Femtosecond laser two-photon living body three-dimensional imaging system based on super-long light needle adaptive depth of field

Abstract

The invention provides a femtosecond laser two-photon living body three-dimensional imaging system based on a super-long light needle adaptive depth of field, and relates to the technical field of two-photon microscopic imaging, the system comprises a femtosecond laser, a spatial light modulator (SLM), a scanning module and a signal acquisition module, wherein the SLM adopts a partition cooperative modulation mechanism of center pixel random phase distribution and peripheral pixel wavefront compensation, and phase distortion correction and scanning path synchronous control are realized by adjusting the pixel proportion of a dynamic balance center area and a compensation area and combining random distribution and a gradient descent optimization algorithm. Through random-deterministic phase collaborative modulation, dynamic depth-of-field matching and light path layered optimization, the limitation of axial resolution and speed of traditional two-photon microscopic imaging is broken through, millisecond-level three-dimensional volume imaging of a living body sample is achieved, and the temporal-spatial resolution detection capacity of the dynamic biological process is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of two-photon microscopy technology, and in particular to an ultra-long light needle adaptive depth-of-field femtosecond laser two-photon in vivo three-dimensional imaging system based on dynamic partitioning coordinated modulation and wavefront compensation of a spatial light modulator (SLM). Background Art

[0002] In the field of two-photon microscopy, existing light needle control technology has significant limitations: the traditional spatial light modulator (SLM) uses a fixed regional phase loading mode, which cannot dynamically adjust the ratio of the core action area to the wavefront compensation area according to the imaging depth, resulting in redundant compensation during shallow imaging and insufficient correction during deep imaging. The existing wavefront compensation algorithm relies on global Zernike polynomial fitting, which makes it difficult to suppress the high-order spatial frequency distortion caused by random phase modulation, and the axial resolution deteriorates sharply with the extension of the depth of field when long light needles are generated. In addition, depth adaptive control requires iterative optimization, and the calculation delay is on the order of hundreds of milliseconds, which cannot meet the needs of real-time imaging of dynamic processes in living organisms.

[0003] The aforementioned technical bottlenecks restrict the application of high-resolution rapid imaging of deep biological samples. Therefore, it is urgent to develop a method to control the axial length of the light needle and adaptively allocate pixels, as well as a method for adaptive depth-controlled pixel partitioning and wavefront compensation based on SLM. Summary of the Invention

[0004] In view of this, in order to solve the above-mentioned technical bottlenecks, the present invention is based on the existing two-photon in vivo imaging system with extended depth of field, and aims to propose a femtosecond laser two-photon in vivo three-dimensional imaging system based on an ultra-long light needle with adaptive depth of field. Through the dynamic phase co-modulation and wavefront compensation technology of the spatial light modulator, it breaks through the axial resolution and speed limitations of traditional imaging, and realizes high-resolution and rapid three-dimensional imaging of deep biological tissues. It aims to achieve high-resolution and high-sensitivity in vivo dynamic optical imaging through the partitioned co-modulation and wavefront compensation of the spatial light modulator, combined with the 4f scanning system, and significantly improve the spatiotemporal resolution detection capability of dynamic biological processes.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Based on the above objectives, in a first aspect, the present invention provides a femtosecond laser two-photon in vivo three-dimensional imaging system based on an ultra-long light needle with adaptive depth of field, comprising the following components:

[0007] Excitation light source module: configured to generate a femtosecond pulse laser beam;

[0008] Optical path adjustment and scanning module: used to guide the light beam path through multi-stage reflectors, and combine with scanning lenses and sleeve lenses to achieve beam collimation and millisecond-level two-dimensional raster scanning;

[0009] Light needle construction and modulation module: used to construct a femtosecond pulsed laser beam into an ultra-long light needle with a length exceeding 100μm and an adaptive depth of field;

[0010] Optical filter module: used to separate the wavelengths of excitation light and fluorescence signals and filter impurity light. Objective lens module: used to focus the modulated laser onto the living sample to excite deep fluorescence signals.

[0011] Optical signal detection and scanning synchronization module: used to convert the fluorescent signal excited by the living sample into an electrical signal and generate a sample image.

[0012] As a further solution of the present invention, the excitation light source module includes a femtosecond laser, a beam expansion and collimating lens group, a half-wave plate and a polarization beam splitter; the beam expansion and collimating lens group includes lens I, lens II, reflector I and reflector II; the femtosecond pulse laser generated by the femtosecond laser is expanded and collimated by lens I and lens II, and then the angle is adjusted by reflector I and reflector II to achieve beam path orientation, and the linear polarization angle of the femtosecond pulse laser is changed by the half-wave plate, and the polarization state of the femtosecond laser pulse is adjusted by the polarization beam splitter to output a beam of vertical polarization state.

[0013] As a further solution of the present invention, the optical path adjustment and scanning module includes a galvanometer mirror, lens III, lens IV, a resonant mirror, a scanning lens and a sleeve lens; the galvanometer mirror, lens III, lens IV and the resonant mirror constitute a 4f system, and the galvanometer mirror serves as the core scanning unit of the 4f system, and is used to perform rapid axial light beam deflection to ensure real-time synchronization between the focal plane of the objective lens and the phase distribution of the spatial light modulator; the lens III and lens IV constitute an optical relay unit of the 4f system, and the lens III serves as a front-focus lens to convert the angular displacement of the galvanometer mirror into spatial displacement, and the lens IV serves as a back-focus lens to refocus the light field to the resonant mirror plane, thereby achieving precise transmission of the light beam wavefront and spatial frequency and eliminating scanning distortion; the resonant mirror serves as the output end of the 4f system, forming an orthogonal scanning axis with the galvanometer mirror, and the resonant frequency of the resonant mirror cooperates with the galvanometer mirror to achieve millisecond-level two-dimensional grating scanning.

[0014] As a further solution of the present invention, the scanning lens and the tube lens cooperate to eliminate field curvature aberration.

