Miniaturized scanning microscopic volume imaging probe and imaging system

Abstract

The application discloses a miniaturized scanning microscopic volume imaging probe and an imaging system. The miniaturized scanning microscopic volume imaging probe comprises a micro light sheet generating unit or a micro light needle generating unit, the micro light sheet generating unit is used for converting an illumination light beam into a two-dimensional light sheet mode, and the micro light needle generating unit is used for converting the illumination light beam into a light needle mode; an imaging light path is used for transmitting the illumination light beam from a light source to an imaging sample, collecting a back signal light beam emitted by the imaging sample and reconstructing the back signal light beam to form an intermediate image; a scanning engine is used for controlling and changing the position of the illumination light beam in the imaging sample, driving the illumination light beam and the back signal light beam to perform scanning movement; and an intermediate image collector is used for receiving the intermediate image and performing pixelization processing on the intermediate image. The application avoids the disadvantage that the imaging light path is bent at right angles at a galvanometer, and greatly improves the compactness and slimness of a front end lens barrel part.

Description

Technical Field

[0001] This invention specifically relates to a miniaturized scanning microscopic volumetric imaging probe and imaging system with optical tomography capabilities, belonging to the fields of optical three-dimensional microscopy, biomedical optics and photonics, and medical imaging instruments. Background Technology

[0002] Currently used diagnostic pathology techniques in clinical practice, including histopathology and intraoperative frozen section pathology, are essentially invasive ex vivo observation methods, prone to sampling bias and unable to provide real-time dynamic information. Next-generation diagnostic pathology calls for imaging devices capable of directly visualizing in situ, in vivo tissues with high spatiotemporal resolution and three-dimensional structure and function; however, existing miniaturized handheld imaging probes have their own limitations:

[0003] 1) Conventional wide-field reflected light (or scattered light) endoscopes can only capture the color and texture of the tissue surface and cannot penetrate deep into the subcutaneous tissue for imaging; even if a fluorescence signal is introduced, its illumination excitation mode cannot distinguish between the in-focus signal and the out-of-focus background, and it lacks depth tomography (i.e., optical sectioning) and three-dimensional resolution.

[0004] 2) Optical coherence tomography (OCT) imaging modality has the ability of depth tomography and three-dimensional imaging. However, its spatial resolution is generally on the order of 5-10 micrometers, and the image contrast comes from the backscattered light of the tissue, which cannot provide effective cell-level tissue microstructure.

[0005] 3) Confocal or two-photon fluorescence point scanning imaging modalities possess micrometer-level spatial resolution and depth tomography capabilities, but are limited by the point-by-point scanning mode, with a maximum two-dimensional frame rate of tens of frames per second. Even with the introduction of axial scanning, the achievable three-dimensional volume imaging rate (i.e., stacking tens to hundreds of two-dimensional images from different depths to form three-dimensional volume data) (hereinafter referred to as volume frame rate) is very limited (typically 0.1-1 volume / second); if a larger area of ​​three-dimensional tissue needs to be explored by translating the imaging probe, the time required is even longer. Considering the inevitable irregular movements in living tissue caused by respiration, heartbeat, or other factors, the aforementioned limited three-dimensional volume frame rate makes the imaging results susceptible to motion artifacts, resulting in irregular three-dimensional deformation of the acquired volume data and preventing the registration of adjacent sets of volume data.

[0006] The relentless pursuit of high spatiotemporal resolution in the field of in vivo microscopy has spurred the emergence of the recently developed swept confocally-aligned planar excitation (SCAPE) microscope. SCAPE microscopy cleverly utilizes the ample aperture angle of high-performance microscope objectives, using a single objective to generate an illumination beam tilted relative to the principal optical axis and collecting the (backward) fluorescence excited by this tilted beam. This combines the excitation and probe objectives of conventional light-sheet microscopy into one, inheriting the inherent optical tomography capabilities and low phototoxicity of light-sheet microscopy while providing an open sample space. It is compatible with model animals of different sizes and various in vivo imaging scenarios, naturally adapting to the optical path architecture of forward-looking imaging probes. More importantly, its confocal probe mode can complete three-dimensional tomographic scanning without moving the objective or sample, avoiding the inherent speed bottleneck and disturbance to the live sample caused by mechanically moving the main objective. Theoretically, its three-dimensional volumetric imaging speed can reach thousands of volumes per second.

[0007] However, existing standard tabletop confocal oblique light scanning microscopes are bulky and have highly complex core optical paths. Miniaturizing them into a lightweight, flexible, and easy-to-manage slender rigid tube-shaped microscopic imaging probe suitable for in situ and in vivo pathological imaging applications faces two prominent difficulties in terms of optical principles: (1) Standard tabletop confocal oblique light scanning microscopes rely on a galvanometer galvanometer mirror conjugate to the back focal plane of the main objective to drive the oblique light scanning, causing the overall optical path to bend at a 90-degree angle with the galvanometer mirror as the inflection point. This is incompatible with the compact and slender shape required for clinical applications; (2) Standard tabletop confocal oblique light scanning microscopes require the intermediate image reconstructed in the focal area of ​​the second objective to be directly magnified onto a research-grade CMOS camera for sampling and recording. This makes it difficult to physically separate the front imaging probe from the bulky camera, greatly restricting the portability and ease of use of the handheld probe.

