femtosecond pulsed laser is split into two by the first beam splitter to form a transmission optical path and a reflection optical path.

[0051] S20. The Gaussian beam in the transmission optical path is incoherently modulated twice by the spatial light modulator to produce a three-dimensional cage beam with zero central intensity.

[0052] S30. The Gaussian beam in the reflected light path adjusts the pulse interval between the Gaussian beam and the three-dimensional cage beam through a corner reflector. The pulse interval is adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used.

[0053] S40. Combine the three-dimensional cage beam modulated by the spatial light modulator and the Gaussian beam adjusted by the corner reflector through the second beam splitter.

[0054] S50. The combined beam is sequentially transmitted through a dichroic mirror, reflected by a galvanometer, transmitted through a scanning lens, and focused by a microscope before being applied to the sample.

[0055] S60. The two-photon fluorescence generated by the sample is returned to the dichroic mirror through the original path. The dichroic mirror reflects the two-photon fluorescence to the photomultiplier tube for photoelectric signal conversion and electrical signal amplification.

[0056] S70, a time-correlated single-photon counter, is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal after fluorescence amplification acquired by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal.

[0057] The S80 imaging processing terminal processes the spatiotemporal information of the two-photon fluorescence signal to form a sample image.

[0058] In step S70, the time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal amplified by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal. Specifically, this includes...

[0059] S71. Two-photon fluorescence generated after two laser pulses is collected by a time-correlated single-photon counter to obtain fluorescence lifetime data;

[0060] S72. Using the time channel where the intensity abrupt change in the fluorescence decay curve is located as the boundary, extract the signal before the channel to form a light cage image, and extract the signal after the channel to form a mixed image. At this time, the mixed image contains some fluorescence signals excited by the light cage beam. The mixing point spread function of the mixed image includes the Gaussian point spread function and the light cage point spread function.

[0061] S73. After obtaining the blended image and the light cage image, perform intensity difference on the blended image and the intensity-enhanced light cage image, i.e., I 超分辨图像 (x,y)=I 混合图像 (x,y)-K×I 光笼图像 (x,y), where K>1;

[0062] S74. Perform three-dimensional imaging on the sample and collect fluorescence lifetime data. After processing all the data, a series of two-dimensional super-resolution images are obtained. After superimposing all the two-dimensional super-resolution images, the final three-dimensional super-resolution image is obtained.

[0063] Step S50 further includes: adjusting the scanning lens and the microscope according to the real-time image of the sample to adjust the light spot so that the focal planes of the three-dimensional cage beam and the Gaussian beam of the combined beam coincide in space.

[0064] like Figure 2 As shown, in this scheme, the time-resolved detection capability (picosecond level) of fluorescence lifetime imaging technology is utilized to acquire the spatiotemporal information of two-photon fluorescent photons via TCSPC. The corner reflector is adjusted so that the time interval between the optical cage and the Gaussian laser pulse is approximately 50%-100% of the fluorescence lifetime of the fluorescent dye used. The optical cage laser pulse must arrive at the sample before the Gaussian laser pulse. At this time, most of the excited-state molecules excited by the optical cage laser spontaneously radiate back to the ground state, and then are excited again by the Gaussian laser, emitting fluorescence. This fluorescence, along with the remaining fluorescence signal excited by the optical cage laser, forms a mixed fluorescence signal, i.e., a mixture of Gaussian and optical cage fluorescence. Therefore, a fluorescence signal is emitted immediately after laser irradiation of the sample. Two-photon fluorescence photons generated after two laser pulses are collected using a TSCPC (black curve in the figure) to obtain fluorescence lifetime data. The time channel where the intensity abrupt change in the fluorescence decay curve occurs is used as the boundary (black dashed line). Signals before the channel are extracted to form an optical cage image, and signals after the channel are extracted to form a mixed image. The mixed image contains some fluorescence signals excited by the optical cage laser; therefore, the (mixed) point spread function of this image includes both a Gaussian point spread function (higher intensity) and an optical cage point spread function (lower intensity). After obtaining the mixed image and the optical cage image, intensity difference is performed between the mixed image and the enhanced optical cage image, i.e., If. 超分辨图像 (x,y)=I 混合图像 (x,y)-K×I 光笼图像(x, y), where K > 1. Since the TCSPC time channel has a sub-nanosecond resolution, it is equivalent to recording the two-photon fluorescence information generated by the Gaussian laser and the photocathode laser excitation of each pixel in the image in real time. Therefore, real-time fluorescence intensity difference between the two beams can be achieved, thus realizing super-resolution imaging. Finally, the sample is three-dimensionally imaged and fluorescence lifetime data is collected. Using the above principle, all data is processed to obtain a series of two-dimensional super-resolution images. These images are then superimposed to obtain the final three-dimensional super-resolution image. Therefore, the method proposed in this invention combines two-photon imaging, signal temporal modulation, and light field manipulation techniques to improve the depth and three-dimensional imaging resolution of optical microscopy, ultimately achieving three-dimensional super-resolution optical imaging at great depths, providing important technical support for research in the biomedical field.