[0015] As a further solution of the present invention, the optical needle construction and modulation module includes:

[0016] Reflecting mirror III, used to reflect the femtosecond pulse laser beam emitted by the tube lens to the reflecting prism;

[0017] The reflecting prism is used to compensate for the optical path difference introduced by the SLM by bending the optical path through the total reflection interface to ensure the spatial symmetry of the 4f system;

[0018] The spatial light modulator (SLM) is used to load a dynamic phase mask and generate a central random phase and a peripheral compensation phase according to the N-value partitioning algorithm to achieve adaptive control of the axial light needle length (100μm level).

[0019] As a further solution of the present invention, the light needle construction and modulation module uses a partitioned cooperative modulation mechanism of a spatial light modulator (SLM) (random phase allocation in the central region + peripheral wavefront compensation) to construct the laser into an adaptive ultra-long light needle with a length exceeding 100 μm. The spatial light modulator (SLM) of the light needle construction and modulation module is partitioned as follows:

[0020] Central area: N×N pixels, using pseudo-random phase allocation algorithm to generate N 2 A dynamic focus array;

[0021] Peripheral compensation area: composed of (4N+4) pixels, used to generate a gradient phase related to the refractive index n of the medium through real-time wavefront reconstruction Eliminate the scattering aberration caused by the change in the penetration depth of the light needle, where k is the wave number, α and β are the aberration compensation coefficients, and n is the refractive index of the medium.

[0022] As a further solution of the present invention, the phase modulation of the microscope objective lens in the objective lens module and the wavefront characteristics of the femtosecond laser satisfy the phase distribution of the microscope objective lens following the formula:

[0023]

[0024] Among them, P Ob is the phase of the microscope objective lens, λ is the laser wavelength, f is the focal length of the microscope objective lens, (x, y) is the spatial coordinate, and n is the refractive index of the sample. The axial displacement of the focus can be achieved by adjusting the laser phase.

[0025] As a further solution of the present invention, the axial length of the ultra-long light needle is adaptively adjusted through discrete phase encoding. The phase modulation function of the ultra-long light needle is defined as:

[0026]

[0027] Where, P DOE is the phase modulation encoding function of the ultra-long optical needle, n is the refractive index of the medium, λ is the laser wavelength, f′(x, y) is the discrete focal length parameter, f0 is the reference focal length of the objective lens without phase modulation, πA·p(x, y) is the coefficient for adjusting the beam diameter, where p(x, y) is an N×N pixel matrix;

[0028] Among them, the discrete focal length parameter f′(x,y) satisfies the formula:

[0029] f′(x,y)=f0+[p(x,y)-1]δf

[0030] Where f0 is the reference focal length of the objective lens without phase modulation, δf is the distance between adjacent focal points, the coefficient πA·p(x,y) is used to adjust the beam diameter, p(x,y) is an N×N pixel matrix, and the phase modulation amount P is DOE ∈[0,2π).

[0031] As a further embodiment of the present invention, the total length of the ultra-long light needle is characterized by Mδf, where M is the total number of focal points, and the spacing between adjacent focal points is constrained by the Rayleigh length formula:

[0032]

[0033] Among them, R L is the constraint distance between adjacent focal points of the ultra-long light needle, r is the Gaussian radius of the incident beam, n is the refractive index of the medium, λ is the laser wavelength, and f is the focal length of the microscope objective lens, ensuring that δf≤R L To achieve focal spot energy continuity.

[0034] As a further solution of the present invention, in order to achieve uniform imaging at a large depth, the light intensity of each section of the light needle is similar, and dynamic phase modulation is used to achieve adaptive matching of imaging depth and optimized energy distribution. The steps are as follows:

[0035] Each active area of ​​the spatial light modulator (SLM) is divided into a (N+2)×(N+2) square pixel array, where the number of active area subsets is In a (N+2)×(N+2) square pixel array, the central area is defined as N×N effective pixels, and a randomization algorithm is used to dynamically allocate the phase or amplitude of the light needle;

[0036] By adjusting the value of N, the size of the central area can be controlled, thereby changing the lateral resolution and energy concentration of the light needle; the (4N+4) pixels outside the central area are defined as the wavefront compensation area, which is used to correct the phase distortion caused by the change of the light needle depth; the number of pixels in the compensation area is linearly related to N (4N+4), and the modulation ability increases as N decreases, which can offset the high-order aberrations in deep imaging.

[0037] It should be noted that the imaging depth is negatively correlated with the value of N. When deep structures need to be observed, the value of N is reduced. At this time, the number of pixels in the peripheral compensation area (4N+4) exceeds the number of pixels in the central area N. 2 The compensation weight is significantly improved, and the wavefront distortion caused by deep scattering is offset by phase superposition, extending the effective focal depth; when it is necessary to observe shallow structures, the N value is increased, the proportion of pixels in the central area is increased, and the energy of the light needle is more concentrated in the near-field area, thereby improving the shallow imaging resolution.

[0038] As a further solution of the present invention, the N value of the spatial light modulator (SLM) is related to the target depth parameter n (n∝1 / N), and an adaptive matching relationship between the light needle characteristics (such as focal spot size and energy density) and the imaging depth is established to achieve closed-loop control of "deep-small N, shallow-large N". During deep imaging, N is reduced to enhance the weight of the compensation area, and during shallow imaging, N is increased to increase the energy density of the central area. A pseudo-random phase allocation algorithm is used in the N×N central area to break the diffraction noise caused by traditional periodic modulation and improve the axial uniformity of the light needle.

[0039] As a further embodiment of the present invention, the imaging system further includes a peripheral compensation area phase gradient. Based on the spatial distribution characteristics of the pixels in the compensation area, a gradient phase distribution function is calculated, which is expressed as:

[0040]

[0041] Among them, φ comp (x, y) is the gradient phase distribution function, k is the wave number, α and β are the aberration compensation coefficients, and n is the refractive index of the medium. By adjusting the n value, the phase gradient is dynamically scaled and compensated to adapt to the wavefront correction requirements of different depths.

[0042] As a further solution of the present invention, the optical filtering module includes:

[0043] The dichroic mirror separates the femtosecond excitation light and the sample fluorescence signal based on its wavelength selection characteristics, suppressing the background noise of the excitation light;

[0044] Filters selectively transmit light of a specific wavelength range while blocking or attenuating light of other wavelengths.

