Double-light-needle intensity gradient regulation and depth mapping two-photon in-vivo imaging system

Abstract

The invention provides a double-light-needle intensity gradient regulation and depth mapping two-photon living body imaging system, which relates to the technical field of biomedical optical imaging and comprises a pumping module, a light modulation module, a light scanning module, a signal acquisition module and a signal detection and control module, wherein the pumping module generates ultrashort pulse laser with a tunable wavelength, the light modulation module encodes laser wavefront through a spatial light phase modulator and modulates an incident collimated laser beam into an ultralong needle-shaped light beam, the light scanning module comprises a high-precision galvanometer and an objective lens group, and through a double-light-needle reverse intensity gradient design, the wavelength of the ultrashort pulse laser is tunable. A calibration model and a synchronous triggering technology are combined, and the axial resolution limit of a traditional light needle is broken through; a spatial light modulator is used for carrying out two-time differential phase modulation on excitation light wavefront, and light needles with opposite axial intensity gradients are respectively generated, namely, the light intensity in the depth direction is gradually increased for the first time; and the light intensity in the depth direction is gradually reduced for the second time, so that the specific imaging depth of the large-depth-of-field optical needle is analyzed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical optical imaging technology, and in particular to a method for achieving Z-axis depth information analysis in large-depth-of-field two-photon in vivo imaging by dual-light needle intensity modulation, which is particularly suitable for three-dimensional high-resolution imaging of cerebral cortical vascular networks. Background Art

[0002] Traditional two-photon microscopy systems typically employ a long-axis light needle scanning strategy to extend the imaging depth of field. However, due to the axial extension of the light needle, its Z-axis resolution is significantly degraded, resulting in a loss of depth information when projecting a three-dimensional vascular network into a two-dimensional image. Existing depth resolution methods based on intensity deconvolution suffer from the lack of a quantitative mapping between the axial intensity distribution of the light needle and depth, and the interference of calibration accuracy with scattering noise from living tissue. A single light intensity distribution cannot distinguish depth differences between overlapping vessels, and scattering noise from living tissue can interfere with calibration accuracy. Summary of the Invention

[0003] In view of this, in order to solve the above-mentioned technical bottlenecks, the purpose of the present invention is to propose a two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping. Through the design of dual light needle reverse intensity gradient, combined with calibration model and synchronous triggering technology, the axial resolution limit of traditional light needles is broken through; the excitation light wavefront is differentially phase modulated twice by a spatial light modulator (SLM) to generate light needles with opposite axial intensity gradients, that is, the light intensity increases in the depth direction for the first time; and the light intensity decreases in the depth direction for the second time, aiming to achieve the resolution of the specific imaging depth of light needles with a large depth of field.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] Based on the above objectives, in a first aspect, the present invention provides a two-photon in vivo imaging system with dual-light needle intensity gradient control and depth mapping, comprising the following components:

[0006] The pump module is used to generate ultrashort pulse lasers with tunable wavelengths and output femtosecond near-infrared laser beams as two-photon excitation light sources;

[0007] The optical modulation module is used to encode the laser wavefront through a spatial light phase modulator (SLM), modulate the incident collimated laser beam into an ultra-long needle-shaped beam with an axial length exceeding 100 μm, and optimize the lateral intensity distribution of the beam to extend the imaging depth of field;

[0008] The optical scanning module, which includes a high-precision galvanometer and objective lens assembly, is used to focus the ultra-long needle-shaped beam onto the surface and deep areas of biological samples, and achieve rapid point-by-point excitation of the sample plane through two-dimensional scanning;

[0009] The signal acquisition module integrates a photomultiplier tube and a lock-in amplifier to collect the fluorescence signal of the sample excited by the laser beam in real time, and reconstructs a two-dimensional projection image of the sample with a high signal-to-noise ratio after photoelectric conversion and noise filtering;

[0010] The signal detection and control module has a built-in synchronous trigger circuit and motion control unit, which coordinates the galvanometer deflection timing of the scanning module, the pulse emission period of the laser module and the data sampling rate of the signal acquisition module. It detects the fluorescence signal through the electronic computer and collector and controls the operation of the scanning module to control the scanning path of the needle beam, ensuring the temporal and spatial consistency of the needle beam scanning path and the fluorescence signal acquisition.

[0011] As a further solution of the present invention, the pump module includes:

[0012] Pump laser, used to generate ultrashort pulse laser with adjustable wavelength, outputting femtosecond near-infrared laser beam as two-photon excitation light source;

[0013] Lens 1 and lens 2 are used to compress and collimate the original laser (femtosecond laser pulse) to output a parallelized laser beam.

