Compressed sensing multi-photon volume imaging device and method, and optical system

Abstract

The present invention provides a compressed sensing multi-photon volume imaging device and method, and an optical system. The optical system according to the present invention includes: a photomultiplier tube, a first converging lens, a filter, a first beam expander, a dichroic mirror, a second beam expander, an objective lens, a first galvanometer, a first collimating lens, a second galvanometer, a second converging lens, a second collimating lens, a third converging lens, and a femtosecond laser; wherein the excitation light path in the light path includes from the femtosecond laser through the third converging lens, the second collimating lens, the second converging lens, the second galvanometer, the first collimating lens, the first galvanometer, the second beam expander, the second dichroic mirror, the objective lens to the sample, and the detection light path in the light path includes from the sample through the objective lens, the second dichroic mirror, the first beam expander, the filter, the first converging lens to the photomultiplier tube.

Description

Technical Field

[0001] The present invention relates to the field of computational imaging, and more particularly to the field of life sciences. More particularly, the present invention relates to a wide-field-of-view, high-resolution, and high-speed compressed sensing multi-photon imaging device and method. Background Art

[0002] Multiphoton microscopy has become an indispensable tool in the life sciences, renowned for its natural tomographic capabilities and high penetration depth. Multiphoton microscopy also offers the advantage of multimodal imaging, enabling it to capture multiple types of signals, including fluorescent labeling, autofluorescence, and second and third harmonic generation (SHG) imaging, providing more sample information. Multiphoton microscopy is developing in four key areas: fast imaging speed, large imaging area, high imaging resolution, and deep imaging depth. However, achieving high performance in all four areas simultaneously is extremely challenging.

[0003] Traditional point-scanning multiphoton imaging microscopes have the advantage of high signal-to-noise ratio, but their general performance limitations are as follows:

[0004] First, a commonly used linear 2D galvanometer mirror captures a 512×512 pixel image at a speed of approximately 1Hz, with a pixel dwell time of ~3000ns. Replacing the linear galvanometer mirror with a resonant galvanometer mirror on one axis of the 2D galvanometer mirror significantly increases image acquisition speed, typically capturing a 512×512 pixel image at 30Hz. Point-scanning multiphoton imaging microscopes typically perform volume imaging by moving the focal plane axially. This means that the speed is significantly reduced when performing volume imaging. For example, capturing volume images from five different planes does not exceed 6Hz.

[0005] Moreover, due to the limitation of the size of the objective lens, it is usually difficult to achieve high performance in both imaging resolution and field of view of the microscope system. The field of view of a typical multiphoton microscope does not exceed 700×700μm. 2 , the lateral resolution can reach 0.3-0.6μm.

[0006] Furthermore, a major factor in the deep penetration capability of multiphoton microscopy is the long excitation wavelength. According to current literature, in microscopic imaging experiments on the mouse cerebral cortex, the maximum imaging depth of a two-photon microscope can reach 1.0 mm, while the maximum imaging depth of a three-photon microscope is ~1.7 mm.

[0007] Currently, multiphoton microscopy requires a fast, large-scale, and high-resolution volume imaging method to expand its applications in life sciences and make more contributions. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a wide-field, high-resolution and high-speed compressed sensing multi-photon imaging device and method in response to the above-mentioned defects in the prior art.

[0009] According to the present invention, a system of a compressed sensing multi-photon volume imaging device is provided, comprising: a point scanning excitation device, a multi-angle excitation device, a compressed sensing module, an optical illumination device, a signal acquisition device, and an image reconstruction module;

[0010] The point scanning excitation device is used to realize laser scanning excitation on the imaging surface, wherein the excitation end does not completely fill the rear focal plane of the objective lens, and the excitation point spread function at the front focal plane of the objective lens is axially elongated, thereby converting the full numerical aperture point excitation to low numerical aperture needle excitation;