[0045] As a further solution of the present invention, the objective lens module includes:

[0046] The microscope objective is used to focus the excitation beam and focus the modulated ultra-long light needle onto the living sample. The numerical aperture of the microscope objective is synergistically optimized with the SLM phase distribution to achieve high two-photon excitation efficiency at great depth.

[0047] As a further solution of the present invention, the living samples are experimental mice purchased from a medical center, and the living mice used in the experiment are subjected to optical imaging experiments under gas anesthesia.

[0048] As a further solution of the present invention, the optical signal detection and scanning synchronization module includes:

[0049] Focusing lens, used to focus the filtered light beam and transmit it to the photomultiplier tube PMT;

[0050] Photomultiplier tube, used to convert the collected fluorescence signal into an electrical signal and transmit it to the signal receiver;

[0051] Fluorescence detection module, this experiment is an electronic computer, and the imaging software that comes with the electronic computer is used to detect fluorescence signals and perform real-time imaging.

[0052] Compared with the existing technology, the femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field proposed in the present invention has the following beneficial effects:

[0053] The femtosecond laser two-photon in vivo three-dimensional imaging system based on the adaptive depth of field of an ultra-long light needle of the present invention solves the depth-resolution contradiction, wavefront compensation redundancy, and lack of real-time performance problems existing in the traditional SLM light needle control method. It is based on the adaptive depth control technology of dynamic pixel partitioning and wavefront collaborative compensation. By establishing an association model of N×N random phase distribution in the central area and the peripheral (4N+4) compensation area, it realizes nonlinear decoupling control of light needle length and wavefront correction. Combining pre-calculation and real-time loading algorithms, it reduces the delay to the millisecond level, while extending the depth of field to 100μm while maintaining submicron axial resolution. The present invention solves the bottlenecks of low compensation efficiency caused by fixed partitioning, the inability of global optimization algorithms to suppress high-order aberrations, and the degradation of axial resolution of long light needles, and provides key technical support for high-temporal and spatial resolution imaging of dynamic biological processes in living organisms.

[0054] These and other aspects of the present application will be more clearly understood in the following description of the embodiments. It should be understood that the above general description and the following detailed description are merely exemplary and explanatory and cannot limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or related technologies, the following briefly introduces the drawings required for the exemplary embodiments or related technical descriptions. The drawings are used to provide a further understanding of the present invention and constitute part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the drawings:

[0056] Figure 1 This is a schematic structural diagram of the femtosecond laser two-photon in vivo three-dimensional imaging system based on an ultra-long light needle and adaptive depth of field disclosed in an embodiment of the present invention.

[0057] Figure 2 This is a structural schematic diagram of one specific implementation of the femtosecond laser two-photon in vivo three-dimensional imaging system based on an ultra-long light needle and adaptive depth of field disclosed in an embodiment of the present invention.

[0058] Figure 3 Schematic diagram of the adaptive depth control pixel partitioning and wavefront compensation method based on SLM of the femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field disclosed in an embodiment of the present invention.

[0059] Reference numerals:

[0060] 100. Excitation light source module; 101. Femtosecond laser; 102. Lens I; 103. Lens II; 104. Reflector I; 105. Reflector II; 106. Half-wave plate; 107. Polarization beam splitter; 200. Optical path adjustment and scanning module; 201. Galvanometer mirror; 202. Lens III; 203. Lens IV; 204. Resonant mirror; 205. Scanning lens; 206. Sleeve lens; 300. Light needle construction and modulation module; 301. Reflector III; 302. Reflecting prism; 303. Spatial light modulator; 400. Optical filtering module; 401. Dichroic mirror; 402. Filter; 500. Objective lens module; 501. Microscope objective lens; 600. Optical signal detection and scanning synchronization module; 601. Focusing lens, 602. Photomultiplier tube; 603. Fluorescence detection module. DETAILED DESCRIPTION

[0061] Below, the present application is further described in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0062] To make the purpose, technical solutions and advantages of the present invention more clearly understood, the following is a further detailed description of the embodiments of the present invention in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0063] It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are intended to distinguish two non-identical entities or non-identical parameters with the same name. Therefore, "first" and "second" are used for convenience of expression only and should not be understood as limitations on the embodiments of the present invention. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, other steps or units inherent to a process, method, system, product, or device that includes a series of steps or units.

[0064] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0065] The flowcharts shown in the accompanying drawings are for illustrative purposes only and do not necessarily include all contents and operations / steps, nor must they be executed in the order described. For example, some operations / steps may be decomposed, combined, or partially merged, so the actual execution order may vary depending on the actual situation.

[0066] The following describes some embodiments of the present application in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features therein may be combined with each other.

[0067] like Figure 2 As shown, an embodiment of the present invention provides a femtosecond laser two-photon in vivo three-dimensional imaging system based on an ultra-long light needle with adaptive depth of field, the imaging system comprising:

[0068] The excitation light source module 100 is used to generate a femtosecond pulse laser beam;

[0069] The optical path adjustment and scanning module 200 is used to guide the light beam path through a multi-stage reflector, and to achieve light beam collimation and millisecond-level two-dimensional raster scanning trajectory control in combination with the scanning lens 205 and the sleeve lens 206;

[0070] The light needle construction and modulation module 300 is used to construct a femtosecond pulse laser into an ultra-long light needle with a length exceeding 100 μm and an adaptive depth of field;

[0071] Light filtering module 400, used to separate the wavelengths of excitation light and fluorescence and filter impurity light;

[0072] Objective lens module 500, used to precisely focus the modulated laser light onto a living sample to stimulate deep-seated fluorescence signals;

[0073] The optical signal detection and scanning synchronization module 600 is used to convert the fluorescence signal excited by the collected living sample into an electrical signal and generate a sample image. The optical signal detection and scanning synchronization module 600 controls the optical path adjustment and scanning module 200 to control the scanning path of the needle beam.