[0008] In summary, scanning oblique light sheet microscopy, with its advantages of open sample space, high resolution, and high-speed 3D imaging (high volume ratio), has enormous application potential in label-free, non-invasive, real-time in situ in vivo pathological imaging. However, its existing tabletop architecture and design concepts cannot be easily transferred to the design of miniaturized, lightweight, flexible, and slender rigid tubular handheld microscopy (and microendoscopic) imaging probes. How to break away from the conventional design of tabletop confocal oblique light sheet scanning microscopes and achieve high spatiotemporal resolution 3D microscopic volumetric imaging within a compact, slender rigid tubular imaging probe framework, thereby enabling highly sensitive and high-resolution visualization of the 3D microstructure and functional dynamics of subepidermal tissues without damaging living tissue and its physiological environment, achieving in situ pathological imaging effects that are "better than slides without the need for slides," is of great significance for promoting precision diagnosis and treatment and advancing the construction of a healthy China. Summary of the Invention

[0009] The main objective of this invention is to provide a miniaturized scanning microscopic volume imaging probe and imaging system. Through innovations in principle architecture and acquisition strategy, it solves the two core difficulties in miniaturizing confocal oblique light scanning microscopes, making it possible to achieve high spatiotemporal resolution and three-dimensional microscopic volume imaging on miniaturized, slender rigid tube-shaped microscopic (or endoscopic) imaging probes, thereby overcoming the shortcomings of existing technologies.

[0010] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0011] This invention provides a miniaturized scanning microscopic volumetric imaging probe, comprising:

[0012] A micro light sheet generating unit or a micro light needle generating unit, wherein the micro light sheet generating unit is used to convert the illumination beam originating from the light source module into a two-dimensional light sheet shape, and the micro light needle generating unit is used to convert the illumination beam originating from the light source module into a light needle shape.

[0013] An imaging optical path is used to transmit an illumination beam from a light source module to an imaging sample, and to collect a back signal beam emitted by the imaging sample and reconstruct the back signal beam to form an intermediate image.

[0014] A scanning engine is used to control and change the position of the illumination beam in the imaging sample, drive the illumination beam to perform scanning motion, and drive the detected back signal beam to perform scanning motion.

[0015] An intermediate image acquisition unit is used to receive the intermediate image and perform pixelation processing on the intermediate image.

[0016] In a typical implementation, the scanning engine includes a first scanning engine and / or a second scanning engine. The first scanning engine works in conjunction with the micro-light sheet generating unit and the intermediate image acquisition unit. The first scanning engine is used to drive the micro-light sheet generating unit and the intermediate image acquisition unit to perform one-dimensional scanning motion synchronously. The second scanning engine works in conjunction with the imaging optical path. The second scanning engine is used to drive the illumination beam and the back signal beam to perform one-dimensional scanning motion.

[0017] Furthermore, the first scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine.

[0018] Furthermore, the first scanning engine includes a piezoelectric driver, a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions, a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes, or a scanning device based on a microelectromechanical system, a linear or rotary motor.

[0019] Furthermore, the second scanning engine includes at least one pair of prisms disposed inside the imaging optical path. The prism pair includes two coaxially arranged prisms that can be deflected relative to each other around their own central axis, thereby driving the illumination beam and the back signal beam to perform one-dimensional scanning motion.

[0020] Furthermore, the prism is a wedge-shaped prism.

[0021] Furthermore, the wedge angles of the two prisms included in the prism pair may be the same or different.

[0022] Furthermore, the two prisms contained in the prism pair are made of the same material.

[0023] Furthermore, when the second scanning engine drives the illumination beam and the back signal beam to perform translational scanning, the prism has the same angular velocity but opposite rotation directions for the two prisms it contains.

[0024] Furthermore, the micro light sheet generating unit includes a light sheet generating component, which is used to directly shape the illumination beam into a light sheet shape.

[0025] Furthermore, the light-generating assembly includes at least one of a focusing element, a diffractive optical element with beam-shaping function, and a metasurface element.

[0026] Furthermore, the focusing element includes a cylindrical lens.

[0027] Furthermore, the cylindrical lens includes a spherical cylindrical lens, a cemented doublet cylindrical lens, a cemented triplicate cylindrical lens, or an aspherical cylindrical lens.

[0028] Furthermore, the micro light sheet generating unit also includes a light sheet angle adjustment element. The light sheet generating component and the light sheet angle adjustment element are sequentially arranged in the optical path of the illumination beam. The light sheet angle adjustment element is used to adjust the incident angle of the illumination beam onto the imaging optical path.

[0029] Furthermore, the light sheet angle adjustment element includes a reflector and / or a polygonal prism.

[0030] Furthermore, the focusing element and the light sheet angle adjustment element are integrated into one unit.

[0031] In a more specific implementation example, the micro light sheet generating unit includes a light needle generating component and a third scanning engine. The light needle generating component is used to shape the illumination beam into a light needle shape. The third scanning engine is connected to the light needle generating component and is used to drive the light needle generating component to scan in a selected direction and form a virtual light sheet shape.

[0032] As a typical implementation example, the light needle generation component includes a reflector for reflecting the illumination beam so that the main ray of the reflected illumination beam is parallel to the propagation direction of the virtual light sheet-shaped illumination beam, and the equivalent light waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

[0033] As another typical implementation, the light needle generation component includes a beam shaping component and a reflector. The beam shaping component is used to change the shape parameters of the illumination beam to shape the illumination beam into a light needle shape. The shape parameters include at least one of numerical aperture, beam waist diameter, and Rayleigh length. The reflector is used to reflect the illumination beam after it has been shaped by the beam shaping component. The equivalent beam waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

[0034] Furthermore, the illumination beam shape of the light needle includes "long straight" or "curved". It should be noted that the illumination beam of the light needle in this invention is generally one-dimensional.

[0035] Furthermore, the illumination beam in the form of a light needle includes a Gaussian beam, a Bessel beam, a Bessel-Gaussian beam, or an Airy beam.

[0036] Furthermore, the beam shaping assembly includes at least one beam shaping lens; preferably, the beam shaping lens includes at least one of a biconvex lens, a single lens having a biconcave, plano-convex, or biconvex spherical or aspherical surface, an achromatic lens, and a graduated refractive index lens.

