Two-photon body imaging method and system based on non-diffraction light needle

Abstract

The invention discloses a two-photon body imaging method and system based on a non-diffraction light needle, and the method comprises the following steps: 1, adjusting the axial focus position distribution and phase shift amount through a quasi-random spatial multiplexing algorithm according to the optical transmission characteristics, penetration depth and spatial resolution requirements of a target biological sample, and obtaining a two-photon body image; generating an optimized composite phase diagram capable of controlling the axial length and the transverse diameter of the light needle; 2, loading the phase diagram to a spatial light modulator, converting a Gaussian beam into a preset non-diffraction light needle, driving a light beam scanning module to realize single two-dimensional scanning of the light needle, and synchronously capturing a fluorescence signal to complete millisecond volume data acquisition; and 3, performing time-space mapping and information reconstruction on the time domain fluorescence signal to generate a two-dimensional projection image containing three-dimensional information. According to the invention, through effective control and rapid modulation of the light beam form, a high-speed three-dimensional imaging capability which is more friendly to a biological sample is realized, and an effective technical means is provided for researching dynamic activities of deep tissues.

Description

Technical Field

[0001] This invention relates to the field of optical microscopy imaging technology, specifically to a two-photon imaging method and system based on a diffraction-free optical needle. Background Technology

[0002] Fluorescence microscopy, with its advantages of being non-contact, highly specific, highly sensitive, and having high spatiotemporal resolution, has become a fundamental and commonly used cell imaging technique in biomedical photonics research. However, the resolution of optical microscopes is limited by diffraction, making it difficult to observe fine subcellular structures. In recent years, super-resolution techniques such as stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM) have broken through the diffraction limitation, but each has its own drawbacks: STED can image thick biological tissues at high resolution, but the high-intensity depletion light can easily damage the sample; STORM has high resolution, but the wide-field excitation and sparse emission mode limit the imaging depth and speed; SIM only improves the resolution by two times, but it is widely used for live cell imaging because it is not limited by fluorescent dyes, does not require high excitation power, and has fast imaging speed.

[0003] Two-photon fluorescence microscopy (TPFM) achieves high-resolution imaging based on the principle of two-photon absorption nonlinear optics. Compared to confocal microscopy, it is more suitable for thick samples, live-cell observation, and targeted photobleaching experiments. It uses femtosecond laser excitation, with the excitation effect localized in the focal region, reducing photobleaching, photodamage, and phototoxicity, and supporting long-term in vivo observation. Near-infrared excitation light penetrates deeper into biological tissues and has spatial compatibility with large samples. It is also easily combined with electrophysiological and optogenetic techniques, making it an important tool in life science and medical research.

[0004] To observe the structure and function of deeper cells and tissues, researchers have been optimizing the excitation light source, beam scanning structure, detection system and imaging strategy of the TPFM system, making progress in imaging speed and depth, and developing a two-photon laser scanning microscope (TPLSM) system. In 2016, Prevedel et al. used light engraving technology to sacrifice part of the spatial resolution to improve the volumetric imaging speed of two-photon calcium imaging of mouse cortical neurons to 60Hz; In 2017, Rongwen et al. combined point spread function engineering to integrate the axially elongated Bessel focus into the TPLSM system, and Guomei Guo of Harbin Institute of Technology developed a microscope with both two-photon fluorescence and coherent anti-Stokes Raman scattering (CARS) imaging capabilities; In 2019, Guozhong Hou et al. built a two-photon microscopy system based on a broadband femtosecond oscillator, and confirmed that the imaging depth can be improved in the long-wavelength detection window of Rhodamine B molecules; In 2020, Kazushi Yamaguchi et al. proposed a method for evaluating the focal volume of the mouse brain in vivo, and Fan et al. used a commercial TPLSM equipped with a Bessel focusing module to realize the imaging of specific volume blood flow dynamics at a frequency of 15Hz at a depth of 600μm in the cerebral cortex of a conscious mouse; In 2021, Yu Huanhuan et al. developed a new type of two-photon multi-focus structured illumination microscope based on a high-speed phase spatial light modulator.