[0014] Half-wave plate (HWP), used to adjust the polarization direction of the incident laser and change the linear polarization angle of the femtosecond pulse laser;

[0015] The polarization beam splitter (PBS) is used to adjust the polarization direction of the incident laser and screen the vertically polarized beam to adapt to the working mode of the subsequent modulation device. The femtosecond pulse laser generated by the pump laser is incident on the half-wave plate through lens 1 and lens 2, and then changes its own linear polarization angle. It is then incident on the polarization beam splitter and outputs a vertically polarized beam.

[0016] As a further solution of the present invention, the optical scanning module includes:

[0017] The galvanometric mirror and the resonant mirror form a resonant-galvanometric composite scanning galvanometer, which drives the ultra-long needle-shaped beam to perform high-frequency two-dimensional scanning in the fast and slow axis directions, realizing point-by-point excitation of the sample plane. The galvanometric mirror realizes linear scanning in the slow axis direction, and the resonant mirror drives high-frequency oscillation in the fast axis direction.

[0018] The scanning galvanometer (Scan Lens) and tube lens (Tube Lens) form a scanning galvanometer group. The scanning galvanometer group and the resonance-galvanometry composite scanning galvanometer form a conjugate imaging system, which converts the beam deflection into the focus point displacement of the sample plane, completing high-speed, large-scale two-dimensional point-by-point scanning, ensuring a constant spot size within the scanning field of view and corrected aberrations;

[0019] The microscope objective is used to focus the excitation beam, focusing the modulated ultra-long needle-shaped beam onto the biological sample, using the two-photon excitation effect to generate nonlinear fluorescence radiation inside the sample, and combined with the reflective prism to achieve light path retracement transmission, ensuring the consistency of excitation efficiency within the long focal depth range.

[0020] As a further solution of the present invention, a multi-reflective surface optical folding component is further provided between the sleeve lens and the microscope objective lens. The multi-reflective surface optical folding component includes at least two reflective surfaces to form a reflective light path. The optical folding component guides the femtosecond pulse laser emitted from the sleeve lens to the spatial light phase modulator for wavefront phase reconstruction. After the modulated laser is transmitted in reverse through the original light path, it is redirected to the microscope objective lens for a second time by another reflective surface of the optical folding component. The laser beam propagation path is guided by the double reflective surfaces to form a closed-loop light path between the SLM and the objective lens, thereby completing the secondary phase modulation.

[0021] As a further solution of the present invention, the phase distribution of the microscope objective lens and the parameters of the femtosecond laser satisfy the following phase matching relationship:

[0022]

[0023] Where, P OB is the phase of the microscope objective lens, λ is the laser wavelength of the femtosecond pulse laser, f is the objective lens focal length of the microscope objective lens, (x, y) are the plane coordinates of the back focal plane of the objective lens, and n is the refractive index of the imaging sample. The spatial light phase modulator can achieve controlled displacement of the needle-shaped beam focus along the optical axis by encoding the phase wavefront and changing the phase of the femtosecond laser pulse through the optical modulation module.

[0024] As a further solution of the present invention, the axial length of the ultra-long needle-shaped beam is realized by programmable control of the number of focal points M, and the discrete phase encoding P is loaded by the SLM. DOM (x,y), the phase modulation function for controlling the axial focus distribution of the ultra-long needle beam satisfies:

[0025]

[0026] f′(x,y)=f0+[p(x,y)-1]δf

[0027]

[0028] Where f0 is the initial focal length of the objective lens, δf is the distance between adjacent focal points, p(x,y) is the N×N phase distribution matrix, and the additional term πA·p(x,y) is used to optimize the lateral distribution of the beam and satisfy the phase range P DOM (x,y)∈(0,2π), total length L=Mδf, adjacent focus interval δf≤R L (Rayleigh length) to ensure consistency in excitation efficiency.

[0029] As a further embodiment of the present invention, the total length of the ultra-long needle-shaped beam is Mδf, where r is the Gaussian radius of the incident light, and the interval length δf between two adjacent focal spots does not exceed one Rayleigh length RL. The constraint condition for the focal spacing δf is:

[0030]

[0031] Among them, R L is the Rayleigh length, r is the radius of the fundamental mode of the incident light, and this constraint ensures the effective energy superposition of adjacent foci.

[0032] As a further solution of the present invention, the signal acquisition module includes a spectroscopic component, a spectral filtering component and a photoelectric conversion component. The spectroscopic component is a dichroic mirror, which is used to separate the excitation light and the sample fluorescence, reflect the excitation light to the objective lens, and transmit the fluorescence to the detection link; the spectral filtering component is a filter, which is used to remove stray light and residual excitation light and improve the signal-to-noise ratio; the photoelectric conversion component includes a focusing lens and a photomultiplier tube, and the focusing lens and the photomultiplier tube (PMT) are used to focus the fluorescence to the photosensitive surface of the photomultiplier tube (PMT), generate a time-series electrical signal through photoelectric conversion, and reconstruct the sample image through phase-locked amplification and analog-to-digital conversion.