[0011] The multi-angle excitation device is used to realize the deflection of different angles of needle excitation, wherein the deflection surface of the multi-angle excitation device is located on the conjugate plane of the front focal plane of the objective lens, that is, on the Fourier plane of the rear focal plane of the objective lens, and the deflection of the deflection surface causes the light spot to shift on the rear focal plane of the objective lens, thereby realizing the excitation of the point spread function at different angles on the excitation surface;

[0012] The compressed sensing module is used to accelerate imaging acquisition, which uses the redundancy of information in the temporal and spatial dimensions of the sample during the imaging process to achieve a sampling frequency lower than the Nyquist sampling theorem;

[0013] The optical illumination device includes a laser, an optical lens, a 4f relay lens, and an objective lens, wherein the laser is used to excite the sample signal, the optical lens and the 4f relay lens are used to adjust the beam diameter, beam collimation, and the conjugate relationship between different focal planes, and the objective lens is used for low numerical aperture excitation and high numerical aperture detection;

[0014] The signal acquisition device is used to detect body imaging signals, including single-channel or multi-channel signal acquisition and imaging;

[0015] The image reconstruction module is used to realize the decoupling and reconstruction of the collected coupling signals.

[0016] Preferably, the signal acquisition module is implemented by a photomultiplier tube.

[0017] Preferably, the compressed sensing multi-photon imaging device includes: a photomultiplier tube, a first converging lens, a filter, a first beam expander, a dichroic mirror, a second beam expander, an objective lens, a first galvanometer, a first collimating lens, a second galvanometer, a second converging lens, a second collimating lens, a third converging lens and a femtosecond laser; wherein, the excitation light path in the light path includes a light path from the femtosecond laser through the third converging lens, the second collimating lens, the second converging lens, the second galvanometer, the first collimating lens, the first galvanometer, the second beam expander, the second dichroic mirror, the objective lens to the sample, and the detection light path in the light path includes a light path from the sample through the objective lens, the second dichroic mirror, the first beam expander, the filter, the first converging lens to the photomultiplier tube.

[0018] Preferably, the photomultiplier tube is used to detect green and / or yellow fluorescence signals, the filter is used to filter photon signals, and the first converging lens and the first beam expander are used to improve the photon collection efficiency.

[0019] Preferably, the second dichroic mirror is used to filter the long-wavelength excitation light and reflect the short-wavelength emission light signal to the end of the photomultiplier tube. The second beam expander is used to adjust the size of the beam diameter to achieve the required low numerical aperture excitation and to eliminate aberrations in the scanning optical path; the first galvanometer is a point scanning excitation device; the first collimating lens adjusts the light beam into a collimated beam, and the second galvanometer is placed at its front focal plane; the second galvanometer is a multi-angle excitation device, placed at the front focal plane of the first collimating lens and the back focal plane of the second converging lens; the second converging lens is used to converge the light beam to the axis center of the two-dimensional deflection axis of the second galvanometer, and the second converging lens and the first collimating lens work together to adjust the beam diameter; the femtosecond laser is used to emit femtosecond pulse excitation light that generates a two-photon signal.

[0020] Preferably, the second collimating lens and the third converging lens are lenses with the same focal length, and a small hole needs to be placed at the focal plane between the second collimating lens and the third converging lens to achieve frequency information expansion of the point spread function.

[0021] According to the present invention, a method for a compressed sensing multi-photon volume imaging device is also provided, which adopts the above-mentioned compressed sensing multi-photon volume imaging device, including: first, deflecting the galvanometer to move the low numerical aperture light beam laterally on the rear focal plane of the objective lens, obtaining an image each time it moves, until all positions are superimposed and fill the rear focal plane of the high numerical aperture objective lens, thereby obtaining an angle-excited image stack; placing the collected image signal into a deconvolution high-resolution reconstruction algorithm for iterative calculation until the predetermined resolution volume imaging result is restored; in the real-time acquisition process, utilizing the sparsity of the image microscopic image information in the discrete cosine transform domain, after compressing the collected samples, reconstructing the volume imaging result through the compressed sensing algorithm to obtain a volume imaging rate above the predetermined frequency.