[0037] As a typical implementation example, the illumination beam in the form of a light needle is a Bessel-Gaussian beam. The beam shaping component includes at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Alternatively, the beam shaping component is formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens with at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Or, the beam shaping component is formed by combining one or more diffractive optical elements with an axial pyramidal holographic phase and / or metasurface lenses.

[0038] As a typical implementation example, the illumination beam in the form of a light needle is an Airy beam, and the beam shaping component has a phase mask with a cubic phase structure. Alternatively, the beam shaping component is mainly formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask having a cubic phase structure.

[0039] Furthermore, the third scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine.

[0040] Furthermore, the third scanning engine includes a piezoelectric driver, a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions, a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes, or a scanning device based on a microelectromechanical system, a linear or rotary motor.

[0041] In another more specific implementation, the scanning engine includes a first scanning engine and / or a second scanning engine. The first scanning engine is driven in conjunction with the micro light needle generating unit and the intermediate image acquisition unit. The first scanning engine is used to drive the micro light needle generating unit and the intermediate image acquisition unit to perform two-dimensional scanning motion synchronously. The second scanning engine is coordinated with the imaging optical path. The second scanning engine is used to drive the illumination beam and the back signal beam to perform two-dimensional scanning motion.

[0042] Furthermore, both the first scanning engine and the second scanning engine are two-dimensional scanning engines.

[0043] Furthermore, the first scanning engine includes a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions, a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes, or a scanning device based on a microelectromechanical system, a linear or rotary motor.

[0044] Furthermore, the second scanning engine includes at least one pair of prisms disposed inside the imaging optical path. The pair of prisms includes two coaxially arranged prisms that can independently deflect relative to each other around their own central axis, thereby driving the illumination beam and the back signal beam to perform two-dimensional scanning motion.

[0045] Furthermore, the prism is a wedge-shaped prism.

[0046] Furthermore, the wedge angles of the two prisms included in the prism pair may be the same or different.

[0047] Furthermore, the two prisms contained in the prism pair are made of the same material.

[0048] In a typical implementation, the micro-light needle generating unit includes a reflector for reflecting the illumination beam. The equivalent light waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

[0049] In another typical implementation, the miniature light needle generating unit includes a beam shaping component and a reflector. The beam shaping component is used to change the shape parameters of the illumination beam to shape the illumination beam into a light needle shape. The shape parameters include at least one of numerical aperture, beam waist diameter, and Rayleigh length. The reflector is used to reflect the illumination beam after it has been shaped by the beam shaping component, so that the principal ray of the reflected illumination beam is parallel to the propagation direction of the virtual light sheet-shaped illumination beam. The equivalent light waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

[0050] Furthermore, the illumination beam shape of the light needle includes "long straight" or "curved".

[0051] Furthermore, the illumination beam in the form of a light needle includes a Gaussian beam, a Bessel beam, a Bessel-Gaussian beam, or an Airy beam.

[0052] Preferably, the beam shaping assembly includes at least one beam shaping lens; preferably, the beam shaping lens includes at least one of a biconvex lens, a single lens having a biconcave, plano-convex, or biconvex spherical or aspherical surface, an achromatic lens, and a graduated refractive index lens.

[0053] In a typical implementation, the illumination beam in the form of a light needle is a Bessel-Gaussian beam. The beam shaping component includes at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Alternatively, the beam shaping component is formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens with at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Or, the beam shaping component is formed by combining one or more diffractive optical elements with an axial pyramidal holographic phase and / or metasurface lenses.

[0054] In a typical implementation, the illumination beam in the form of a light needle is an Airy beam, and the beam shaping component has a phase mask with a cubic phase structure. Alternatively, the beam shaping component is mainly formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask having a cubic phase structure.

[0055] Furthermore, the imaging optical path includes a first objective lens, a relay optical system, and a second objective lens arranged sequentially. The front end of the first objective lens faces the imaging sample, and the front end of the second objective lens faces the intermediate image acquisition device. The second scanning engine is disposed between the first objective lens and the second objective lens. The relay optical system is used to relay the illumination beam and the scanning motion of the second scanning engine to the imaging sample located at the front end of the first objective lens and to transmit the back signal beam collected by the first objective lens to the second objective lens, and to reconstruct the intermediate image in the focal area of ​​the second objective lens.

[0056] Furthermore, the imaging optical path is coaxially arranged with the prism included in the second scanning engine.

[0057] Furthermore, the second scanning engine is conjugately coupled to the back focal plane of the first objective lens.

[0058] Furthermore, the relay optical system includes a 4F system.

[0059] Furthermore, the intermediate image acquisition device is used to sample the intermediate image at the optical level and then transmit it to an external image sensor to complete the digital recording of the intermediate image. Alternatively, the intermediate image acquisition device itself has photoelectric conversion function and can directly complete the pixelation and digital recording of the intermediate image.

[0060] Furthermore, the intermediate image acquisition device includes an optical fiber image transmission bundle with a beveled receiving end face, and the intermediate image formed in the imaging optical path can be projected onto the beveled receiving end face of the optical fiber image transmission bundle, thereby realizing the reception and optical sampling of the intermediate image.

[0061] Furthermore, the propagation direction of the illumination beam / sheet, after being adjusted by the light sheet angle adjustment element, is parallel to the oblique receiving end face of the optical fiber image bundle, and the equivalent optical waist of the illumination beam falls on the receiving end face of the optical fiber image bundle.

[0062] Furthermore, the equivalent optical waist of the illumination beam coincides with the focal point of the second objective lens.

[0063] Furthermore, the first scanning engine is located outside the imaging optical path, and the micro light sheet generating unit or the micro light needle generating unit is fixedly coupled with the intermediate image acquisition unit. The micro light sheet generating unit or the micro light needle generating unit and the intermediate image acquisition unit can be driven by the first scanning engine to move synchronously, and the intermediate image and the intermediate image acquisition unit move synchronously and remain relatively stationary.