[0074] The femtosecond laser light generated by the excitation light source module 100 is incident on the optical path adjustment and scanning module 200. Three-dimensional spatial encoding is achieved through the composite scanning mechanism of the galvanometer mirror 201 and the resonant mirror 204. Dynamic compensation of optical aberrations is achieved in conjunction with the lens group to establish a high-precision scanning optical path. Under the control of the optical needle construction and modulation 300, the expanded light beam is loaded with a diffraction phase pattern in real time based on a reflective spatial light modulator (SLM) to achieve an ultra-long needle-shaped beam. The beam then passes through the optical filtering module 400 to achieve efficient separation of the excitation light and the fluorescence signal. The excitation light is focused onto the sample through the objective lens module 500 and achieves point scanning. When the ultra-long needle-shaped laser beam is irradiated onto the sample, it achieves two-dimensional scanning excitation of the sample and generates a fluorescence signal. The fluorescence signal generated by the sample forms a reverse-reflected fluorescence signal. The optical signal detection and scanning synchronization module 600 captures the reflected fluorescence signal and converts it into an electrical signal acceptable to a computer. The control module 600 can control the operation of the optical path adjustment and scanning module 200.

[0075] In this embodiment, the femtosecond laser generated by the laser module 100 is incident on the beam modulation module 200, and is modulated into an ultra-long needle-shaped beam by the beam modulation module 200. The ultra-long needle-shaped beam is focused on the sample under the control of the scanning module 300 and realizes point scanning. When the ultra-long needle-shaped beam laser is irradiated on the sample, it realizes two-dimensional scanning excitation of the sample and generates a fluorescence signal. The fluorescence signal generated by the sample forms a reverse reflected fluorescence signal. The signal acquisition module 400 captures the reflected fluorescence signal and converts it into an electrical signal that can be received by a computer. The control module 500 controls the operation of the scanning module 300.

[0076] In this embodiment, the imaging depth of field of the ultra-long needle-shaped light beam is extended by hundreds of times relative to that of Gaussian light; by dynamically adjusting the axial propagation characteristics of the light beam, an ultra-long light needle with adjustable axial length is generated, breaking through the depth of field limitation of traditional optical imaging and achieving high-resolution, high-sensitivity imaging of deep biological tissues such as neuronal networks in living brain regions. This method optimizes the adaptive allocation of pixel density, dynamically balancing resolution and imaging efficiency according to sample characteristics during the scanning process, avoiding image blur caused by axial light intensity attenuation or defocusing, and at the same time utilizing the extension of the axial range of action of the light needle to reduce the laser power density, significantly reducing the risk of light damage to living samples, and providing reliable support for long-term dynamic observation of cell migration or neural activity. Moreover, due to the use of an ultra-long needle-shaped light beam with a large depth of field, volume excitation can be achieved without the need for step-by-step scanning along the longitudinal depth, thereby improving the speed of three-dimensional volume imaging.

[0077] The SLM-based adaptive depth-controlled pixel partitioning and wavefront compensation method disclosed in the present invention enables real-time three-dimensional imaging of living organisms, as well as control and information processing of the brain nervous system, and multi-parameter quantitative characterization and analysis of cerebral blood volume, blood flow, and blood oxygen level-dependent signals. It dynamically controls the wavefront phase distribution of the laser beam and, in real time, corrects wavefront distortion caused by sample refractive index differences, optical component distortion, or environmental interference, thereby achieving high-precision beam focusing and stable propagation in complex imaging scenarios. This method leverages the programmable nature of the SLM and combines it with an adaptive algorithm to perform axial depth partitioning of the imaging area. By loading phase masks specific to each partition, it independently optimizes the beam focusing intensity and pixel density distribution at each depth layer, ensuring consistent imaging resolution and contrast from the surface to deep layers. Furthermore, a multi-stage filtering design utilizing polarization beam splitters, dichroic mirrors, and fluorescence detection modules, combined with noise reduction optimization of the photomultiplier tube and filters, effectively suppresses background scattered light and random noise, improving weak signal detection capabilities. This method provides flexible and stable technical support for high-precision dynamic observation of living organisms, laser micromachining, and complex optical communication scenarios.

[0078] The embodiments of the present invention significantly improve the depth, resolution and adaptability of two-photon in vivo imaging by breaking through the depth of field limitation and dynamic wavefront control of traditional optical imaging. For deep living tissues, the ultra-long light needle technology combined with pixel adaptive allocation can penetrate hundreds of microns to millimeters in depth, achieve high signal-to-noise ratio imaging under low phototoxicity, and solve the problem of light damage and image blur in long-term dynamic observation; and the SLM-based depth partitioning and real-time wavefront compensation technology can maintain cross-depth resolution consistency in transparent samples, organoids or in vivo models by correcting the aberrations caused by refractive index gradients or tissue heterogeneity, which is particularly suitable for high-precision research such as synapse tracking and cell migration analysis. The two work together to further expand the multi-scale imaging capability, and can clearly capture everything from single-cell substructures to organ-level dynamic processes, providing a more reliable real-time imaging tool for disease mechanism analysis, drug screening and precision medicine.

[0079] For details, see Figures 1 to 3As shown, as a specific embodiment of the present invention, in this example, the excitation light source module 100 includes a femtosecond laser 101, a beam expanding and collimating lens group, a half-wave plate 106 and a polarization beam splitter 107; the beam expanding and collimating lens group includes lens I 102, lens II 103, reflector I 104 and reflector II 105; the femtosecond pulse laser generated by the femtosecond laser 101 is expanded and collimated by lens I 102 and lens II 103, and then the angle is adjusted by reflector I 104 and reflector II 105 to realize the orientation of the beam path, and the linear polarization angle of the femtosecond pulse laser is changed by the half-wave plate 106, and the polarization state of the femtosecond laser pulse is adjusted by the polarization beam splitter 107 to output a beam of vertical polarization state. In this embodiment, the pulse width of the femtosecond pulse laser is in the femtosecond range (e.g., 100 fs) and the wavelength is 800-1200 nm. The half-wave plate 106 is used to adjust the linear polarization angle of the femtosecond laser, and the polarization beam splitter 107 is used to split the incident laser into a vertically polarized beam and a horizontally polarized beam. The femtosecond pulse laser output by the femtosecond laser 101 is collimated by lens I 102 and lens I 103 in sequence before being incident on the half-wave plate 106. After adjusting its linear polarization angle, it enters the polarization beam splitter 107. The output vertically polarized beam directly enters the scanning module, while the horizontally polarized beam is dynamically phase modulated by the reflective spatial light modulator 303 to generate an axially extended ultra-long light needle. The linkage between the polarization beam splitter 107 and the half-wave plate 106 can precisely control the power distribution of the output beam.