[0033] As a further solution of the present invention, fluorescent beads with a size smaller than the Rayleigh length of the light needle are used as calibration objects. Their axial position is accurately scanned by a Z-axis electric translation stage, and the mapping relationship between the fluorescence intensity ratio under two light intensity distributions and the known depth is recorded to construct a fluorescence calibration model.

[0034] As a further solution of the present invention, dual-light needle scanning is performed on the living imaging target (such as the cerebral cortical blood vessels), and the absolute depth value is inverted by matching the intensity ratio of the pixels at the same position with the calibration curve to achieve an improvement in the Z-axis resolution.

[0035] As a further solution of the present invention, the light needle intensity of the two-photon in vivo imaging system can be flexibly controlled by phase encoding to perform light needle intensity gradient design: the first SLM modulation makes the light needle axial intensity meet the following requirements:

[0036] I1(z)=I0exp(-(z-z0) / L)

[0037] The second modulation generates a reverse gradient that satisfies:

[0038] I2(z)=I0exp(-(z-z0) / L)

[0039] During the calibration process, the non-light needle mode (point scanning) is used to obtain the three-dimensional distribution of fluorescent beads as a benchmark to eliminate the influence of the light needle's own intensity distribution on the calibration;

[0040] The fluorescent bead-agar model was fixed on the translation stage, and its Z-axis coordinate was obtained in point scanning mode. Then, the optical needle mode was switched to record the intensity values I1 and I2 of each fluorescent bead under the two SLM modulations, and the intensity values I1 and I2 of each fluorescent bead were calculated. A linear fitting curve with a known depth was obtained; a dual-light needle scan was performed on a living sample (such as the cranial window area of anesthetized mice) to simultaneously collect I1 and I2 images of the vascular network; pixel-by-pixel calculation was performed The value is substituted into the calibration curve to analyze the absolute depth and reconstruct the three-dimensional vascular topology; the two optical needle scans of the living sample are triggered in time synchronization to eliminate the pixel misalignment error caused by blood flow movement.

[0041] Compared with existing technologies, the two-photon in vivo imaging system with dual-light needle intensity gradient control and depth mapping proposed in this invention has the following beneficial effects by constructing a three-dimensional dynamic imaging module for living samples, a neural circuit control and information processing module, and a multi-parameter simultaneous quantitative analysis module:

[0042] The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping of the present invention breaks through the Z-axis resolution limit of traditional light needles through the combination of dual-gradient light needle scanning and fluorescence calibration; establishes a nonlinear mapping model of intensity ratio-depth, significantly improving the noise resistance in the living environment; and is compatible with existing two-photon microscopy systems, and can realize depth mapping function by simply adding an SLM module and calibration protocol.

[0043] 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

[0044] 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:

[0045] Figure 1 The invention discloses a two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping.

[0046] Figure 2 This is a structural diagram of a specific implementation of the two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping disclosed in an embodiment of the present invention.

[0047] Reference numerals:

[0048] 100. Pump module; 101. Pump laser; 200. Optical modulation module; 201. Lens 1; 202. Lens 2; 203. Half-wave plate; 204. Polarization beam splitter; 205. Spatial light modulator; 206. Reflection prism; 300. Optical scanning module; 301. Galvanometer galvanometer; 302. Resonant mirror; 303. Scanning galvanometer; 304. Sleeve lens; 400. Signal acquisition module; 401. Microscope objective; 500. Signal detection and control module; 501. Binary mirror; 502. Focusing lens; 503. Filter; 504. Photomultiplier tube; 505. Signal detection and control terminal. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] like Figure 1 and Figure 2 As shown, an embodiment of the present invention provides a two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping, the imaging system comprising:

[0056] The pump module 100 is used to generate ultrashort pulse laser with tunable wavelength and output a femtosecond near-infrared laser beam as a two-photon excitation light source, providing a reference light source for the construction of the optical needle.

[0057] The optical modulation module 200 is used to encode the laser wavefront through a spatial light phase modulator (SLM), modulate the incident collimated laser beam into an ultra-long needle-shaped beam with an axial length exceeding 100 μm, and optimize the lateral light intensity distribution of the beam to extend the imaging depth of field. The optical modulation module 200 collimates and expands the light beam, changes the power and modulates the laser into a long light needle beam with a length exceeding 100 μm.