[0022] According to the present invention, an optical system is also provided, which includes: a photomultiplier tube, a first converging lens, a filter, a first beam expander, a dichroic mirror, a second beam expander, an objective lens, a first galvanometer, a first collimating lens, a second galvanometer, a second converging lens, a second collimating lens, a third converging lens and a femtosecond laser; wherein the excitation light path in the light path includes a path from the femtosecond laser through the third converging lens, the second collimating lens, the second converging lens, the second galvanometer, the first collimating lens, the first galvanometer, the second beam expander, the second dichroic mirror, the objective lens to the sample, and the detection light path in the light path includes a path from the sample through the objective lens, the second dichroic mirror, the first beam expander, the filter, the first converging lens to the photomultiplier tube.

[0023] <Technical Effects>

[0024] The present invention realizes a fast, large-scale and high-resolution multi-photon volume imaging method. It is embodied in the following aspects:

[0025] In terms of achieving volumetric imaging, the present invention breaks away from the limitations of point scanning and single-point excitation, extending the axial range of the optical system's point spread function (PSF), extending it from point scanning to needle scanning. Ordinary point scanning multiphoton microscopes have an axial resolution in the range of 1μm-3μm. The present invention uses a low numerical aperture (NA) excitation method to extend the axial range of the point spread function (PSF) to 100μm and above, from the traditional excitation of a single point to the excitation of all axial content within a depth range of more than 100μm. This achieves the transition from traditional single-frame single-layer images to single-frame image excitation of volumetric information acquisition in a depth range greater than 100μm.

[0026] In terms of achieving high resolution, the present invention uses a high numerical aperture (NA) objective lens to collect emitted light, and after multi-angle excitation, an algorithm is used to reconstruct and restore high resolution. Using a high numerical aperture (NA) objective lens for collection can improve collection efficiency. Under the conditions of low numerical aperture (NA) excitation and high numerical aperture (NA) acquisition, the ratio of in-focus signal to out-of-focus background signal can be significantly improved, thereby increasing the imaging depth of the multiphoton microscopy system. By combining the multi-angle excitation and acquisition of the imaging volume after illumination from different directions with the deconvolution algorithm of the point spread function (PSF), the present invention can not only reconstruct the axial information of different layers of out-of-body imaging, but also restore the resolution of the imaging system under the high numerical aperture (NA) objective lens.

[0027] In order to realize the large field of view imaging area, the present invention independently designs the excitation and collection multiplexed multi-photon objective lens of the system device. By eliminating the optical aberration and correcting the distortion, the 1×1mm 2 The high lateral resolution of 0.2 μm is maintained under the imaging area.

[0028] In terms of achieving fast volumetric imaging, the present invention utilizes the sparsity of microscopic image information in the discrete cosine transform domain and uses a compressed sensing method to perform multi-angle needle excitation in a target area of ​​1mm×1mm×>100μm. After sparsely collecting multi-photon signals excited by low numerical aperture NA at different angles, the image is reconstructed to obtain 30Hz volumetric imaging capability, achieving resolution preservation and isotropy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] A more complete understanding of the present invention and its attendant advantages and features will be more readily appreciated by reference to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0030] Figure 1 The optical system of the compressed sensing multi-photon volume imaging method according to a preferred embodiment of the present invention is schematically shown.

[0031] Figure 2 The figure schematically shows a reconstruction flow chart of a compressed sensing multi-photon volume imaging method according to a preferred embodiment of the present invention.

[0032] Figure 3 The figure schematically shows a coordinate diagram of a compressed sensing multi-photon volume imaging method according to a preferred embodiment of the present invention.