[0080] The optical path adjustment and scanning module 200 includes a galvanometer mirror 201, a lens III 202, a lens IV 203, a resonant mirror 204, a scanning lens 205 and a sleeve lens 206; the galvanometer mirror 201, the lens III 202, the lens IV 203 and the resonant mirror 204 form a 4f system, and the galvanometer mirror 201 is used as the core scanning unit of the 4f system to perform fast axial beam deflection to ensure that the focal plane of the microscope objective lens 501 and the phase distribution of the spatial light modulator 303 are synchronized in real time; the lens III 202 and the lens IV 20 3 constitutes the optical relay unit of the 4f system. Lens III 202 acts as a front-focus lens, converting the angular displacement of the galvanometer mirror 201 into spatial displacement. Lens IV 203 acts as a back-focus lens, refocusing the light field onto the plane of the resonant mirror 204, achieving precise transmission of the beam wavefront and spatial frequency and eliminating scanning distortion. The resonant mirror 204, serving as the output of the 4f system, forms an orthogonal scanning axis with the galvanometer mirror 201. The resonant frequency of the resonant mirror 204 cooperates with the galvanometer mirror 201 to achieve millisecond-level two-dimensional raster scanning. The scanning lens 205 and the tube lens 206 work together to eliminate field curvature aberrations. The optical path adjustment and scanning module 200 is used to achieve rapid two-dimensional scanning of the laser beam in the X / Y directions. The scanning lens 205 and the tube lens 206 work together to maintain the stability of the spot size within the scanning field of view and correct field curvature aberrations. The microscope objective 501 is used to focus the modulated ultra-long light needle deep into the sample. After passing through the galvanometer mirror 201, lens III 202, resonant mirror 204, scanning lens 205, tube lens 206, and microscope objective 501, the femtosecond laser beam achieves high-precision excitation scanning on the sample plane. The spatial light modulator 303 modulates the wavefront of the horizontally polarized beam by loading a dynamic phase mask. Combined with the light filtering of the reflecting prism 302 and dichroic mirror 401 and the focusing of the objective lens, it generates a focused light needle with adjustable axial length. The photomultiplier tube 601, filter 402, and lens assembly in the fluorescence detection module 603 enable high signal-to-noise ratio two-photon signal acquisition from deep samples.

[0081] As a specific embodiment of the present invention, the optical modulation module, based on the phase control mechanism of femtosecond pulsed lasers, applies a preset phase distribution to the incident light beam via a spatial light modulator 303 or an optical phase plate, causing the laser focal points to form a periodic array along the optical axis Z, thereby constructing an axially extended ultra-long needle-shaped beam. The axial length of the ultra-long needle-shaped beam is dynamically controlled by adjusting the distribution density of the focal array, specifically by increasing or decreasing the number of focal points to correspondingly expand or compress the beam's range.

[0082] Furthermore, a reflector assembly 301 with a dual-reflecting surface structure is provided between the sleeve lens 206 and the microscope objective lens 501 to construct a folded optical path for the laser beam. The reflector assembly first reflects the femtosecond pulse laser emitted by the sleeve lens 206 to the spatial light modulator SLM 303. The phase-modulated laser beam is then reflected twice by the second reflecting surface of the reflector assembly 301 and guided to the microscope objective lens 501, thereby completing the closed-loop modulation and path optimization of the light beam. It should be noted that the total reflector structure between the microscope objective lens 501 and the reflector assembly (104, 105, 301) in the illustrated embodiment is only used for adjusting the direction of the optical path. Its presence or absence does not affect the realization of the core function. In actual application, it can be retained or removed according to the system layout requirements.

[0083] In this embodiment, the optical needle construction and modulation module 300 includes:

[0084] Reflecting mirror III 301 is used to reflect the femtosecond pulse laser beam emitted by the tube lens 206 to the reflecting prism 302;

[0085] The spatial light modulator 303 (SLM) is used to load a dynamic phase mask and generate a central random phase and a peripheral compensation phase according to an N-value partitioning algorithm to achieve adaptive control of the axial light needle length (100 μm level);

[0086] The reflecting prism 302 is used to compensate for the optical path difference introduced by the SLM by bending the optical path through the total reflection interface, thereby ensuring the spatial symmetry of the 4f system.

[0087] The light needle construction and modulation module 300 uses a partitioned cooperative modulation mechanism (random phase allocation in the central region + peripheral wavefront compensation) of the spatial light modulator 303 (SLM) to construct the laser into an adaptive ultra-long light needle with a length exceeding 100 μm. The spatial light modulator 303 (SLM) of the light needle construction and modulation module 300 is partitioned as follows:

[0088] Central area: N×N pixels, using pseudo-random phase allocation algorithm to generate N 2 A dynamic focus array;

[0089] Peripheral compensation area: composed of (4N+4) pixels, used to generate a gradient phase related to the refractive index n of the medium through real-time wavefront reconstruction Eliminate the scattering aberration caused by the change of light needle penetration depth, where φ comp (x,y)k is the wave number, α and β are the aberration compensation coefficients, and n is the refractive index of the medium.