[0058] The optical scanning module 300 includes a high-precision galvanometer and an objective lens group, which is used to focus the ultra-long needle-shaped light beam onto the surface and deep areas of the biological sample, and achieve rapid point-by-point excitation of the sample plane through two-dimensional scanning.

[0059] The signal acquisition module 400 integrates a photomultiplier tube 504 and a lock-in amplifier, and is used to collect the fluorescence signal of the sample excited by the laser beam in real time, and reconstruct a two-dimensional projection image of the sample with a high signal-to-noise ratio after photoelectric conversion and noise filtering.

[0060] The signal detection and control module 500 has a built-in synchronous trigger circuit and motion control unit, which coordinates the galvanometer deflection timing of the scanning module, the pulse emission period of the laser module and the data sampling rate of the signal acquisition module 400. It detects the fluorescence signal through the electronic computer and collector and controls the operation of the scanning module to control the scanning path of the needle beam, ensuring the temporal and spatial consistency of the needle beam scanning path and the fluorescence signal acquisition.

[0061] Among them, the femtosecond laser output by the pump module 100 is wavefront phase encoded by the spatial light modulator 205 to generate an ultra-long needle-shaped beam with an axial extension of more than 100μm; the focusing position of this beam is dynamically controlled by the scanning module through a high-precision galvanometer, and point-by-point two-dimensional scanning excitation is performed on the sample plane to trigger the two-photon nonlinear fluorescence effect; the generated back-fluorescence signal is separated by the binary mirror 501, and then spectrally filtered, light intensity converted and electrical signal reconstructed by the focusing lens 502 in the signal acquisition module 400 and the photoelectric detector of the signal detection and control terminal 505, and the laser emission period, galvanometer deflection timing and signal sampling rate are synchronously coordinated to form a closed-loop control link of excitation-scanning-detection, realizing high-resolution real-time imaging of a large range of living tissues.

[0062] In this embodiment, the imaging depth of field of the ultra-long needle-shaped beam is extended a hundredfold compared to a Gaussian beam, and 50-100 times greater than that of traditional two-photon microscopy systems. Compared to Bessel beam technology, its axial energy distribution can be dynamically controlled through phase encoding, ensuring uniform excitation efficiency across a long focal depth. Due to this large depth of field, the system can capture a 3D imaging volume in a single scan, eliminating the need for layer-by-layer axial scanning. This increases the 3D imaging rate compared to traditional point-by-point focusing methods while also avoiding timing errors and photobleaching effects introduced by mechanical motion.

[0063] The large depth of field light needle depth resolution technology involved in the present invention significantly improves the depth resolution capability of biomedical three-dimensional imaging through a dual-intensity gradient controlled light needle resolution method. This technology utilizes the differential signal generated by two light intensity gradient scans (deep-shallow / shallow-deep mode) combined with the depth response curve calibrated by fluorescent beads to break through the physical constraint of the Z-axis resolution in traditional light sheet microscopy being limited by the length of the light needle. It can achieve submicron depth positioning accuracy in living samples (such as the mouse brain vascular system), improving the spatial resolution capability by more than 3 times compared to traditional methods. In addition, the integration of the light intensity-depth conversion algorithm and the three-dimensional reconstruction module supports the automatic quantitative analysis of vascular morphological parameters, providing a non-invasive and highly reliable three-dimensional imaging solution for the fields of neuroscience, oncology, and drug development.

[0064] In this embodiment, see Figure 1 and Figure 2 As shown, the pump module 100 includes:

[0065] Pump laser 101 is used to generate ultrashort pulse laser with adjustable wavelength and output femtosecond near-infrared laser beam as a two-photon excitation light source;

[0066] Lens 1 201 and lens 2 202 are used to perform beam waist compression and collimation on the original laser (femtosecond laser pulse) to output a parallelized laser beam;

[0067] A half-wave plate 203 (HWP) is used to adjust the polarization direction of the incident laser and change the linear polarization angle of the femtosecond pulse laser;

[0068] The polarization beam splitter 204 (PBS) is used to adjust the polarization direction of the incident laser and screen the vertically polarized beam to adapt to the working mode of the subsequent modulation device. The femtosecond pulse laser generated by the pump laser 101 is incident on the half-wave plate 203 through lens 1 201 and lens 2 202, and then changes its own linear polarization angle. It is then incident on the polarization beam splitter 204 and outputs a vertically polarized beam.

[0069] Among them, Figure 2 As shown, after the pump module 100 of this embodiment emits a light beam through the pump laser 101, it is composed of a collimation link and a polarization control unit of the optical modulation module 200: the femtosecond laser outputs a near-infrared pulse laser with a pulse width of 100fs and a wavelength of 800-1200nm, which is shaped into a parallel light beam through the collimation link composed of lens 1 201 and lens 2 202; the half-wave plate 203 adjusts the polarization direction of the incident laser, and the polarization beam splitter 204 (PBS) filters the vertically polarized light beam to input the optical modulation module. The remaining horizontally polarized light beam realizes dynamic laser power control through polarization state conversion.