[0033] It should be noted that the accompanying drawings are intended to illustrate the present invention, not to limit it. Note that the accompanying drawings showing structures may not be drawn to scale. Furthermore, in the accompanying drawings, identical or similar elements are labeled with identical or similar reference numerals. DETAILED DESCRIPTION

[0034] In order to make the contents of the present invention clearer and easier to understand, the contents of the present invention are described in detail below with reference to specific embodiments and drawings.

[0035] The system of the compressed sensing multi-photon volume imaging device according to a preferred embodiment of the present invention includes: a point scanning excitation device, a multi-angle excitation device, a compressed sensing module, an optical illumination device, a signal acquisition device and an image reconstruction module.

[0036] Among them, the point scanning excitation device is used to realize laser scanning excitation on the imaging surface. Since the excitation end does not fully fill the rear focal plane of the objective lens, the excitation point spread function at the front focal plane of the objective lens is axially elongated, and the point excitation of the full numerical aperture NA is transformed into the needle excitation of the low numerical aperture NA. The point scanning device is used to realize the movement of the excitation point spread function PSF on the focal plane, thereby exciting the entire imaging surface. The devices that can be used for the point scanning device include: galvanometer galvanometer, resonant galvanometer and acousto-optic modulator, etc., usually two-dimensional grid scanning. In the example of the present invention, a galvanometer galvanometer can be used to realize the point spread function PSF scanning in the X and Y directions on the imaging surface. Among them, X and Y are the coordinate values ​​of two mutually perpendicular coordinate systems on the imaging surface, such as Figure 3 shown.

[0037] The multi-angle excitation device is used to realize the deflection of needle excitation at different angles, and its deflection surface needs to be placed on the conjugate plane of the front focal plane of the objective lens, that is, the Fourier plane of the rear focal plane of the objective lens. The multi-angle excitation device can be realized by using a two-dimensional galvanometer (including a galvanometer galvanometer and a resonant galvanometer), a micro-electromechanical device (MEMS) and a swing mirror. Its deflection can make the light spot translate on the rear focal plane of the objective lens, thereby realizing the excitation of different U and V angles of the point spread function PSF on the excitation surface. Collect information on excitation at different angles to facilitate the reconstruction of volume imaging data on the image reconstruction module. In this example of the present invention, MEMS can be used to realize the excitation of different U and V angles of the point spread function PSF. Among them, U and V are the coordinate values ​​of two mutually perpendicular coordinate systems on the rear focal plane of the objective lens, and U and V jointly determine the tilt angle of PSF, such as Figure 3 shown.

[0038] The compressed sensing module is used to accelerate the imaging acquisition process. Compressed sensing can utilize the redundancy of information in the time and space dimensions of the sample during the imaging process to achieve a sampling frequency lower than the Nyquist sampling theorem, thereby realizing a fast volume imaging solution. In the example of the present invention, the scenarios in which the compressed sensing algorithm can be used include two parts: (1) When the UV tilt angle is fixed, the selection of the scanning trajectory on the XY imaging plane can achieve fast single-frame imaging by utilizing the redundancy of spatial information and the time-space decoupling algorithm; (2) The determination of different UV tilt angles, how many different UV tilt angles need to be collected, and the determination of the specific values ​​of U and V, and the selection of as few UV tilt angles as possible while ensuring the reconstruction resolution, saving the time of volume imaging.

[0039] The optical illumination device includes a laser, an optical lens, a 4f relay lens, and an objective lens. The laser is used to excite the sample signal, and the optical lens and 4f relay lens are used to adjust the beam diameter, beam collimation, and the conjugate relationship between different focal planes. The objective lens is used for low numerical aperture NA excitation and high numerical aperture NA detection, while ensuring high-resolution imaging in a large field of view. The specific parameter settings and model selection of the embodiments of the present invention can be referred to Figure 1 .