[0090] In a specific embodiment of the present invention, the generation of an ultra-long needle-shaped beam is based on the phase modulation principle of the axial focus sequence. Specifically, a preset wavefront phase distribution is applied to the femtosecond laser pulse by the spatial light modulator 303, and a periodic focus array with a preset interval is generated along the optical axis Z direction. Specifically, the partitioned wavefront control mechanism of the spatial light modulator (SLM) constructs a deep adaptive wavefront control system. The core of the system is to achieve dynamic depth of focus adjustment and aberration correction through the partitioned coordination mechanism of (N+2)×(N+2) pixel blocks. Figure 3 As shown, each In the modular control unit, the central N×N pixel uses a pseudo-random phase allocation algorithm to generate N 2 A dynamic focus array breaks the diffraction limit to form an axially extended light needle structure, and the compensation area composed of the peripheral (4N+4) pixels generates a gradient phase related to the depth parameter n through real-time wavefront reconstruction. Effectively eliminate the scattering aberration caused by the change of light needle penetration depth. The focal spot array f1-f9 on the right side intuitively shows the axial layered imaging effect under different N value control: when the N value is reduced, the pixel ratio of the compensation area increases (4N+4>N 2 ), by enhancing the phase superposition effect to suppress deep high-order aberrations and extend the effective focal depth to the micron level; when the N value is increased, the high-density pixels in the central area focus on the near-field area, which improves the lateral resolution of the light needle to the sub-micron level. The parallel operation of three independent control modules realizes the dynamic matching of light needle energy density, focal spot size and target depth, achieving high temporal and spatial resolution in vivo microscopic imaging while maintaining axial uniformity, overcoming the technical bottleneck of the mutual restriction between focal depth and resolution in traditional methods.

[0091] In this embodiment, the phase modulation of the microscope objective lens 501 in the objective lens module 500 and the wavefront characteristics of the femtosecond laser satisfy the phase distribution of the microscope objective lens 501 following the formula:

[0092]

[0093] Among them, P OB is the phase of the microscope objective lens 501, λ is the laser wavelength, f is the focal length of the microscope objective lens 501, (x, y) is the spatial coordinate, and n is the refractive index of the sample. The axial displacement of the focus can be achieved by adjusting the laser phase.

[0094] The axial length of the ultra-long light needle is adaptively adjusted through discrete phase encoding. The phase modulation function of the ultra-long light needle is defined as:

[0095]

[0096] Where, P DOEis the phase modulation encoding function of the ultra-long optical needle, n is the refractive index of the medium, λ is the laser wavelength, f′(x, y) is the discrete focal length parameter, f0 is the reference focal length of the objective lens without phase modulation, πA·p(x, y) is the coefficient for adjusting the beam diameter, where is an N×N pixel matrix;

[0097] Among them, the discrete focal length parameter f′(x,y) satisfies the formula:

[0098] f′(x,y)=f0+[p(x,y)-1]δf

[0099] Where f0 is the reference focal length of the microscope objective lens 501 without phase modulation, δf is the distance between adjacent focal points, the coefficient πA·p(x,y) is used to adjust the beam diameter, p(x,y) is an N×N pixel matrix, and the phase modulation amount P is DOE ∈[0,2π).

[0100] The total length of the ultra-long light needle is characterized by Mδf, where M is the total number of focal points, and the spacing between adjacent focal points is constrained by the Rayleigh length formula:

[0101]

[0102] Among them, R L is the constraint distance between adjacent focal points of the ultra-long light needle, r is the Gaussian radius of the incident light beam, n is the refractive index of the medium, λ is the laser wavelength, and f is the focal length of the microscope objective lens 501, ensuring that δf≤R L To achieve focal spot energy continuity.

[0103] During this process, the numerical aperture of microscope objective 501 and the wavefront phase distribution of the incident light beam must satisfy the Abbe sine condition. This means that by dynamically adjusting the phase parameters of the femtosecond laser pulse, the beam propagation characteristics at the focal plane of microscope objective 501 are precisely controlled, ensuring a strict correspondence between each focal position and the amount of phase modulation. This phase modulation can be achieved by loading a discrete phase mask or a continuous phase gradient distribution, ultimately forming an axially extended, ultra-long needle-shaped beam in the back focal region of the objective lens. The Abbe sine condition is as follows:

[0104]

[0105] Among them, P OB is the phase of the microscope objective lens 501, λ is the laser wavelength, f is the focal length of the microscope objective lens 501, (x, y) is the spatial coordinate, and n is the refractive index of the sample. The axial displacement of the focus can be achieved by adjusting the laser phase.

[0106] Furthermore, the axial length of the ultra-long light needle is adaptively adjusted through discrete phase encoding, and its phase modulation function is defined by formula (2):

[0107]

[0108] The discrete focal length parameter f′(x,y) satisfies formula (3):

[0109] f′(x,y)=f0+[p(x,y)-1]δf#(3)

[0110] Where f0 is the reference focal length of the microscope objective lens 501 without phase modulation, δf is the distance between adjacent focal points, M is the total number of focal points, the coefficient πA·p(x,y) is used to adjust the beam diameter, p(x,y) is an N×N pixel matrix, and the phase modulation amount P is DOE ∈[0,2π).

[0111] The total length of the light needle is characterized by Mδf, and the distance between adjacent focal points is constrained by the Rayleigh length formula

[0112]

[0113] Where r is the Gaussian radius of the incident beam, ensuring that δf ≤ R L To achieve focal spot energy continuity.

[0114] In order to achieve uniform imaging at large depths, the light intensity of each section of the light needle is similar. Dynamic phase modulation is used to achieve adaptive matching of imaging depth and optimized energy distribution. The steps are as follows:

[0115] Each active area of ​​the spatial light modulator 303 (SLM) is divided into a square pixel array of (N+2)×(N+2), where the number of active area subsets is In a (N+2)×(N+2) square pixel array, the central area is defined as N×N effective pixels, and a randomization algorithm is used to dynamically allocate the phase or amplitude of the light needle;

[0116] By adjusting the value of N, the size of the central area can be controlled, thereby changing the lateral resolution and energy concentration of the light needle; the (4N+4) pixels outside the central area are defined as the wavefront compensation area, which is used to correct the phase distortion caused by the change of the light needle depth; the number of pixels in the compensation area is linearly related to N (4N+4), and the modulation ability increases as N decreases, which can offset the high-order aberrations in deep imaging.

[0117] It should be noted that the imaging depth is negatively correlated with the value of N. When deep structures need to be observed, the value of N is reduced. At this time, the number of pixels in the peripheral compensation area (4N+4) exceeds the number of pixels in the central area N. 2 The compensation weight is significantly improved, and the wavefront distortion caused by deep scattering is offset by phase superposition, extending the effective focal depth; when it is necessary to observe shallow structures, the N value is increased, the proportion of pixels in the central area is increased, and the energy of the light needle is more concentrated in the near-field area, thereby improving the shallow imaging resolution.