[0064] Furthermore, the intermediate image remains relatively stationary with respect to the oblique receiving end face of the optical fiber image bundle.

[0065] Furthermore, both the intermediate image and the intermediate image acquisition device are stationary.

[0066] Another aspect of the present invention provides a miniaturized three-dimensional volumetric imaging microscope system, including the aforementioned miniaturized scanning microscopic volumetric imaging probe.

[0067] Furthermore, the miniaturized three-dimensional volumetric imaging microscope system also includes a light source module for providing an illumination beam.

[0068] Furthermore, the miniaturized three-dimensional volumetric imaging microscope system also includes an external image sensor, which is used to receive intermediate images obtained by the intermediate image acquisition unit.

[0069] Furthermore, the miniaturized three-dimensional volumetric imaging microscope system also includes a filter element, which is disposed between the external image sensor and the intermediate image acquisition unit, and is used to filter out light signals within a specific wavelength range.

[0070] Furthermore, the filtering element includes at least one filter.

[0071] Compared with the prior art, the advantages of the present invention include:

[0072] 1) The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which has an innovative illumination beam (or light sheet) scanning engine and scanning strategy. It breaks away from the design convention of standard tabletop oblique light sheet scanning microscopes that rely on reflective galvanometer scanning mirrors or microelectromechanical systems (MEMS) scanning mirrors. It avoids the disadvantage of the imaging optical path being bent at a right angle at the mirror, and allows the main objective, relay lens group, second objective, etc. to be placed coaxially, which greatly improves the compactness and slimness of the front end tube.

[0073] 2) The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which features an innovative intermediate image acquisition device design and a corresponding descan detection strategy, enabling the bulky camera to be separated from the imaging probe. This achieves high-speed three-dimensional microscopic volume imaging while ensuring the small size and lightweight flexibility of the imaging probe.

[0074] 3) Compared to wide-field illumination endoscopes, the miniaturized microscopic imaging probe provided by this invention possesses depth tomography (i.e., optical slicing capability) and three-dimensional volumetric imaging capabilities, enabling clear visualization of the three-dimensional microstructure of subepidermal tissues. Compared to optical coherence tomography (OCT) imaging modality based on backscattered light, the miniaturized three-dimensional microscopic volumetric imaging probe provided by this invention can utilize fluorescence signals for imaging, providing molecular sensitivity, subcellular spatial resolution, and richer image contrast information.

[0075] 4) Compared with endoscopic imaging probes based on point scanning imaging modes that rely on tightly focused excitation light, such as confocal fluorescence, two-photon fluorescence, or coherent Raman, the miniaturized volumetric imaging probe provided by this invention adopts a light sheet illumination and parallel detection imaging strategy. It can perform three-dimensional microscopic imaging without mechanically moving the imaging probe or the imaging sample, resulting in an order-of-magnitude improvement in the three-dimensional volume ratio. Even if there is irregular axial relative movement between the living tissue and the probe, there is still sufficient three-dimensional overlap between the adjacent sets of volumetric data blocks acquired by the imaging probe provided by this invention. Therefore, the three-dimensional misalignment between adjacent volumetric data blocks can be intuitively and accurately estimated from the obtained high-speed "three-dimensional volumetric data stream". Then, after registration and fusion, a panoramic three-dimensional image covering a range of several millimeters is generated, providing richer and more accurate in vivo in situ pathological feedback for clinical applications.

[0076] 5) Compared with the desktop confocal oblique light sheet scanning microscope, this invention maintains its high-speed three-dimensional volumetric imaging advantage while avoiding the disadvantage of the former's imaging optical path being bent at a right angle at the galvanometer through an innovative illumination beam (or light sheet) scanning engine and scanning strategy. Furthermore, through innovative intermediate image and descan detection strategies, the bulky camera can be separated from the imaging probe, greatly improving the compactness and slimness of the imaging probe. Thus, it achieves high spatiotemporal resolution and in situ in vivo pathological imaging that is "better than a slice" without the need for a slice on a miniaturized, slender rigid tube-shaped microscopic (or endoscopic) imaging probe. Attached Figure Description

[0077] Figure 1 This is a schematic diagram of a miniaturized three-dimensional volumetric imaging microscope system provided in a typical embodiment of the present invention;

[0078] Figure 2a This is a schematic diagram of a miniaturized volumetric imaging probe based on a sheet-like illumination beam and a rear-mounted light sheet scanning engine.

[0079] Figure 2b To explain the working principle of the micro-light-generating unit based on the negative diopter cylindrical lens and its overall design and structural diagram of its assembly with the intermediate image acquisition unit and scanning engine;

[0080] Figure 2c To explain the working principle of the micro-light-generating unit based on positive diopter cylindrical lens and its overall design and structural diagram of its assembly with intermediate image acquisition unit and scanning engine;

[0081] Figure 2d To explain the working principle of the micro-light sheet generation unit based on negative diopter cylindrical lens and polygonal prism, and its overall design and structural diagram of its assembly and combination with intermediate image acquisition unit and scanning engine;

[0082] Figure 2e To explain the working principle of the micro-light sheet generation unit that integrates a negative refractive power cylinder with a polygonal prism, and its overall design and structural diagram of its assembly and combination with the intermediate image acquisition unit and scanning engine;

[0083] Figure 3a This is a schematic diagram of the system structure of a miniaturized volumetric imaging probe based on a sheet-like illumination beam and a centrally positioned wedge prism for scanning. It also shows the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique light sheet is scanned to the negative y limit position.

[0084] Figure 3b For the corresponding Figure 3a The design is illustrated in the diagrams of the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique light plate is scanned to the center position of the y axis.

[0085] Figure 3c For the corresponding Figure 3a The design is illustrated in the diagrams of the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique light plate is scanned to the positive y limit position.