[0065] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A deep-depth two-photon three-dimensional super-resolution imaging system, characterized in that, include: Femtosecond pulsed laser, first beam splitter, spatial light modulator, corner reflector, second beam splitter, dichroic mirror, photomultiplier tube, time-correlated single-photon counter, imaging processing terminal, galvanometer, microscope and three-dimensional moving platform; The first beam splitter is located on the output optical path of the femtosecond pulse laser and is used to split the Gaussian beam emitted by the femtosecond pulse laser into two, forming a transmission optical path and a reflection optical path; the spatial light modulator is located on the transmission optical path and is used to convert the transmission laser beam into a three-dimensional cage beam with zero central intensity after two incoherent modulations. The corner reflector is located on the reflected light path and is used to adjust the pulse interval between the Gaussian beam and the three-dimensional cage beam. The pulse interval is adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used. The second beam splitter is used to combine the three-dimensional cage beam and the Gaussian beam from the corner reflector. The dichroic mirror, galvanometer, microscope, and three-dimensional moving platform are sequentially positioned in the output light path of the combined beam from the second beam splitter. The dichroic mirror forms a 45° angle with the combined beam. The combined beam passes sequentially through the galvanometer and microscope to the three-dimensional moving platform, illuminating the sample placed on the platform. The two-photon fluorescence generated by the sample returns to the dichroic mirror along the original path, and the dichroic mirror reflects the two-photon fluorescence to a photomultiplier tube for photoelectric signal conversion and electrical signal amplification. The photomultiplier tube, time-correlated single-photon counter, and imaging processing terminal are connected by electrical signals in sequence. The time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal after fluorescence amplification acquired by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal. The imaging processing terminal is used to process the spatiotemporal information of the photon fluorescence signal to form a sample image.

2. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 1, characterized in that: The first beam splitter is provided with a half-glass slide and a first reflector. The half-glass slide is used to adjust the polarization of the transmitted laser beam. The first reflector is used to direct the adjusted transmitted laser beam onto the spatial light modulator with a 45° polarization direction.

3. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 2, characterized in that: The transmission light path is also provided with a quarter glass plate, a first lens, and a second reflector; the liquid crystal surface of the spatial light modulator is divided into two equal regions. A vortex phase is loaded in the left region of the liquid crystal surface of the spatial light modulator to modulate the polarization p component of the incident beam. The reflected light passes through the first lens and the quarter glass plate in sequence, is reflected by the second reflector, and then passes through the first lens and the quarter glass plate again to reach the right region of the liquid crystal surface of the spatial light modulator. A 0 / π phase is loaded in the right region of the liquid crystal surface of the spatial light modulator to modulate the other vertical component of the incident beam. The quarter glass plate rotates the polarization vector of the beam by 90°, allowing the vertical component of the incident light, i.e., the s component, to be modulated through the right side of the spatial light modulator; the second reflector and the light modulator are respectively located at the front and rear focal points of the first lens to maintain the conjugate modulation of the laser wavefront.

4. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 3, characterized in that: The transmission light path is also provided with a third reflecting mirror and a fourth reflecting mirror. The third reflecting mirror reflects the outgoing light beam from the right side region of the liquid crystal surface of the spatial light modulator to the fourth reflecting mirror. The fourth reflecting mirror reflects the outgoing light beam reflected by the third reflecting mirror to the second beam splitter.

5. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 4, characterized in that: The reflected light path is provided with a fifth reflector and a sixth reflector. The fifth reflector reflects the reflected light from the first beam splitter onto the corner reflector, and the sixth reflector reflects the Gaussian beam from the corner reflector onto the second beam splitter.

6. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 5, characterized in that: A second lens and a filter are also provided between the dichroic mirror and the photomultiplier tube.

7. The deep-depth two-photon three-dimensional super-resolution imaging system as described in claim 6, characterized in that: The microscope comprises, in sequence, a scanning lens, a tube lens, and an objective lens.

8. A deep-depth two-photon three-dimensional super-resolution imaging method, applied to the deep-depth two-photon three-dimensional super-resolution imaging system according to any one of claims 1-7, characterized in that, include: The Gaussian beam emitted by the femtosecond pulsed laser is split into two by the first beam splitter to form a transmission optical path and a reflection optical path. The Gaussian beam in the transmission optical path is incoherently modulated twice by the spatial light modulator to produce a three-dimensional cage beam with zero central intensity. The Gaussian beam in the reflected light path is adjusted by a corner reflector to change the pulse interval between the Gaussian beam and the three-dimensional cage beam. The pulse interval is adjusted to 50%-100% of the fluorescence lifetime of the fluorescent dye used. The three-dimensional cage beam modulated by the spatial light modulator and the Gaussian beam adjusted by the corner reflector are combined through the second beam splitter. The combined beam is sequentially transmitted through a dichroic mirror, reflected by a galvanometer, transmitted through a scanning lens, and focused by a microscope before illuminating the sample. The two-photon fluorescence generated by the sample is returned to the dichroic mirror, which reflects the two-photon fluorescence to the photomultiplier tube for photoelectric signal conversion and electrical signal amplification. The time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from the femtosecond pulsed laser and the two-photon fluorescence signal after fluorescence amplification acquired by the photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal; The imaging processing terminal processes the spatiotemporal information of the two-photon fluorescence signal to form a sample image.

9. The deep two-photon three-dimensional super-resolution imaging method as described in claim 8, characterized in that: A time-correlated single-photon counter is used to simultaneously receive the pulse synchronization signal input from a femtosecond pulsed laser and the amplified two-photon fluorescence signal acquired by a photomultiplier tube, and to measure and record the spatiotemporal information of the two-photon fluorescence signal, specifically including... Fluorescence lifetime data were obtained by acquiring two-photon fluorescence generated after two laser pulses using a time-correlated single-photon counter. Using the time channel where the intensity abrupt change in the fluorescence decay curve is located as the boundary, the signal before the channel is extracted to form the light cage image, and the signal after the channel is extracted to form the mixed image. At this time, the mixed image contains some fluorescence signals excited by the light cage beam. The mixing point spread function of the mixed image includes the Gaussian point spread function and the light cage point spread function. After obtaining the blended image and the light cage image, intensity difference is performed on the blended image and the intensity-enhanced light cage image, i.e., I 超分辨图像 (x,y)=I 混合图像 (x,y)-K×I 光笼图像 (x,y), where K>1; The sample was subjected to three-dimensional imaging and fluorescence lifetime data was collected. After processing all the data, a series of two-dimensional super-resolution images were obtained. The final three-dimensional super-resolution image was obtained by superimposing all the two-dimensional super-resolution images.

10. The deep-depth two-photon three-dimensional super-resolution imaging method as described in claim 8, characterized in that: Also includes: The scanning lens and the microscope are adjusted according to the real-time image of the sample to adjust the light spot, so that the focal planes of the three-dimensional cage beam and the Gaussian beam of the combined beam coincide in space.

Images