[0040] The signal acquisition device is used to detect volume imaging signals, including single-channel or multi-channel signal acquisition and imaging. This is typically achieved using a photomultiplier tube (PMT). Placing two lenses between the PMT and the objective lens can enhance photon collection efficiency. In this embodiment, four PMTs can be configured to collect the second harmonic generation signal, green / yellow fluorescence signal, red fluorescence signal, and infrared fluorescence signal, respectively.

[0041] The image reconstruction module is used to realize the decoupling reconstruction of the collected coupling signals and reconstruct the volume information through computer algorithms.

[0042] A multi-angle excitation device module and a compressed sensing acquisition module are embedded in a conventional point-scanning multiphoton microscopy system, where S represents the sample. It should be noted that the improved multiphoton imaging system does not fully fill the rear focal plane of the objective lens, resulting in low numerical aperture (NA) excitation. The excitation beam diameter in the embodiments of the present invention is typically 1 / 10 of the full NA. When a high-NA objective lens is used to achieve low-NA excitation, the original point scanning also becomes a needle scanning excitation. The compressed sensing module enables fast and efficient multi-angle excitation and acquisition while ensuring the ability to restore high NA resolution, aiming to achieve a volume imaging rate of 30 Hz. The signal acquisition module typically uses a photomultiplier tube (PMT) to collect light intensity signals. Notably, the present invention allows the simultaneous acquisition of different types of sample signals using one or more PMTs. These different types of signals can be separated by selecting appropriate dichroic mirrors and filters, and collected by the PMTs in their respective channels. Finally, the image reconstruction module produces large-scale, high-resolution, and real-time multiphoton volume imaging results.

[0043] <Optical System>

[0044] Figure 1 The optical system of the compressed sensing multi-photon volume imaging method according to the preferred embodiment of the present invention is schematically shown. The schematic diagram of the optical system device includes a point scanning excitation device, a multi-angle excitation device, an optical illumination module, and a signal acquisition device. The solid line part in the optical path is the excitation optical path: from the femtosecond laser to the sample, and the dotted line part in the optical path is the detection optical path: from the sample to the photomultiplier tube. Of course, Figure 1 The optical system shown can also be used as a stand-alone optical system.

[0045] like Figure 1As shown, the optical system of the compressed sensing multi-photon imaging method according to a preferred embodiment of the present invention includes: a photomultiplier tube 1, a first converging lens 2, a filter 3, a first beam expander 4, a second dichroic mirror 5, a second beam expander 6, an objective lens 7, a first galvanometer 8, a first collimating lens 9, a second galvanometer 10, a second converging lens 11, a second collimating lens 12, a third converging lens 13 and a femtosecond laser 14.

[0046] Among them, the excitation light path in the optical path includes from the femtosecond laser 14 through the third converging lens 13, the second collimating lens 12, the second converging lens 11, the second galvanometer 10, the first collimating lens 9, the first galvanometer 8, the second beam expander 6, the second dichroic mirror 5, the objective lens 7 to the sample S, and the detection light path in the optical path includes from the sample S through the objective lens 7, the second dichroic mirror 5, the first beam expander 4, the filter 3, the first converging lens 2 to the photomultiplier tube 1.