[0005] However, as the core performance indicator of the two-photon fluorescence imaging system, the imaging speed directly determines its dynamic capture capability and application boundary. The Gaussian beam excitation mode of the traditional TPFM needs to rely on axial mechanical scanning to collect images layer by layer to realize three-dimensional volumetric imaging, which greatly prolongs the imaging time and makes it difficult to capture the millisecond-level neural activity dynamics in the living brain. The performance of the beam scanning structure becomes the key bottleneck limiting the imaging speed; Although the non-diffracting beam (such as Bessel beam) has extended focal depth and constant lateral resolution, it can theoretically project three-dimensional volumetric information onto a two-dimensional plane to realize single-exposure acquisition of volume data and improve the imaging rate, but the existing Bessel beam-based scheme has strong sidelobe effect, which will introduce background noise and artifacts, and usually needs complex correction optical elements (such as ring masks) to compensate for aberrations, increasing the system complexity and light energy loss.

[0006] Therefore, we propose a two-photon volumetric imaging method and system based on a non-diffracting light needle. SUMMARY

[0007] The purpose of the present application is to provide a two-photon volumetric imaging method and system based on a non-diffracting light needle, which solves the problems proposed in the background art.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a two-photon volumetric imaging method based on a non-diffracting light needle, comprising the following method steps: Step 1: Based on the optical transmission characteristics of the target biological sample, the penetration depth requirement and the spatial resolution requirement, the axial focal point position distribution and the phase shift amount between focal points are adjusted through a quasi-random spatial multiplexing algorithm to dynamically generate an optimized composite phase map which can simultaneously control the axial length and the lateral diameter of the light needle; Step 2: The composite phase map generated in step 1 is loaded into a spatial light modulator, so that the incident Gaussian light beam is converted into a non-diffractive light needle with preset parameters; by controlling the software to drive the light beam scanning module, the non-diffractive light needle performs a single two-dimensional scan on the sample plane, and at the same time, the photodetection module synchronously captures the fluorescence signal generated by the sample being excited, thereby realizing millisecond-level volume data acquisition; Step 3: The signal acquisition module performs time-space mapping and information reconstruction on the time-domain fluorescence signal captured by the photodetection module, directly generating a two-dimensional projection image containing three-dimensional spatial information, without the need for axial mechanical scanning to analyze the depth structure of the sample.

[0009] As a preferred embodiment of the present application, in step 1, the quasi-random spatial multiplexing algorithm randomly allocates the N×N pixel units of the phase mask to different focal point groups through a unit cell method, so that the pixel positions of each focal point are irregularly distributed on the entire phase mask, thereby suppressing high-order diffraction without the need for additional spatial filtering devices.

[0010] As a preferred embodiment of the present application, in step 1, when generating the composite phase map, the focal point positions need to be optimized through an iterative algorithm, and the composite phase map is quickly switched through the control program Blink Overdrive Plus of the spatial light modulator.

[0011] As a preferred embodiment of the present application, in step 2, the incident Gaussian light beam needs to be adjusted in power by a power adjustment unit, which is composed of a half-wave plate and a polarization beam splitter prism. By rotating the half-wave plate, the proportion of the two mutually perpendicular linearly polarized lights emitted by the polarization beam splitter prism is changed, and the P-polarized light is selected as the source of the Gaussian light beam for two-photon excitation.

[0012] As a preferred embodiment of the present application, in step 2, the core of the photodetection module is a photomultiplier tube (PMT); or the PMT can be replaced by an EMCCD camera or an sCMOS camera, and a double-helix phase plate is added in the detection light path for depth information decoding, combined with a depth diffraction array needle-shaped light beam phase map, to realize multi-focal point super-resolution volume imaging.

[0013] As a preferred embodiment of the present application, in step 2, the control software is an open-source Matlab program ScanImage running on a computer terminal, which is used to synchronously control the scanning action of the light beam scanning module and the signal capture of the photodetection module, and the imaging parameters such as the number of pixels, the pixel residence time and the image saving path can be set through the software.