[0118] In this embodiment, the N value of the spatial light modulator 303 (SLM) is related to the target depth parameter n (n∝1 / N), and an adaptive matching relationship between the light needle characteristics (such as focal spot size and energy density) and the imaging depth is established to achieve a closed-loop control of "deep-small N, shallow-large N". When deep imaging is performed, N is reduced to enhance the weight of the compensation area, and when shallow imaging is performed, N is increased to increase the energy density of the central area. A pseudo-random phase allocation algorithm is used in the N×N central area to break the diffraction noise caused by traditional periodic modulation and improve the axial uniformity of the light needle.

[0119] In some embodiments, the imaging system further includes a peripheral compensation region phase gradient, and based on the spatial distribution characteristics of the pixels in the compensation region, a gradient phase distribution function is calculated, which is expressed as:

[0120]

[0121] Among them, φ comp (x, y) is the gradient phase distribution function, k is the wave number, α and β are the aberration compensation coefficients, and n is the refractive index of the medium. By adjusting the n value, the phase gradient is dynamically scaled and compensated to adapt to the wavefront correction requirements of different depths.

[0122] In this embodiment, the optical filtering module 400 includes:

[0123] The dichroic mirror 401 separates the femtosecond excitation light and the sample fluorescence signal based on its wavelength selection characteristics, thereby suppressing the background noise of the excitation light;

[0124] The filter 402 selectively transmits light within a specific wavelength range while blocking or attenuating light of other wavelengths.

[0125] In this embodiment, the objective lens module 500 includes:

[0126] The microscope objective lens 501 is used to focus the excitation beam and focus the modulated ultra-long light needle onto the living sample. The numerical aperture of the microscope objective lens 501 is synergistically optimized with the SLM phase distribution to achieve high-depth two-photon excitation efficiency.

[0127] The living samples were experimental mice purchased from a medical center, and the living mice used in the experiment were subjected to optical imaging experiments under gas anesthesia.

[0128] In this embodiment, the optical signal detection and scanning synchronization module 600 includes:

[0129] Focusing lens 601, used to focus the filtered light beam and transmit it to the photomultiplier tube 602PMT;

[0130] The photomultiplier tube 602 is used to convert the collected fluorescence signal into an electrical signal and transmit it to a signal receiver;

[0131] Fluorescence detection module 603, this experiment is an electronic computer, and the imaging software provided by the electronic computer is used to detect the fluorescence signal and perform real-time imaging.

[0132] As a specific embodiment of the present invention, the optical filtering module and optical signal detection and scanning synchronization module include a dichroic mirror 401, a filter 402, a focusing lens 601, and a photomultiplier tube 602. The dichroic mirror 401 is configured to separate the incident excitation light from the sample fluorescence signal. Specifically, the excitation light beam is transmitted through the dichroic mirror 401 and incident on the microscope objective 501. The fluorescence excited by the sample is reflected by the dichroic mirror 401, spectrally filtered by the filter 402, and then focused by the focusing lens 601 onto the photosensitive target surface of the photomultiplier tube 602. Ultimately, the fluorescence photons are converted into electrical signals and transmitted to the computer terminal 603 for data reconstruction.

[0133] The control module 600 is composed of a multifunctional data acquisition card and a synchronous controller, wherein the input end of the data acquisition card is connected to the analog signal output end of the photomultiplier tube 602, and the output end of the synchronous controller drives the resonant-galvanometer scanning mirror 205 in the scanning module 200 to perform X / Y axial scanning motion. Furthermore, the synchronous controller implements the timing synchronization control of signal output and acquisition through an integrated hardware programming platform (such as Labview or Matlab), ensuring that the scanning positioning accuracy matches the signal acquisition rate. It should be noted that the controller controls the axial displacement of the scanning lens 205, the sleeve lens 206, the reflector group 301 and the microscope objective lens 501, and adopts the synchronous control method in the prior art to achieve dynamic coordination with the propagation of the excitation beam. Its specific implementation method is not repeated here.

[0134] In summary, the femtosecond laser two-photon in vivo three-dimensional imaging system based on the ultra-long light needle adaptive depth of field of the present invention solves the depth-resolution contradiction, wavefront compensation redundancy and lack of real-time performance problems existing in the traditional SLM light needle control method. It is based on the adaptive depth control technology of dynamic pixel partitioning and wavefront collaborative compensation. By establishing an association model of N×N random phase distribution in the central area and the peripheral (4N+4) compensation area, it realizes nonlinear decoupling control of light needle length and wavefront correction. Combining pre-calculation and real-time loading algorithms, it reduces the delay to millisecond level, and maintains submicron axial resolution while extending the depth of field to 100μm. The present invention solves the bottlenecks of low compensation efficiency caused by fixed partitioning, the inability of global optimization algorithms to suppress high-order aberrations and the degradation of axial resolution of long light needles, and provides key technical support for high-temporal and spatial resolution imaging of dynamic biological processes in living organisms.

[0135] The above are exemplary embodiments disclosed in the present invention, but it should be noted that various changes and modifications may be made without departing from the scope of the embodiments disclosed in the claims. The functions, steps and / or actions of the method claims according to the disclosed embodiments described herein do not need to be performed in any particular order. In addition, although the elements disclosed in the embodiments of the present invention may be described or required in individual form, they may also be understood as multiple unless expressly limited to the singular.

[0136] It should be understood that, as used herein, the singular form "a" or "an" is intended to include the plural form as well, unless the context clearly supports an exception. It should also be understood that, as used herein, "and / or" refers to any and all possible combinations of one or more of the items listed in association. The serial numbers of the embodiments disclosed in the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0137] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the disclosure of the embodiments of the present invention (including the claims) is limited to these examples. Within the spirit of the embodiments of the present invention, the technical features of the above embodiments or different embodiments may be combined, and there are many other variations of different aspects of the above embodiments of the present invention, which are not provided in detail for the sake of simplicity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present invention should be included in the scope of protection of the embodiments of the present invention.