[0047] Among them, the photomultiplier tube 1 is used to detect green and / or yellow fluorescence signals, and the filter 3 is used to filter out photon signals of unnecessary wavelengths. In this solution, only information with a wavelength of 529±20nm is passed, that is, photons in the green / yellow band are collected. It is worth mentioning that the number of photomultiplier tubes can be increased according to demand, and multi-channel imaging information can be collected by selecting appropriate filters and dichroic mirrors. The first converging lens 2 and the first beam expander 4 are used to improve the efficiency of photon collection. The second dichroic mirror 5 is used to filter long-wavelength excitation light and reflect short-wavelength emission light signals to the photomultiplier tube end. The second beam expander 6 has two functions: (1) adjusting the size of the beam diameter to achieve the required low-NA excitation; (2) eliminating aberrations in the scanning light path. It can be composed of a scanning lens and a tube lens. The first galvanometer 8 is a point scanning excitation device, which is used to realize signal excitation in the X and Y directions of a single frame image. The first collimating lens 9 adjusts the light beam to a collimated beam, and the second galvanometer 10 is placed at its front focal plane. The second galvanometer 10 is a multi-angle excitation device that realizes the change of different angles U and V. It is placed at the front focal plane of the first collimating lens 9 and the back focal plane of the second converging lens 11. Since its position is conjugate with the sample plane, its angular deflection can realize the translation of the position of the light beam at the back focal plane of the objective lens, thereby realizing the change of different U and V inclination angles of the excitation PSF. The second converging lens 11 is used to converge the light beam onto the axis of the two-dimensional deflection axis of the second galvanometer 10. It works together with the first collimating lens 9 to adjust the beam diameter. The second collimating lens 12 and the third converging lens 13 can use lenses with the same focal length. A small hole needs to be placed at the focal plane between the two to realize the expansion of the PSF information in frequency, thereby ensuring the successful implementation of the reconstruction algorithm. The femtosecond laser 14 is used to emit femtosecond pulse excitation light that generates a two-photon signal.

[0048] The optional parameters of the femtosecond laser are: average energy 1-2W, repetition rate 80MHz, pulse width 150fs, wavelength 920nm, and laser exit beam diameter 1.2mm. A first set of equal focal length converging lenses and collimating lenses are placed at the laser exit to ensure the parallelism of the exit laser, and a small hole is placed at the focal plane between the two. The second set of converging lenses and collimating lenses scales the beam to 0.432mm. The focal length of this set of lenses is f 会聚 =500mm, f 准直 =180mm. In this embodiment, the second galvanometer 10 is a MEMS two-dimensional galvanometer, using the product A7M8.1 of Mirrorcle Technology, and the scanning speed can reach up to 6kHz. The second galvanometer 10 is located at the conjugate plane of the image plane, and the rotation of its two axes (U axis and V axis) can make the low NA excitation light beam move laterally on the rear focal plane of the objective lens. The function of this galvanometer is to realize multi-angle tilted needle excitation in the sample. The first galvanometer 8 is a resonant galvanometer set, which can achieve frame rate imaging of more than 30Hz. The first galvanometer 8 is located at the conjugate plane of the rear focal plane of the objective lens, and the rotation of its two axes (X axis and Y axis) can make the focus below the objective lens move laterally on the sample, scanning the excitation imaging area. The dichroic mirror uses a long-pass short-reflection filter with a cut-off wavelength of 750nm to separate the excitation light from the emission light. The emission light is collected by the high NA of the objective lens, and then detected by the PMT after passing through the beam expander, the appropriate bandwidth filter and the converging lens to form an image signal.

[0049] The design of the 4f relay lens and objective lens needs to meet the requirements of distortion-free scanning field, large field of view and high resolution.

[0050] <Reconstruction>

[0051] The first acquisition requires deflecting the second galvanometer 10 to move the low NA light beam laterally on the rear focal plane of the objective lens. An image is obtained by moving one position until all positions are superimposed and filled with the high NA rear focal plane of the objective lens. An image stack of all angles of excitation is obtained. The acquired image signal is put into the deconvolution high-resolution reconstruction algorithm for iterative calculation until the volume imaging result of the predetermined resolution (for example, 0.2m resolution) is restored. In the real-time acquisition process, the sparsity of the discrete cosine transform domain of the image microscopic image information is utilized, and the volume imaging result is reconstructed by the compressed sensing algorithm after the compressed acquisition sample to obtain a volume imaging rate above the predetermined frequency (for example, 30Hz).

[0052] It can be seen that the embodiments of the present invention can achieve multiphoton imaging results at 30Hz, with a field of view of >1mm×1mm×100m and a resolution of 0.2m. It is worth noting that low-NA excitation effectively resists sample scattering, achieving a deeper imaging depth than high-NA excitation. In scattering samples, at the same imaging resolution, the solution of the present invention can achieve a deeper maximum imaging depth.