[0014] The application also relates to a two-photon body imaging system based on a non-diffraction light needle, which comprises an excitation light adjusting module, a light beam scanning module, a microscopic imaging module, a photoelectric detection module and a signal acquisition module; the excitation light adjusting module is used for generating a non-diffraction light needle, the light beam scanning module is used for driving the non-diffraction light needle to scan, the microscopic imaging module is used for forming a scanning light needle and collecting a fluorescent signal, the photoelectric detection module is used for capturing the fluorescent signal, and the signal acquisition module is used for synchronous control and signal processing.

[0015] As a preferred embodiment of the application, the excitation light adjusting module comprises a femtosecond pulse laser, a half-wave plate, a polarization beam splitting prism, a beam expander lens group (L1, L2), a spatial light modulator, a lens group (L3, L4) and an adjustable diaphragm; the femtosecond pulse laser has a power of 4W, a pulse width of 145fs and an output wavelength of 1036nm; the beam expander lens group (L1, L2) is used for expanding the beam after power adjustment; the lens group (L3, L4) is used for conjugating the phase distribution of the liquid crystal working surface of the spatial light modulator to the back focal plane of the objective lens of the microscopic imaging module; the adjustable diaphragm is arranged between the lens group (L3, L4) and is used for filtering out the first-order diffraction light.

[0016] As a preferred embodiment of the application, the light beam scanning module comprises a lens group (L5, L6), an XY scanning galvanometer and a scanning lens (L7); the lens group (L5, L6) is used for adjusting the beam diameter of the non-diffraction light needle; the XY scanning galvanometer is used for driving the light beam to perform two-dimensional scanning on the sample plane; and the scanning lens (L7) is used for guiding the scanned light beam to the microscopic imaging module.

[0017] As a preferred embodiment of the application, the microscopic imaging module comprises a tube lens, an objective lens and a sample stage; the objective lens has a numerical aperture of 1.1 and a working distance of 2mm, is used for converging the non-diffraction light needle into a scanning light needle and collecting a sample fluorescent signal, and the signal acquisition module comprises a data acquisition card and a computer terminal, is used for acquiring the electric signal output by the photoelectric detection module, and the computer terminal runs ScanImage software to realize signal processing and image generation.

[0018] Compared with the prior art, the application has the following beneficial effects: The application effectively solves the key limitations of traditional methods in high-speed, deep, and in-vivo imaging by combining single-beam adjustable non-diffracting light needle excitation and single-exposure volume data acquisition. The technical solution only needs to use a spatial light modulator to load a complex phase mask, which can directly convert the incident Gaussian beam into a parameter-adjustable light needle, and realize flexible control of the axial length and transverse diameter of the light beam by quickly switching the mask. The design combines a photomultiplier tube (PMT) to directly collect fluorescence signals, significantly improving the acquisition rate of three-dimensional volume data, replacing the time-consuming mechanical axial layer-by-layer scanning process in traditional technology.

[0019] In terms of imaging performance, the uniformity of the axial energy distribution of the light needle enables single-exposure to cover a larger depth range and maintain relatively consistent transverse resolution throughout the imaging area. This uniformity is mainly achieved by optimizing the axial focal position and phase shift, without the need for complex amplitude modulation processes, simplifying system operation. This feature significantly reduces the light damage and phototoxicity effects on in-vivo biological samples, especially deep tissues, providing more favorable conditions for long-term dynamic observation.

[0020] System compatibility and practicality are fully demonstrated: the phase mask technology used can be directly integrated into existing microscope systems for functional upgrades, and the scheme mainly relies on phase modulation, avoiding the complexity of joint modulation and simplifying the operation process. BRIEF DESCRIPTION OF DRAWINGS

[0021] Other features, objects, and advantages of the application will become more apparent after reading the detailed description of non-limiting embodiments with reference to the following drawings: Figure 1 A schematic diagram of each module of the system of the application; Figure 2 A flowchart of the generation algorithm of the needle-shaped light beam of the application; Figure 3 A schematic diagram of the iterative algorithm of the application; Figure 4 A schematic diagram of the generation of the needle-shaped light beam of the application; Figure 5 An operation interface diagram of the ScanImage program of the application; Figure 6 An implementation result diagram of the application. DETAILED DESCRIPTION

[0022] To make the technical means, creative features, purposes and effects achieved by the application easy to understand, the following further describes the application in conjunction with specific embodiments.