[0086] Figure 4a This is a schematic diagram of the system structure of a miniaturized volumetric imaging probe based on a virtual illumination beam sheet formed by needle-shaped illumination beam scanning and a rear-mounted two-dimensional scanning engine.

[0087] Figure 4b To explain the working principle of the illumination beam needle generation unit that directly uses the light emitted from a single-mode fiber, and its overall design and structural diagram of its assembly with the slow-axis scanner, intermediate image acquisition unit, and fast-axis scanning engine;

[0088] Figure 4c To explain the working principle of the illumination beam needle generation unit with the introduction of a beam shaping lens, and its overall design and structural diagram of the assembly and combination with the slow axis scanner, intermediate image acquisition unit and fast axis scanning engine;

[0089] Figure 4d To explain the working principle of the illumination beam needle generation unit that uses GRIN lens and cone lens to generate Bessel beam, and its overall design and structural diagram of assembly and combination with slow axis scanner, intermediate image acquisition unit and fast axis scanning engine;

[0090] Figure 4eTo explain the working principle of the illumination beam needle generation unit that uses a GRIN lens and a cubic phase mask to generate an Airy beam, and its overall design and structural diagram of its assembly with a slow-axis scanner, an intermediate image acquisition unit, and a fast-axis scanning engine;

[0091] Figure 5a This is a schematic diagram of the system structure of a miniaturized volumetric imaging probe based on a virtual illumination beam sheet and a centrally positioned wedge prism for scanning. It also shows schematic diagrams of the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique beam sheet is scanned to the negative y limit position.

[0092] Figure 5b The corresponding Figure 5a The design is illustrated in the diagrams of the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique light plate is scanned to the center position of the y axis.

[0093] Figure 5c The corresponding Figure 5a The design is illustrated in the diagrams of the illumination beam path and the back signal beam path (upper half) in the yz plane view and the illumination beam path (lower half) in the xy plane view when the oblique light plate is scanned to the positive y limit position.

[0094] Figure 6 This is a schematic diagram of the system structure of a miniaturized volumetric imaging probe based on a needle-shaped illumination beam and a rear-mounted two-dimensional independently controlled scanning engine, as well as a possible example of a two-dimensional scanning trajectory.

[0095] Figure 7 This is a schematic diagram of the system structure of a miniaturized three-dimensional microscopic volumetric imaging probe based on a centrally located asynchronous scanning wedge prism pair using two-dimensional optical needle scanning. An example of the scanning position of the illumination beam and the corresponding back signal beam path (marked only in the yz plane) are shown in the yz plane view (upper half) and the xy plane view (lower half). Detailed Implementation

[0096] In view of the shortcomings of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The following will further explain and illustrate this technical solution, its implementation process, and principles in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely for explaining the invention and not for limiting it. Furthermore, it should be noted that, for ease of description, the accompanying drawings only show the parts relevant to the invention, not all structures. It should also be noted that the various optical components used in this invention are known to those skilled in the art and can all be commercially available; therefore, no specific product models are limited here.

[0097] Please see Figure 1 , Figure 1 This is a miniaturized three-dimensional volumetric imaging microscope system, including a light source module, a front-end imaging probe (i.e., the aforementioned miniaturized scanning microscopic volumetric imaging probe, hereinafter the same) and an external image sensor (optional). The arrows in the figure represent the illumination beam and the transmission path of the backlight signal / beam.

[0098] The core of a miniaturized three-dimensional volumetric imaging microscope system is the front-end imaging probe, which directly contacts the imaging sample (especially living tissue) and performs volumetric microscopy imaging. This front-end imaging probe generally includes the following modules:

[0099] Miniature light sheet generation unit: used to convert the illumination beam from the light source module into a two-dimensional light sheet shape (morphology), the illumination beam in the light sheet shape can also be called an illumination light sheet; this miniature light sheet generation unit can use cylindrical lenses such as cylindrical lenses and Powell lenses to directly generate sheet-like or layered illumination beams, or it can use fast-scanning needle-like illumination beams (i.e., light needles, specifically including Gaussian beams, Bessel beams, Airy beams, etc.) to form virtual light sheets (also known as digital light sheets). It should be noted that the specific shape of the virtual light sheet generated by the second method is related to the trajectory of the fast scan;

[0100] The scanning engine, a non-galvanometer scanning engine, is primarily used to control and change the position of the illumination beam / sheet in the imaging sample, drive the illumination beam / sheet to perform scanning motion, and scan the detected back-facing signal beam. It should be noted that "non-galvanometer" means that unlike traditional galvanometers or MEMS scanning mirrors that achieve beam scanning by changing the angle of a reflective surface, this scanning engine uses a different working principle to achieve the scanning motion of the beam or sheet in the downstream imaging optical path. This scanning engine can be installed in two ways: "central" and "rear." "Central" means that the scanning engine is placed between the first objective lens (i.e., the main objective lens) and the second objective lens, usually conjugately coupled to the back focal plane of the main objective lens; "rear" means that the scanning engine is located outside the imaging optical path.

[0101] Imaging optical path: It is used to transmit the illumination beam to the imaging sample, collect the backward signal beam (fluorescence or scattered light) emitted by the imaging sample, and transmit the backward signal beam back to one end far away from the imaging sample for acquisition; this imaging optical path generally includes a first objective lens (hereinafter referred to as the "main objective lens"), a second objective lens, and a relay optical system supporting the two. The front end of the main objective lens directly faces the imaging sample, and the front end of the second objective lens faces the image acquisition module. The relay optical system generally selects a 4f-system with an appropriate magnification; the relay optical system is used to relay the illumination beam / sheet and the scanning movement of the scanning engine to the front end of the main objective lens (i.e., in the imaging sample), and to transmit the backward signal beam collected by the main objective lens to the second objective lens; when a mid-position scanning engine is used, the optical path of the mid-position scanning engine may also become part of the relay optical system;

[0102] It should be noted that the overall optical design of the imaging optical path should minimize aberration as much as possible, so as to transmit the illumination beam or light sheet to the imaging sample with high fidelity, and to collect and transmit the backward signal beam generated by the illuminated area of the illumination beam / light sheet in the imaging sample back to the second objective lens with high fidelity, and reconstruct an intermediate (optical) image with as little aberration as possible in the focal area of the second objective lens.