[0053] <Technical Effects>

[0054] The present invention realizes a fast, large-scale and high-resolution multi-photon volume imaging method. It is embodied in the following aspects:

[0055] In terms of achieving volumetric imaging, the present invention breaks away from the limitations of point scanning and single-point excitation, extending the axial range of the optical system's point spread function (PSF), extending it from point scanning to needle scanning. Ordinary point scanning multiphoton microscopes have an axial resolution in the range of 1μm-3μm. The present invention uses a low numerical aperture (NA) excitation method to extend the axial range of the PSF to 100μm and above, from the traditional excitation of a single point to the excitation of all axial content within a depth range of more than 100μm. This achieves the transition from traditional single-frame single-layer images to single-frame image excitation of volumetric information acquisition in a depth range greater than 100μm.

[0056] In terms of achieving high resolution, the present invention uses a high numerical aperture NA (>1.0) objective lens to collect emitted light, and after multi-angle excitation, an algorithm is used to reconstruct and restore high resolution. Using a high numerical aperture NA objective lens for collection can improve collection efficiency. Under the conditions of low numerical aperture NA excitation and high numerical aperture NA collection, the ratio of in-focus signal to out-of-focus background signal can be significantly improved, increasing the imaging depth of the multiphoton microscopy system. By collecting the imaging volume after illumination from different directions through multi-angle excitation and combining it with the PSF deconvolution algorithm, the present invention can not only reconstruct the axial information of different layers of out-of-body imaging, but also restore the imaging system resolution to that under the high numerical aperture NA objective lens.

[0057] In order to realize the large field of view imaging area, the present invention independently designs the excitation and collection multiplexed multi-photon objective lens of the system device. By eliminating the optical aberration and correcting the distortion, the 1×1mm 2 The high lateral resolution of 0.2 μm is maintained under the imaging area.

[0058] In terms of achieving fast volumetric imaging, the present invention utilizes the sparsity of microscopic image information in the discrete cosine transform domain and uses a compressed sensing method to perform multi-angle needle excitation in a target area of ​​1mm×1mm×>100μm. After sparsely collecting multi-photon signals excited by low numerical aperture NA at different angles, the image is reconstructed to obtain 30Hz volumetric imaging capability, achieving resolution preservation and isotropy.

[0059] It should be noted that, unless otherwise specified, the terms "first", "second", "third", etc. in the specification are only used to distinguish the various components, elements, steps, etc. in the specification, and are not used to indicate the logical relationship or sequential relationship between the various components, elements, steps, etc.