[0023] As shown in Figures 1-6 , a two-photon volume imaging method and system based on non-diffracting light needles; The system is composed of the following modules: excitation light adjustment module, beam scanning module, microscopic imaging module, photoelectric detection module and signal acquisition module. The excitation light adjustment module includes a femtosecond pulse laser, two half-wave plates, a polarizing beam splitter, an expansion lens group (L1 and L2) and a spatial light modulator (SLM). The excitation light source is a laser with a power of 4 W, a pulse width of 145 fs and an output wavelength of 1036 nm (AnYang, FemtoYL-6). In order to realize the continuous adjustment of the excitation light power, a 1 / 2 wave plate (Thorlabs, AHWP05M-980) and a polarized light beam splitter (Thorlabs, CCM1-PBS252) are used to form a power adjustment module. By rotating the 1 / 2 wave plate behind the PBS, the proportion of the two mutually perpendicular linearly polarized lights of the PBS is changed. Here, the P-polarized light emitted from the PBS is used as the subsequent two-photon excitation light source. Then, the light beam is expanded by the lens group composed of L1 and L2, and is obliquely incident (about 10°) to the working surface of a high-speed near-infrared SLM (Meadowlark Optics, NIRHSP1920-1152, with a pixel number of 1920x1152 and a single pixel size of 9.2μm x 9.2μm) through a mirror.

[0024] By loading a phase map of the SLM to produce a needle-shaped beam without diffraction needles, the incident Gaussian beam is directly converted into a needle-shaped beam with adjustable axial length and controllable lateral diameter. Then, the phase distribution of the SLM liquid crystal working surface is conjugated to the back focal plane of the objective lens using lens groups L3 and L4. Adjustable apertures (Iris, Thorlabs, ID25) are placed between the lens groups for spatial filtering to filter out the first-order diffraction light required for subsequent optical paths. The beam scanning module consists of lens groups (L5 and L6), XY scanning mirrors (Thorlabs, GVS002), and scanning lens L7. The laser beam, modulated and shaped by the excitation light adjustment module, is incident on the X-axis mirror after the beam diameter is adjusted by lens groups L5 and L6. Reflected by the X-axis mirror, it is incident on the Y-axis mirror, and then, after reflection, passes sequentially through scanning lens L7 and a microscopic imaging module consisting of a tube lens, objective lens, and sample surface. The designed scanning needle is formed at the back focal plane of the objective lens (Nikon, CFIApo LWD25XW, 1.1NA and 2mm WD). The excited sample fluorescence signal is collected by the same objective lens and returned via the tube lens, then reflected by a dichroic mirror into the detection light path (i.e., the photodetector module). The fluorescence signal is focused onto the detection surface of the photomultiplier tube (PMT) and recorded. The signal acquisition module includes a data acquisition card (DAQ, NI-USB6363), a computer terminal (PC), and the open-source Matlab program ScanImage running on it. It is used for the synchronization of beam scanning and fluorescence signal acquisition, and performs data segmentation and reconstruction on the time-series signals acquired by the PMT to achieve real-time image display.