[0070] In this embodiment, the optical scanning module 300 includes:

[0071] The galvanometric mirror and the resonant mirror 302 form a resonant-galvanometric composite scanning galvanometer 303, which drives the ultra-long needle-shaped beam to perform high-frequency two-dimensional scanning in the fast and slow axis directions, realizing point-by-point excitation of the sample plane. The galvanometric mirror realizes linear scanning in the slow axis direction, and the resonant mirror 302 drives high-frequency oscillation in the fast axis direction.

[0072] Scanning galvanometer mirror 303 (Scan Lens) and tube lens 304 (Tube Lens), the scanning galvanometer mirror 303 and tube lens 304 form a scanning galvanometer mirror group 303, and the scanning galvanometer mirror group 303 and the resonance-galvanometry composite scanning galvanometer mirror 303 form a conjugate imaging system, which converts the amount of light beam deflection into the focus point displacement of the sample plane, completing high-speed, large-scale two-dimensional point-by-point scanning, ensuring a constant spot size within the scanning field of view and corrected aberrations;

[0073] The microscope objective lens 401 is used to focus the excitation beam, focusing the modulated ultra-long needle-shaped beam onto the biological sample, using the two-photon excitation effect to generate nonlinear fluorescence radiation inside the sample, and combined with the reflective prism 206 to realize light path retracement transmission to ensure the consistency of excitation efficiency within the long focal depth range.

[0074] The optical scanning module 300 integrates a resonant mirror 302 and a galvanometer galvanometer mirror 301, driving fast and slow axis scanning, respectively. Combined with a scanning galvanometer mirror 303 and a tube lens 304, the 4f conjugate imaging system converts beam deflection into focal point displacement on the sample plane. The microscope objective 401 focuses the ultra-long needle-shaped beam onto the sample for point-by-point excitation. The beam propagation path sequentially passes through the galvanometer mirror, the scanning galvanometer mirror 303, and the microscope objective 401, ensuring that spot size fluctuations within the field of view are within a certain range and correcting for spherical aberration and astigmatism, enabling high-speed two-dimensional scanning excitation.

[0075] In this embodiment, a multi-reflective surface optical folding component is further provided between the sleeve lens 304 and the microscope objective lens 401. The multi-reflective surface optical folding component includes at least two reflective surfaces to form a reflective light path. The optical folding component guides the femtosecond pulse laser emitted from the sleeve lens 304 to the spatial light phase modulator for wavefront phase reconstruction. After the modulated laser is transmitted in reverse through the original light path, it is redirected to the microscope objective lens 401 for a second time by another reflective surface of the optical folding component. The laser beam propagation path is guided by the double reflective surfaces to form a closed-loop light path between the SLM and the objective lens, thereby completing the secondary phase modulation.

[0076] Among them, a reflecting prism 206 with a double-reflecting surface structure is arranged between the sleeve lens 304 and the microscope objective lens 401. Its first reflecting surface reflects the femtosecond pulse laser emitted by the sleeve lens 304 to the optical modulation module 200 for wavefront phase encoding. The modulated light beam is reflected for the second time by the second reflecting surface of the reflecting prism 206 to the focal plane of the microscope objective lens 401, forming a closed-loop phase correction link.

[0077] In this embodiment, the formation of the ultra-long needle-shaped beam is based on phase encoding technology, which can generate a focal distribution with specific intervals along the axial direction. Specifically, by modulating the phase of the femtosecond pulse laser, the focal position of the femtosecond pulse laser after passing through the microscope objective 401 in the light scanning module 300 can be changed. During this process, the relationship between the phase of the microscope objective 401 and the femtosecond pulse laser conforms to the Abbe sine condition, wherein the phase distribution of the microscope objective 401 and the femtosecond laser parameters satisfy the following phase matching relationship:

[0078]

[0079] Where, P OB is the phase of the microscope objective lens 401, λ is the laser wavelength of the femtosecond pulse laser, f is the objective lens focal length of the microscope objective lens 401, (x, y) are the plane coordinates of the back focal plane of the objective lens, and n is the refractive index of the imaging sample. The spatial light phase modulator can achieve controlled displacement of the needle-shaped light beam focus along the axial direction of the optical axis by encoding the phase wavefront and changing the phase of the femtosecond laser pulse through the optical modulation module, that is, achieving axial movement of the needle-shaped light beam focus.