[0103] Intermediate image collector (module): It is used to receive the intermediate (optical) image reconstructed by the second objective lens and perform sampling (i.e., pixelization).

[0104] In some embodiments, the intermediate image collector samples the intermediate image at the optical level. For example, special fiber optic elements can be used to distinguish and collect photons corresponding to each pixel, and then transmit them to an external image sensor located outside the imaging probe and physically separated from the imaging probe to complete the digital recording of the intermediate image.

[0105] In some other embodiments, the intermediate image collector itself can have a photoelectric conversion function. For example, a miniaturized image sensor is used to directly complete the pixelization and digital recording of the intermediate image.

[0106] In some embodiments, the intermediate image reconstructed in the focal area of the second objective lens is stationary. In some other embodiments, the intermediate image is not stationary. At this time, the intermediate image collector moves synchronously with the intermediate image and remains relatively stationary, so as to achieve efficient "de-scanning" acquisition of the intermediate image.

[0107] Specifically, the light source module preferably uses a laser light source, but it can also use light-emitting diodes, lamps, and various light sources emitted after being transmitted through optical fibers. The light source module can contain multiple physically separated light source devices, can contain multiple wavelength components, or directly use broadband light sources such as supercontinuum lasers, superluminescent diodes, and swept lasers. When the light source module is physically separated from the front-end imaging probe, the light source module can also include one or more optical fibers for transmitting the illumination beam from the light source to the front-end imaging probe.

[0108] Specifically, when the intermediate image acquisition device of the aforementioned front-end imaging probe does not have photoelectric conversion function, an external image sensor separate from the imaging probe is required. Specifically, the external image sensor includes, but is not limited to, charge-coupled devices (CCD), complementary metal-oxide semiconductors (CMOS), single photon avalanche diodes (SPAD) or SPAD arrays, photomultipliers (PMT) or PMT arrays, silicon photomultipliers (SiPM) or SiPM arrays. Depending on the optical design of the front-end imaging probe and the size of the intermediate image, the external image sensor used can be a single-point detector or a one-dimensional or two-dimensional array detector.

[0109] In a first embodiment of the present invention, the micro-light sheet generating unit directly generates a sheet-shaped illumination beam. The illumination beam / sheet is relayed to the front end of the probe via the imaging optical path and incident on the sample to be imaged. Under the drive of the scanning engine, it is scanned in translation. The excited back signal beam (fluorescence or reflected light, etc.) is scanned by the scanning engine to generate an intermediate image that is stationary relative to the intermediate image acquisition module.

[0110] Example 1

[0111] Figure 2a A miniaturized oblique-slab three-dimensional microscopic volumetric imaging probe (hereinafter referred to as the probe) based on a rear-mounted fiber optic scanning engine is demonstrated.

[0112] In this embodiment, the illumination beam is transmitted to the micro-light sheet generation unit 12 at the rear end of the probe through a single-mode optical fiber 11. The thin light sheet emitted from the micro-light sheet generation unit 12 enters the imaging optical path composed of the second objective lens 131, the relay optical system (lenses 132-135) and the first objective lens (i.e., the main objective lens) 136 at a certain tilt angle relative to the main optical axis. An tilted illumination beam / sheet is generated in the focal area of ​​the first objective lens 136. The excited back signal beam (fluorescence or scattered light, etc.) returns through the imaging optical path, forming a tilted intermediate image in the focal area of ​​the second objective lens 131, which is received by the intermediate image acquisition unit.

[0113] In this embodiment, the intermediate image acquisition device uses an optical fiber image bundle 14 with a beveled receiving end face. By adjusting the placement of the optical fiber image bundle 14, the intermediate image formed in the focal area of ​​the second objective lens 131 can be accurately projected onto the beveled receiving end face of the optical fiber image bundle 14. The optical fiber array completes the reception and optical sampling of the intermediate image, and then transmits it back to the camera and host located at the remote end for image recording, display and processing.

[0114] To achieve optical sheet scanning and de-scanning detection, this embodiment binds the miniature optical sheet generation unit 12 and the fiber optic image transmission bundle 14 together via a scanning cantilever 15, and uses a piezoelectric actuator (i.e., scanning engine) 16 to drive their synchronous movement; this achieves the illumination beam / sheet moving along the y-axis direction (see...). Figure 2a (As shown by the dashed arrow) scanning; secondly, since the optical fiber image bundle 14 and the micro-optical sheet generating unit 12 scan synchronously, according to the principle of optical path reversibility, the intermediate image formed in the focal area of ​​the second objective lens 131 remains relatively stationary with the oblique receiving end face of the optical fiber image bundle 14 during the scanning process, thus cleverly ensuring the de-scanning acquisition and reception of the intermediate image.

[0115] In this type of embodiment, the core function of the micro-optical sheet generation unit 12 is to convert single-mode light from the fiber core of the optical fiber 11 into a thin optical sheet, which can be implemented in various ways.