[0025] The specific steps for generating a non-diffractive optical needle are as follows: A needle-shaped beam is a beam with an extremely long focal depth and uniform axial intensity distribution. Its algorithm flowchart is shown below. Figure 2 As shown. The generation principle of the needle-shaped beam is based on spatial multiplexing phase modulation technology. A phase mask is used to transform the single focus of a traditional Gaussian beam into a series of densely arranged focal arrays along the optical axis. The phase modes of these focal points are superimposed to form an extended depth-of-focus beam with a needle-like distribution. Then, an additional phase modulation P is introduced... m (x,y,f m f) Achieve focus shift from the original position f to the target position f m The axial offset. The phase mask pixels are divided into M groups, each group corresponding to a specific focal position f. m, the "unit cell" method (N x N pixel units) is used to randomly assign each unit pixel to different focal points. This random assignment enables uniform energy distribution, and its Fourier transform characteristics effectively suppress high-order diffraction, suppressing sidelobe interference without additional spatial filtering devices. M densely arranged focal points are generated by a phase mask and a lens, and the output light field is approximately a linear superposition of each focal point light field. The focal point spacing is dynamically adjusted by an iterative algorithm: the spacing between adjacent focal points is increased when the local intensity is higher than the average value, and the spacing is decreased when the local intensity is lower than the average value. Finally, a needle-shaped light beam with a preset length and diameter is obtained.

[0026] The core technology of the algorithm for forming a non-diffracted light needle is as follows: (1) Focal point shift function: The focal point shift function is used to generate densely spaced focal points along the axial direction, and its mathematical expression is:

[0027] where P Obj is the phase distribution of the objective lens that satisfies the Abbe sine condition. Through this function, the focal point can be moved from the f axis to a new position f m .

[0028] (2) Phase mask pixel assignment: In the design of the phase mask of the needle-shaped light beam, each focal point is modulated by multiple pixels, and the pixel position of each focal point is irregularly distributed on the entire phase mask. This quasi-random spatial multiplexing suppresses high-order diffraction.

[0029] (3) Focal point position optimization: An iterative algorithm (as shown in Figure 3 ) is used to adjust the positions of the focal points to achieve uniform intensity distribution of the light needle in the axial direction and reduce the maximum diameter of the light needle, thereby improving the imaging accuracy.

[0030] (4) Compound phase mask generation: The phase patterns of all focal points are superimposed to generate the final phase mask, and finally a light needle is formed through a lens, as shown in Figure 4 .

[0031] Figure 1 The English labels in the above figures are defined as follows: Laser: laser; HWP: half-wave plate; PBS: polarizing beam splitter prism; mirror: mirror; L1-L7: lens; SLM: spatial light modulator; Iris: adjustable iris; Galvo XY: two-dimensional scanning galvanometer; dichroic mirror: dichroic mirror; PMT: photomultiplier tube; tube lens: tube lens; Objective: objective lens; Sample: sample (located on the stage); DAQ: data acquisition card; PC: computer terminal.

[0032] Refer to Figure 1, the laser beam emitted by the femtosecond pulse laser (Laser) first passes through the power adjustment unit composed of a half-wave plate (HWP) and a polarization beam splitter prism (PBS) for continuous adjustment of the excitation light power. The adjusted light beam then passes through the beam expander lens group (L1 and L2) for beam expansion. The expanded light beam is directed by a mirror and incident on the working surface of the spatial light modulator (SLM) at a certain angle. By loading a specific complex phase mask (needle beam phase diagram) on the SLM, the incident Gaussian beam is directly converted into a non-diffracted light needle with adjustable axial length and controllable transverse diameter. The phase distribution of the SLM liquid crystal working surface is then conjugated to the back focal plane position of the subsequent objective lens through another lens group (L3 and L4). An adjustable iris (Iris) is placed between the L3 and L4 lens groups for spatial filtering to select the required first-order diffracted light. Subsequently, the modulated and shaped light needle beam passes through the lens group (L5 and L6) to adjust the beam diameter, and then is incident on the XY scanning galvanometer system. The light beam is reflected and deflected by the X-axis galvanometer and the Y-axis galvanometer in turn. The deflected light beam passes through the scanning lens (L7), and the light needle beam guided by L7 and the sleeve lens is finally focused on the sample surface by the high numerical aperture objective lens, forming the expected designed scanning light needle for two-photon excitation of the sample. The fluorescence signal excited by the sample is collected by the same objective lens, returns along the original path, is reflected by the dichroic mirror (Dichroic mirror), and enters the photoelectric detection light path. The fluorescence signal is finally focused on the detection surface of the photomultiplier tube (PMT) for high-sensitivity detection. The control software (ScanImage, etc.) running on the computer is used to synchronize the control of the light beam scanning (galvanometer and SLM phase updating) and the fluorescence signal acquisition. The data acquisition card collects the timing fluorescence signals output by the PMT in real time, and the control software performs data segmentation and recombination on these signals, finally realizing real-time reconstruction and display of three-dimensional images.