[0080] In this embodiment, the axial length of the ultra-long needle beam is achieved by programmable control of the number of focal points M, and the discrete phase encoding P is loaded by the SLM. DOM (x,y), the phase modulation function for controlling the axial focus distribution of the ultra-long needle beam satisfies:

[0081]

[0082] f ′ (x,y)=f0+[p(x,y)-1]δf

[0083]

[0084] Where f0 is the initial focal length of the objective lens, δf is the distance between adjacent focal points, p(x,y) is the N×N phase distribution matrix, and the additional term πA·p(x,y) is used to optimize the lateral distribution of the beam and satisfy the phase range P DOM (x,y)∈(0,2π), total length L=Mδf, adjacent focus interval δf≤R L (Rayleigh length) to ensure consistency in excitation efficiency.

[0085] In this embodiment, the total length of the ultra-long needle-shaped beam is Mδf, where r is the Gaussian radius of the incident light. The interval length δf between two adjacent focal spots does not exceed one Rayleigh length RL. The constraint condition for the focal spacing δf is:

[0086]

[0087] Among them, R L is the Rayleigh length, r is the radius of the fundamental mode of the incident light, and this constraint ensures the effective energy superposition of adjacent foci.

[0088] In this embodiment, the signal acquisition module 400 includes a spectroscopic component, a spectral filtering component and a photoelectric conversion component. The spectroscopic component is a dichroic mirror 501, which is used to separate the excitation light and the sample fluorescence, reflect the excitation light to the objective lens, and transmit the fluorescence to the detection link; the spectral filtering component is a filter 503, which is used to remove stray light and residual excitation light and improve the signal-to-noise ratio; the photoelectric conversion component includes a focusing lens 502 and a photomultiplier tube 504. The focusing lens 502 and the photomultiplier tube 504 (PMT) are used to focus the fluorescence onto the photosensitive surface of the photomultiplier tube 504 (PMT), generate a time-series electrical signal through photoelectric conversion, and reconstruct the sample image through phase-locked amplification and analog-to-digital conversion.

[0089] In this embodiment, the signal detection and control module is composed of a bisection mirror 501, a focusing lens 502, a filter 503, and a photomultiplier tube 504. The function of the bisection mirror 501 is to separate the laser light from the fluorescence generated by the sample excitation. The laser light reflected by the reflecting prism 206 can penetrate the bisection mirror 501 and further enter the microscope objective 401; while the fluorescence excited by the sample is separated by the bisection mirror 501 and reflected to the focusing lens 502. The focusing lens 502 and the filter 503 work together to filter and focus the reflected fluorescence signal, which is then received by the photosensitive surface of the photomultiplier tube 504. The photomultiplier tube 504 converts the received fluorescence signal into an electrical signal and transmits the electrical signal to the signal detection and control terminal 505.

[0090] The signal detection and control module 500 includes a multifunctional acquisition card and a controller. The input of the multifunctional acquisition card is used to collect the voltage signal output by the photomultiplier tube 504; the output of the controller is used to drive the resonant-galvanometer scanning mirror 303 in the scanning module to perform XY scanning operations. The controller uses hardware programming to synchronize the signal output and acquisition process.

[0091] It should be noted that the controller is also responsible for controlling the scanning galvanometer 303, the tube lens 304, the reflective prism 206, the microscope objective lens 401 to move synchronously with the femtosecond pulse laser. This control method belongs to the scope of the existing technology.

[0092] In this embodiment, fluorescent beads with a size smaller than the Rayleigh length of the light needle are used as calibration objects. Their axial position is accurately scanned by a Z-axis electric translation stage, and the mapping relationship between the fluorescence intensity ratio under two light intensity distributions and the known depth is recorded to construct a fluorescence calibration model.

[0093] In this embodiment, dual-light needle scanning is performed on a living imaging target (such as cerebral cortical blood vessels), and the absolute depth value is inverted by matching the intensity ratio of pixels at the same position with the calibration curve to achieve an improvement in the Z-axis resolution.

[0094] In this embodiment, the light needle intensity of the two-photon in vivo imaging system can be flexibly controlled by phase encoding to perform light needle intensity gradient design: the first SLM modulation makes the light needle axial intensity meet the following requirements:

[0095] I1(z)=I0exp(-(z-z0) / L)

[0096] The second modulation generates a reverse gradient that satisfies:

[0097] i2(z)=I0exp(-(z-z0) / L)

[0098] During the calibration process, the non-light needle mode (point scanning) is used to obtain the three-dimensional distribution of fluorescent beads as a benchmark to eliminate the influence of the light needle's own intensity distribution on the calibration;

[0099] The fluorescent bead-agar model was fixed on the translation stage, and its Z-axis coordinate was obtained in point scanning mode. Then, the optical needle mode was switched to record the intensity values I1 and I2 of each fluorescent bead under the two SLM modulations, and the intensity values I1 and I2 of each fluorescent bead were calculated. A linear fitting curve with a known depth was obtained; a dual-light needle scan was performed on a living sample (such as the cranial window area of anesthetized mice) to simultaneously collect I1 and I2 images of the vascular network; pixel-by-pixel calculation was performed The value is substituted into the calibration curve to analyze the absolute depth and reconstruct the three-dimensional vascular topology; the two optical needle scans of the living sample are triggered in time synchronization to eliminate the pixel misalignment error caused by blood flow movement.