[0116] Figure 2b A specific implementation of the micro-light sheet generation unit 12 is shown. The illumination beam emitted from the single-mode fiber 11 is first collimated by the graded refractive index (GRIN) lens 121, then focused into a light sheet shape by the micro cylindrical mirror 122, and finally reflected by the micro mirror 123 to achieve a suitable tilt angle relative to the optical axis of the imaging optical path. Figure 2b The miniature cylindrical mirror 122 shown has a negative refractive power and can diverge the collimated illumination beam emitted from the graded refractive index lens 121 along the y-axis direction shown in the figure, forming an equivalent thin sheet with a width along the x-axis; the installation orientation of the miniature reflector 123 must ensure that: (1) the propagation direction of the reflected illumination beam / sheet is parallel to the oblique receiving end face of the optical fiber image bundle 14, and the equivalent optical waist ( Figure 2b (1) The point where the dashed lines converge falls on the receiving end face of the fiber optic image bundle 14 (in other words, the illumination beam / sheet coincides with the receiving end face of the intermediate image), optimizing the clarity of the intermediate image acquisition; (2) The equivalent optical waist coincides with the focal point of the second objective lens 131, optimizing the spatial resolution of the system.

[0117] Figure 2c Another specific implementation scheme of the micro-light sheet generation unit 12 is shown. This scheme uses a cylindrical mirror 124 with positive diopter, that is, focusing along the y-axis to generate a thin light sheet. By designing and optimizing the relative position of the positive diopter cylindrical mirror 124 and the micro-reflector 123, the illumination beam / sheet can also be made to coincide with the receiving end face of the fiber optic image bundle 14, and the equivalent optical waist can overlap with the focal point (or front focal plane) of the second objective lens 131.

[0118] In alternative implementations, the micro-light sheet generation unit 12 can also employ spherical cylindrical mirrors with plano-concave, plano-convex, biconcave, biconvex, or meniscus shapes, or cemented doublet or cemented triplet cylindrical mirrors (to better eliminate chromatic aberration), and introduce aspherical cylindrical mirrors including Powell lenses and flat-top light shapers, or combinations of the above-mentioned different types of cylindrical mirrors, to generate illumination beams / sheets and optimize their uniformity. In alternative implementations, the micro-light sheet generation unit 12 can also employ a combination of a pyramidal cylindrical mirror and a slit mask, or other designs familiar to those skilled in the art, to generate Bezier sheets with extended depth of field.

[0119] In alternative implementations, the micro-light sheet generation unit 12 can also incorporate diffractive optical elements or metasurface elements / lenses with beam shaping functions, and can combine diffractive elements or metasurface lenses with the aforementioned cylindrical mirrors or axial prism mirrors to optimize and control the geometry of the generated thin light sheet, thereby meeting the needs of practical imaging applications.

[0120] Figure 2b and Figure 2c The miniature light sheet generating unit 12 shown in the diagram all employs a miniature reflector 123 to control the emission angle of the light sheet. The reflective surface of the reflector can be coated with a metal or dielectric reflective film, and the shape of its base portion can be polygonal, cylindrical, or other arbitrary shapes, depending on the requirements of assembly and scanning load.

[0121] Figure 2dAnother specific implementation of the micro light sheet generating unit 12 is shown, which uses a polygonal prism 125 to adjust the tilt angle of the illumination beam / sheet (relative to the principal optical axis); its reflective surface can be based on a total internal reflection mechanism, or it can utilize a surface coated with metal or a dielectric film; in the corresponding alternative implementation, the polygonal prism can have an arbitrary shape, the light sheet can undergo one or more reflections inside the prism, and can undergo refraction when emitted, the specific design is based on achieving the required orientation of the emitted light sheet for the imaging system.

[0122] Figure 2e Another specific implementation scheme of the micro-light sheet generation unit 12 is shown, which scheme will Figure 2d The micro cylindrical mirror 122 used to form the light sheet and the polygonal prism 125 used to adjust the tilt angle of the light sheet are integrated into a single device 126. In a corresponding alternative implementation, the refractive cylinder and other reflective prisms of the device 126 can also be designed as conic cylindrical surfaces or generalized aspherical surfaces to further adjust the emission orientation, divergence degree, and position of the equivalent waist of the illumination beam / sheet.

[0123] In such Figures 2b-2e In all the implementation schemes and alternative implementation schemes of the micro-optical sheet generation units shown, the graded refractive index lens 121 used for collimation can be replaced with a graded refractive index fiber, which can be directly fused with the single-mode fiber 11; it can also be replaced with a micro-spherical or aspherical lens, a diffractive optical element, or a metasurface lens, etc., to achieve the purpose of beam shaping. It should be particularly noted that, in the alternative embodiments, the light emitted from the single-mode fiber, after passing through the graded refractive index lens 121, the graded refractive index fiber, the micro-spherical or aspherical lens, the diffractive optical element, or the metasurface lens, etc., may not be completely collimated, but may have a certain degree of convergence or divergence, as long as it can produce an illumination beam / sheet that meets the application requirements after being used in conjunction with subsequent optical elements.

[0124] In such Figures 2a-2e In the illustrated embodiments or implementation schemes of the micro-optical sheet generation unit, the scanning engine preferably uses a piezoelectric driver 16. Specifically, this can be a strip-shaped piezoelectric bimorph, or a ring-shaped or cylindrical piezoelectric transistor, or any type of piezoelectric bend, etc., capable of driving the scanning cantilever and thus driving the micro-optical sheet generation unit and the fiber optic image bundle to scan synchronously. In alternative embodiments, the scanning engine can also employ a microelectromechanical system (MEMS) scanner, a micromotor, or other driving devices based on various other transduction principles, to meet the one-dimensional linear motion required for optical sheet scanning.