[0060] It is understood that although the present invention has been disclosed above with reference to preferred embodiments, the above embodiments are not intended to limit the present invention. For any person skilled in the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or to modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A compressed sensing multi-photon volume imaging device, Features include: Point scanning excitation device, multi-angle excitation device, compressed sensing module, optical lighting device, signal acquisition device and image reconstruction module; Wherein, the point scanning excitation device is used to realize laser scanning excitation on the imaging surface, wherein the excitation end does not completely fill the rear focal plane of the objective lens, and the excitation point spread function at the front focal plane of the objective lens is axially elongated, thereby converting from full numerical aperture point excitation to low numerical aperture needle excitation; The multi-angle excitation device is used to realize the deflection of different angles of needle excitation, wherein the deflection surface of the multi-angle excitation device is on the conjugate surface of the front focal plane of the objective lens, that is, on the Fourier plane of the rear focal plane of the objective lens, and the deflection of the deflection surface causes the light spot to translate on the rear focal plane of the objective lens, thereby realizing the excitation of the point spread function at different angles on the excitation surface; The compressed sensing module is used to accelerate imaging acquisition. It uses the redundancy of information in the time and space dimensions of the sample during the imaging process to achieve a sampling frequency lower than the Nyquist sampling theorem. In the real-time acquisition process, the sparsity of the image microscopic image information in the discrete cosine transform domain is used to reconstruct the volume imaging results through the compressed sensing algorithm after the compressed acquisition samples to obtain a volume imaging rate above the predetermined frequency; The optical illumination device comprises a laser, an optical lens, a 4f relay lens and an objective lens, wherein the laser is used to excite the sample signal, the optical lens and the 4f relay lens are used to adjust the beam diameter, the beam collimation and the conjugate relationship between different focal planes, and the objective lens is used for low numerical aperture excitation and high numerical aperture detection; The signal acquisition device is used to detect body imaging signals, including single-channel or multi-channel signal acquisition and imaging; The image reconstruction module is used to realize the decoupling and reconstruction of the collected coupling signals; The signal acquisition module is realized by a photomultiplier tube; The compressed sensing multi-photon volume imaging device comprises: a photomultiplier tube, a first converging lens, a filter, a first beam expander, a dichroic mirror, a second beam expander, an objective lens, a first galvanometer, a first collimating lens, a second galvanometer, a second converging lens, a second collimating lens, a third converging lens and a femtosecond laser; wherein the excitation light path in the light path comprises a path from the femtosecond laser through the third converging lens, the second collimating lens, the second converging lens, the second galvanometer, the first collimating lens, the first galvanometer, the second beam expander, the second dichroic mirror, the objective lens to the sample, and the detection light path in the light path comprises a path from the sample through the objective lens, the second dichroic mirror, the first beam expander, the filter, the first converging lens to the photomultiplier tube; Moreover, the second dichroic mirror is used to filter the long-wavelength excitation light and reflect the short-wavelength emission light signal to the end of the photomultiplier tube; the second beam expander is used to adjust the size of the light beam diameter to achieve the required low numerical aperture excitation and to eliminate the aberration in the scanning light path; the first galvanometer is a point scanning excitation device; the first collimating lens adjusts the light beam to a collimated light beam, and the second galvanometer is placed at its front focal plane; the second galvanometer is a multi-angle excitation device, which is placed at the front focal plane of the first collimating lens and at the back focal plane of the second converging lens; the second converging lens is used to converge the light beam onto the axis of the two-dimensional deflection axis of the second galvanometer, and the second converging lens and the first collimating lens work together to adjust the beam diameter; the femtosecond laser is used to emit femtosecond pulse excitation light that generates a two-photon signal.

2. The compressed sensing multi-photon volume imaging device according to claim 1, It is characterized in that The photomultiplier tube is used to detect green and / or yellow fluorescence signals, the filter is used to filter photon signals, and the first converging lens and the first beam expander are used to improve the photon collection efficiency.

3. The compressed sensing multi-photon volume imaging device according to claim 1, It is characterized in that The second collimating lens and the third converging lens are lenses with the same focal length, and a small hole needs to be placed at the focal plane between the second collimating lens and the third converging lens to achieve the expansion of the point spread function information in frequency.

4. A compressed sensing multi-photon volume imaging method, Features A compressed sensing multi-photon volume imaging device as described in one of claims 1 to 3 is adopted, comprising: firstly, a galvanometer is deflected to move a low numerical aperture light beam laterally on the rear focal plane of the objective lens, and an image is obtained each time it is moved, until all positions are superimposed and fill the rear focal plane of the high numerical aperture objective lens, thereby obtaining an angle-excited image stack; the collected image signal is put into a deconvolution high-resolution reconstruction algorithm for iterative calculation until a volume imaging result with a predetermined resolution is restored; in the real-time acquisition process, the sparsity of the image microscopic image information in the discrete cosine transform domain is utilized, and after the compressed acquisition samples, the volume imaging result is reconstructed by a compressed sensing algorithm to obtain a volume imaging rate above a predetermined frequency.

Images