[0033] Referring to Figure 5 , the user can modify the imaging parameters in the ScanImage program: pixel number, pixel residence time, and image automatic storage path, etc., among which the pixel residence time directly affects the galvanometer scanning speed and imaging speed. Finally, the two-dimensional projection image containing three-dimensional spatial information is displayed in the "Chanel1" window.

[0034] Taking the imaging experiment of 500nm fluorescent beads as an example, the experimental steps are as follows: Step 1: Use the bright field light to illuminate the sample, observe the sample through the microscope eyepiece, adjust the stage position up and down to find the focal plane, and adjust the stage position forward and backward to find the cell region of interest.

[0035] Step 2: Turn on the controller power of the laser, SLM, DAQ card, galvanometer, PMT, displacement stage, open the corresponding control software on the computer, adjust the laser power through the laser control software, load the required phase pattern on the SLM control software (in this experiment, Gaussian single-point phase pattern and 10 μm light needle phase pattern are loaded for comparison), open the PMT control software to make it in the state of waiting for triggering.

[0036] Step 3: Image in the ScanImage program, select appropriate imaging parameters (in this experiment, the number of pixels is 512x512, the pixel residence time is 4000 ns, and the magnification is 4), click the "FOCUS" button of the "MAIN CONTROLS" window to trigger the galvanometer scanning and real-time imaging, and fine-tune up and down and left and right in the displacement stage control program to select the appropriate imaging focal plane and field of view; then click the "GRAB" button to capture a scan image, if automatic saving is required, check the "Save" item before capturing the image and set the save path in advance. The imaging experimental results of the 500 nm fluorescent bead depth sample are shown in FIGS. 1 and 2. Figure 6 Figure 6 (a) is the superposition of the Gaussian imaging layer scanning image in the axial 10 μm depth range, and (b) is the projection of the light needle single scanning imaging with a length of 10 μm, it can be seen directly that the light needle scanning can obtain the corresponding depth information of the sample in a single time, and has a good signal-to-noise ratio.

[0037] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It is obvious for those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be realized in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting, the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.

[0038] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description manner of the specification is only for the sake of clarity, those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.​

Claims

1. A two-photon volume imaging method based on a non-diffracting light needle, characterized by: The method comprises the following steps: Step 1: Based on the optical transmission characteristics of the target biological sample, the penetration depth requirement and the spatial resolution requirement, the axial focal point position distribution and the phase shift amount between focal points are adjusted through a quasi-random spatial multiplexing algorithm to dynamically generate an optimized composite phase map which can control the axial length and the lateral diameter of the light needle simultaneously; Step 2: The composite phase map generated in step 1 is loaded into a spatial light modulator, so that the incident Gaussian light beam is converted into a non-diffractive light needle with preset parameters; Through the control software, the light beam scanning module is driven to make the non-diffractive light needle perform single two-dimensional scanning on the sample plane, and the photoelectric detection module is used to synchronously capture the fluorescence signal generated by the sample being excited, so that millisecond-level data acquisition is realized; Step 3: The time-space mapping and information reconstruction are performed on the time-domain fluorescence signal captured by the photoelectric detection module by using the signal acquisition module, so that a two-dimensional projection image containing three-dimensional spatial information is directly generated, and the depth structure of the sample can be analyzed without axial mechanical scanning.

2. The two-photon tomography method and system based on non-diffracting light needle according to claim 1, characterized in that: In step 1, the quasi-random spatial multiplexing algorithm randomly allocates the N×N pixel units of the phase mask to different focal point groups through a unit cell method, so that the pixel positions of each focal point are irregularly distributed on the entire phase mask, thereby suppressing high-order diffraction without the need of an additional spatial filtering device.