[0100] Compared with existing technologies, the two-photon in vivo imaging system with dual-light needle intensity gradient control and depth mapping proposed in this invention has the following beneficial effects by constructing a three-dimensional dynamic imaging module for living samples, a neural circuit control and information processing module, and a multi-parameter simultaneous quantitative analysis module:

[0101] The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping of the present invention breaks through the Z-axis resolution limit of traditional light needles through the combination of dual-gradient light needle scanning and fluorescence calibration; establishes a nonlinear mapping model of intensity ratio-depth, significantly improving the noise resistance in the living environment; and is compatible with existing two-photon microscopy systems, and can realize depth mapping function by simply adding an SLM module and calibration protocol.

[0102] 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.

[0103] 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.

[0104] 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 two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping, characterized by: Includes the following components: The pump module is used to generate ultrashort pulse lasers with tunable wavelengths and output femtosecond near-infrared laser beams as two-photon excitation light sources; The optical modulation module is used to encode the laser wavefront through a spatial optical phase modulator, modulate the incident collimated laser beam into an ultra-long needle-shaped beam with an axial length exceeding 100 μm, and optimize the lateral light intensity distribution of the beam to extend the imaging depth of field; The optical scanning module, which includes a high-precision galvanometer and objective lens assembly, is used to focus the ultra-long needle-shaped beam onto the surface and deep areas of biological samples, and achieve rapid point-by-point excitation of the sample plane through two-dimensional scanning; The signal acquisition module integrates a photomultiplier tube and a lock-in amplifier to collect the fluorescence signal of the sample excited by the laser beam in real time, and reconstructs a two-dimensional projection image of the sample with a high signal-to-noise ratio after photoelectric conversion and noise filtering; The signal detection and control module has a built-in synchronous trigger circuit and motion control unit, which coordinates the galvanometer deflection timing of the scanning module, the pulse emission period of the laser module and the data sampling rate of the signal acquisition module. It detects the fluorescence signal through the electronic computer and collector and controls the operation of the scanning module to control the scanning path of the needle beam.

2. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 1, characterized in that: The pump module includes: Pump laser, used to generate ultrashort pulse laser with adjustable wavelength, outputting femtosecond near-infrared laser beam as two-photon excitation light source; Lens 1 and Lens 2 are used to perform beam waist compression and collimation on the femtosecond laser pulse and output a parallelized laser beam; Half-wave plate, used to adjust the polarization direction of the incident laser and change the linear polarization angle of the femtosecond pulse laser; The polarization beam splitter is used to adjust the polarization direction of the incident laser and screen the vertically polarized beam to adapt to the working mode of the subsequent modulation device. The femtosecond pulse laser generated by the pump laser is incident on the half-wave plate through lens 1 and lens 2, and then changes its own linear polarization angle. It is then incident on the polarization beam splitter and outputs a vertically polarized beam.

3. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 2, characterized in that: The optical scanning module includes: The galvanometer mirror and the resonant mirror form a resonant-galvanometer composite scanning galvanometer, which drives the ultra-long needle-shaped beam to perform high-frequency two-dimensional scanning in the fast and slow axis directions, realizing point-by-point excitation of the sample plane. The galvanometer mirror realizes linear scanning in the slow axis direction, and the resonant mirror drives high-frequency oscillation in the fast axis direction. The scanning galvanometer and the sleeve lens form a scanning galvanometer group. The scanning galvanometer group and the resonance-galvanometry composite scanning galvanometer form a conjugate imaging system, which converts the beam deflection into the focus point displacement of the sample plane, completing high-speed, large-scale two-dimensional point-by-point scanning. The microscope objective is used to focus the excitation beam, focusing the modulated ultra-long needle-shaped beam onto the biological sample, using the two-photon excitation effect to generate nonlinear fluorescence radiation inside the sample, and combining with the reflective prism to realize the light path retracement transmission.

4. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 3, characterized in that: A multi-reflective surface optical folding component is also provided between the sleeve lens and the microscope objective lens. The multi-reflective surface optical folding component includes at least two reflective surfaces to form a reflective light path. The optical folding component guides the femtosecond pulse laser emitted from the sleeve lens to the spatial light phase modulator for wavefront phase reconstruction. After the modulated laser is transmitted in reverse through the original light path, it is redirected to the microscope objective lens for a second time by another reflective surface of the optical folding component. The laser beam propagation path is guided by the double reflective surfaces to form a closed-loop light path between the SLM and the objective lens, thereby completing the secondary phase modulation.

5. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 4, characterized in that: The phase distribution of the microscope objective lens and the parameters of the femtosecond laser satisfy the following phase matching relationship: Where, P OB is the phase of the microscope objective lens, λ is the laser wavelength of the femtosecond pulse laser, f is the objective lens focal length of the microscope objective lens, (x, y) are the plane coordinates of the back focal plane of the objective lens, and n is the refractive index of the imaging sample. The spatial light phase modulator can achieve controlled displacement of the needle-shaped beam focus along the optical axis by encoding the phase wavefront and changing the phase of the femtosecond laser pulse through the optical modulation module.

6. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 5, characterized in that: The axial length of the ultra-long needle beam is achieved by programmable control of the number of focal points M, and the discrete phase encoding P is loaded by SLM. DOM (x,y), the phase modulation function for controlling the axial focus distribution of the ultra-long needle beam satisfies: f′(x,y)=f0+[p(x,y)-1]δf Where f0 is the initial focal length of the objective lens, δf is the distance between adjacent focal points, p(x,y) is the N×N phase distribution matrix, and the additional term πA·p(x,y) is used to optimize the lateral distribution of the beam and satisfy the phase range P DOM (x,y)∈(0,2π), total length L=Mδf, adjacent focus interval δf≤R L , ensuring consistency in excitation efficiency.

7. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping according to claim 6, characterized in that: The total length of the ultra-long needle-shaped beam is Mδf, where r is the Gaussian radius of the incident light. The interval length δf between two adjacent focal spots does not exceed one Rayleigh length RL. The constraint condition for the focal spacing δf is: Among them, R L is the Rayleigh length, r is the radius of the fundamental mode of the incident light, and this constraint ensures the effective energy superposition of adjacent foci.

8. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 7, characterized in that: The signal acquisition module includes a spectroscopic component, a spectral filtering component and a photoelectric conversion component. The spectroscopic component is a dichroic mirror, which is used to separate the excitation light and the sample fluorescence, reflect the excitation light to the objective lens, and transmit the fluorescence to the detection link; the spectral filtering component is a filter, which is used to remove stray light and residual excitation light and improve the signal-to-noise ratio; the photoelectric conversion component includes a focusing lens and a photomultiplier tube, which are used to focus the fluorescence onto the photosensitive surface of the photomultiplier tube, generate a time-series electrical signal through photoelectric conversion, and reconstruct the sample image through phase-locked amplification and analog-to-digital conversion.

9. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 8, characterized in that: Fluorescent beads with a size smaller than the sharp length of the light needle are used as calibration objects. Their axial position is accurately scanned by a Z-axis electric translation stage, and the mapping relationship between the fluorescence intensity ratio under two light intensity distributions and the known depth is recorded to construct a fluorescence calibration model.

10. The two-photon in vivo imaging system with dual light needle intensity gradient control and depth mapping as claimed in claim 1, characterized in that: The light needle intensity of the two-photon in vivo imaging system is flexibly controlled by phase encoding, and the light needle intensity gradient is designed: the first SLM modulation makes the light needle axial intensity meet the following requirements: I1(z)=I0exp(-(z-z0) / L) The second modulation generates a reverse gradient that satisfies: I2(z)=I0exp(-(z-z0) / L) During the calibration process, the non-light needle mode is used to obtain the three-dimensional distribution of fluorescent beads as a benchmark to eliminate the influence of the light needle's own intensity distribution on the calibration; The fluorescent bead-agar model was fixed on the translation stage, and its Z-axis coordinate was obtained in point scanning mode. Then, the optical needle mode was switched to record the intensity values I1 and I2 of each fluorescent bead under the two SLM modulations, and the intensity values I1 and I2 of each fluorescent bead were calculated. A linear fitting curve with a known depth was obtained; a dual-light needle scan was performed on a living sample (such as the cranial window area of anesthetized mice) to simultaneously collect I1 and I2 images of the vascular network; pixel-by-pixel calculation was performed The value is substituted into the calibration curve to analyze the absolute depth and reconstruct the three-dimensional vascular topology; the two optical needle scans of the living sample are triggered in time synchronization to eliminate the pixel misalignment error caused by blood flow movement.

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