Claims

1. A femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field, characterized by: Includes the following components: Excitation light source module: configured to generate a femtosecond pulse laser beam; Optical path adjustment and scanning module: used to guide the light beam path through multi-stage reflectors, and combine with scanning lenses and sleeve lenses to achieve beam collimation and millisecond-level two-dimensional raster scanning; Light needle construction and modulation module: used to construct a femtosecond pulsed laser beam into an ultra-long light needle with a length exceeding 100μm and an adaptive depth of field; Optical filter module: used to separate the wavelengths of excitation light and fluorescence signal and filter impurity light Objective lens module: used to focus the modulated laser onto the living sample to stimulate deep fluorescence signals; Optical signal detection and scanning synchronization module: used to convert the fluorescent signal excited by the living sample into an electrical signal and generate a sample image.

2. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field according to claim 1, characterized in that: The excitation light source module includes a femtosecond laser, a beam expansion and collimating lens group, a half-wave plate and a polarization beam splitter; the beam expansion and collimating lens group includes lens I, lens II, reflector I and reflector II; the femtosecond pulse laser generated by the femtosecond laser is expanded and collimated by lenses I and II, and then the angle is adjusted by reflectors I and II to achieve beam path orientation, and the linear polarization angle of the femtosecond pulse laser is changed by the half-wave plate, and the polarization state of the femtosecond laser pulse is adjusted by the polarization beam splitter to output a beam with a vertical polarization state.

3. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field according to claim 2, characterized in that: The optical path adjustment and scanning module includes a galvanometer mirror, lens III, lens IV, a resonant mirror, a scanning lens and a sleeve lens; the galvanometer mirror, lens III, lens IV and the resonant mirror constitute a 4f system, and the galvanometer mirror serves as the core scanning unit of the 4f system, and is used to perform rapid axial light beam deflection to ensure real-time synchronization between the focal plane of the objective lens and the phase distribution of the spatial light modulator; the lenses III and IV constitute the optical relay unit of the 4f system, and the lens III serves as a front-focus lens to convert the angular displacement of the galvanometer mirror into spatial displacement, and the lens IV serves as a back-focus lens to refocus the light field onto the resonant mirror plane, thereby achieving precise transmission of the light beam wavefront and spatial frequency and eliminating scanning distortion; the resonant mirror serves as the output end of the 4f system and forms an orthogonal scanning axis with the galvanometer mirror. The resonant frequency of the resonant mirror cooperates with the galvanometer mirror to achieve millisecond-level two-dimensional grating scanning.

4. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long optical needle adaptive depth of field according to claim 3, characterized in that: The scanning lens and the tube lens cooperate to eliminate field curvature aberration.

5. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field according to claim 3, characterized in that: The optical needle construction and modulation module includes: Reflecting mirror III, used to reflect the femtosecond pulse laser beam emitted by the tube lens to the reflecting prism; The reflecting prism is used to compensate for the optical path difference introduced by the spatial light modulator by bending the optical path through the total reflection interface to ensure the spatial symmetry of the 4f system; The spatial light modulator is used to load a dynamic phase mask and generate a central random phase and a peripheral compensation phase according to the N-value partitioning algorithm to achieve adaptive control of the axial light needle length.

6. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long optical needle adaptive depth of field according to claim 5, characterized in that: The light needle construction and modulation module uses a partitioned cooperative modulation mechanism of a spatial light modulator to construct the laser into an adaptive ultra-long light needle with a length exceeding 100 μm. The spatial light modulator of the light needle construction and modulation module is partitioned as follows: Central area: N×N pixels, using pseudo-random phase allocation algorithm to generate N 2 A dynamic focus array; Peripheral compensation area: composed of 4N+4 pixels, used to generate a gradient phase related to the refractive index n of the medium through real-time wavefront reconstruction Eliminate the scattering aberration caused by the change in the penetration depth of the light needle, where k is the wave number, α and β are the aberration compensation coefficients, and n is the refractive index of the medium.

7. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field according to claim 6, characterized in that: The phase modulation of the microscope objective lens in the objective lens module and the wavefront characteristics of the femtosecond laser satisfy the phase distribution of the microscope objective lens following the formula: Among them, P OB is the phase of the microscope objective lens, λ is the laser wavelength, f is the focal length of the microscope objective lens, (x, y) is the spatial coordinate, and n is the refractive index of the sample. The axial displacement of the focus can be achieved by adjusting the laser phase.

8. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long optical needle adaptive depth of field according to claim 7, characterized in that: The axial length of the ultra-long light needle is adaptively adjusted through discrete phase encoding. The phase modulation function of the ultra-long light needle is defined as: Where, P DOE is the phase modulation encoding function of the ultra-long optical needle, n is the refractive index of the medium, λ is the laser wavelength, and f ′ (x, y) is the discrete focal length parameter, f0 is the reference focal length of the objective lens without phase modulation, πA·p(x, y) is the coefficient for adjusting the beam diameter, where is an N×N pixel matrix; Among them, the discrete focal length parameter f ′ (x,y) satisfies the formula: f′(x,y)=f0+[p(x,y)-1]δf Where f0 is the reference focal length of the objective lens without phase modulation, δf is the distance between adjacent focal points, the coefficient πA·p(x,y) is used to adjust the beam diameter, p(x,y) is an N×N pixel matrix, and the phase modulation amount P is DOE ∈[0,2π).

9. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long optical needle adaptive depth of field according to claim 8, characterized in that: The total length of the ultra-long light needle is characterized by Mδf, where M is the total number of focal points, and the spacing between adjacent focal points is constrained by the Rayleigh length formula: Among them, R L is the constraint distance between adjacent focal points of the ultra-long light needle, r is the Gaussian radius of the incident beam, n is the refractive index of the medium, λ is the laser wavelength, and f is the focal length of the microscope objective lens, ensuring that δf≤R L To achieve focal spot energy continuity.

10. The femtosecond laser two-photon in vivo three-dimensional imaging system based on ultra-long light needle adaptive depth of field according to claim 1, characterized in that: The optical filtering module includes: The dichroic mirror separates the femtosecond excitation light and the sample fluorescence signal based on its wavelength selection characteristics, suppressing the background noise of the excitation light; Filters selectively transmit light of a specific wavelength range while blocking or attenuating light of other wavelengths.

Images