3. The two-photon tomography method and system based on non-diffracting light needle according to claim 1, characterized in that: In step 1, the focal point position needs to be optimized through an iterative algorithm, and the control program Blink Overdrive Plus of the spatial light modulator is used to realize the rapid switching of the composite phase map.

4. The two-photon tomography method and system based on non-diffracting light needle according to claim 1, characterized in that: In step 2, the incident Gaussian light beam needs to be adjusted in power by a power adjustment unit, the power adjustment unit is composed of a half-wave plate and a polarization beam splitting prism, the proportion of two mutually perpendicular linearly polarized lights emitted by the polarization beam splitting prism is changed by rotating the half-wave plate, and the P-polarized light is selected as the source of the Gaussian light beam for two-photon excitation.

5. The two-photon tomography method and system based on non-diffracting light needle according to claim 1, characterized in that: In step 2, the core of the photoelectric detection module is a photomultiplier tube (PMT), or the PMT can be replaced by an EMCCD camera or an sCMOS camera, and a double-helix phase plate is added in the detection light path to decode the depth information, and a depth diffraction array needle-shaped light beam phase map is loaded to realize multi-focal point super-resolution body imaging.

6. The two-photon tomography method and system based on non-diffracting light needle according to claim 1, characterized in that: In step 2, the control software is an open-source Matlab program ScanImage running on a computer terminal, which is used to synchronously control the scanning action of the light beam scanning module and the signal capture of the photoelectric detection module, and the imaging parameters such as the number of pixels, the pixel residence time and the image saving path can be set through the software.

7. A two-photon tomography system based on non-diffracting optical needles, suitable for use in the two-photon tomography method based on non-diffracting optical needles according to any one of claims 1-6, characterized in that: The system comprises an excitation light adjustment module, a light beam scanning module, a microscopic imaging module, a photoelectric detection module and a signal acquisition module; the excitation light adjustment module is used to generate a non-diffractive light needle, the light beam scanning module is used to drive the non-diffractive light needle to scan, the microscopic imaging module is used to form a scanning light needle and collect a fluorescence signal, the photoelectric detection module is used to capture the fluorescence signal, and the signal acquisition module is used for synchronous control and signal processing.

8. The two-photon tomographic imaging system based on non-diffracting optical needle according to claim 7, characterized in that: The excitation light adjusting module comprises a femtosecond pulse laser, a half-wave plate, a polarization beam splitting prism, a beam expander lens set (L1, L2), a spatial light modulator, a lens set (L3, L4) and an adjustable diaphragm; the femtosecond pulse laser has a power of 4W, a pulse width of 145fs and an output wavelength of 1036nm; the beam expander lens set (L1, L2) is used for expanding the beam after power adjustment; the lens set (L3, L4) is used for conjugating the phase distribution of the liquid crystal working surface of the spatial light modulator to the back focal plane of the objective lens of the microscopic imaging module; the adjustable diaphragm is arranged between the lens set (L3, L4) and is used for filtering out the first-order diffraction light.

9. The two-photon endomicroscopy system based on non-diffracting optical needle according to claim 7, characterized in that: The light beam scanning module comprises a lens set (L5, L6), an XY scanning galvanometer and a scanning lens (L7); the lens set (L5, L6) is used for adjusting the beam diameter of the non-diffraction light needle; the XY scanning galvanometer is used for driving the light beam to perform two-dimensional scanning on the sample plane; and the scanning lens (L7) is used for guiding the scanned light beam to the microscopic imaging module.

10. The two-photon endomicroscopy system based on non-diffracting optical needle according to claim 7, wherein: The microscopic imaging module comprises a tube lens, an objective lens and a sample stage; the objective lens has a numerical aperture of 1.1 and a working distance of 2mm and is used for converging the non-diffraction light needle into a scanning light needle and collecting the sample fluorescence signal; and the signal acquisition module comprises a data acquisition card and a computer terminal and is used for acquiring the electrical signal output by the photoelectric detection module; the computer terminal runs ScanImage software to realize signal processing and image generation.

Images