[0125] In such Figures 2a-2eIn the illustrated embodiment or the implementation scheme of the micro-optical sheet generation unit, the intermediate image acquisition unit preferably uses an optical fiber image bundle 14 with a beveled receiving end face. The tilt angle of its beveled receiving end face relative to the optical axis of the imaging optical path (hereinafter referred to as the main optical axis) should be consistent with the tilt angle of the illumination beam / sheet relative to the main optical axis. It is particularly important to note that the tilt angle of the beveled receiving end face of the optical fiber image bundle relative to the main optical axis and the bevel angle of the beveled receiving end face itself (i.e., the tilt angle of the beveled receiving end face relative to the central axis of the optical fiber image bundle, or the bevel angle of the end face of each component fiber in the optical fiber image bundle) can be controlled independently. Given that the bevel angle of the end face of a single-mode or multi-mode fiber waveguide affects the direction of its transmitting and receiving light cones, in alternative embodiments, the bevel angle of the end face of the optical fiber image bundle can be optimized to maximize the collection efficiency of the back-facing signal beam while ensuring that the entire receiving end face coincides with the intermediate image.

[0126] In alternative embodiments, the intermediate image acquisition unit can also employ any other type of array detector of suitable size (generally small enough), including charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), electron-magnified CCDs (EMCCDs), or complementary metal-oxide-semiconductor (CMOS) image sensors, as well as area arrays of photomultiplier tubes (PMTs), silicon photomultiplier tubes (Si-PMTs), or avalanche photodiode (APD) arrays. Furthermore, microlens arrays can be fitted in front of these area array detectors for focusing light or resolving light field information.

[0127] In such Figure 2a In the illustrated embodiment, the second objective lens 131 and the first objective lens 136 are relayed and coupled through two sets of 4f systems. In an alternative embodiment, the second objective lens 131 and the first objective lens 136 may include any number of relay 4f systems to flexibly adjust the length of the lens barrel and adapt to the needs of actual imaging scenarios. The relay 4f systems involved may consist of a single lens, an achromatic lens, or a gradient index lens (GRIN lens), or may employ a Hopkins rod lens relay system, etc.

[0128] Example 2

[0129] Figures 3a-3cA miniaturized oblique light sheet three-dimensional microscopic volume imaging probe based on a centrally positioned wedge prism pair is demonstrated.

[0130] In this embodiment, the illumination beam emitted from the light source module is input to the rear end of the probe through the single-mode fiber 11, and enters the micro-optical sheet generation unit 12 from the end of the single-mode fiber. The thin optical sheet emitted from the micro-optical sheet generation unit 12 enters the imaging optical path composed of the second objective lens 231, the second barrel lens 232, the second scanning lens 233, the wedge prism pair scanner 21, the first scanning lens 234, the first barrel lens 235 and the main objective lens (i.e., the first objective lens) 236 at a certain tilt angle relative to the main optical axis. The wedge prism pair scanner 21 (i.e., the optical wedge) is composed of two coaxially arranged first wedge prisms (also called optical wedges, Risley prisms) 211 and second wedge prisms (also called optical wedges, Risley prisms) 212 with the same wedge angle. By controlling the relative rotation of the two wedge prisms 211 and 212 around the central axis, the deflection of the emitted beam can be controlled.

[0131] In this embodiment, the two wedge prisms rotate at the same angular velocity but in opposite directions, thereby controlling the beam's direction. Figures 3a-3c The deflection occurs in the yz plane as shown. Specifically, when the two wedge prisms are rotated until their thickest and thinnest points are aligned (as shown in the image), the deflection occurs within the yz plane. Figure 3a and Figure 3c As shown), the entire wedge prism effectively forms an isosceles prism with a apex angle equal to the sum of the two wedge angles for the scanner 21. At this time, the deflection angle of the illumination beam behind the prism reaches the positive y-axis (as shown). Figure 3c ) or negative ( Figure 3a The maximum value of ); when the two are rotated to the point where the thickest (thinnest) part of the first wedge prism 211 aligns with the thinnest (thickest) part of the second wedge prism 212, the wedge faces of the two wedge prisms are parallel to each other, and the entire wedge prism pair of scanner 21 can be equivalent to parallel glass plates. The beam does not undergo angular deflection when passing through the prism pair (although there is a certain degree of vertical axis translation, see Figure 3b When the two rotate to other intermediate states, the degree of beam deflection and the final scanning displacement of the focal area of ​​the first objective lens 236 are between two extreme values. By designing the wedge angle and rotation speed (phase) of the first wedge prism 211 and the second wedge prism 212, the scanning angle and scanning speed of the outgoing beam and the illumination beam / sheet in the focal area of ​​the first objective lens 236 can be controlled.

[0132] In this embodiment, the backscattered signal beam (fluorescence or backscattered photons) emitted from the illumination beam / sheet passes through the main objective lens 236, the first barrel lens 235, and the first scanning lens 234 in the opposite order to the excitation light, and then enters the wedge prism scanner 21, where it is de-scanned. After passing through the second scanning lens 233, the second barrel lens 232, and the second objective lens 231, a static intermediate image is formed in the focal area of ​​the second objective lens 231, and is received by the intermediate image acquisition device 17. It should be noted that in this embodiment, both the intermediate image and the intermediate image acquisition device 17 are stationary.

[0133] It should be noted that, depending on the specific scanning requirements, the wedge prism can be driven to rotate synchronously and continuously relative to the scanner 21, i.e., undergoing... Figures 3a to 3b Again Figure 3c All states; can also drive the wedge prism to scan the 21 from such Figure 3a The state shown evolves to the point where... Figure 3b After reaching the state shown, reverse the rotation direction of the two wedge prisms, from the state shown... Figure 3b The state shown returns to as follows Figure 3a The state shown; or any desired wedge prism pair with steering and rotation angle range can be specified according to application requirements.

[0134] In this embodiment, the first wedge prism 211 and the second wedge prism 212 are mounted in a plane-to-plane manner. In an alternative embodiment, they can also be mounted in a plane-to-wedge or wedge-to-wedge manner to achieve the same beam deflection and light sheet scanning effect.

[0135] exist Figures 3a-3c In this embodiment, the second objective lens 231 is coupled to the wedge prism scanner 21, and the wedge prism scanner 21 is coupled to the first objective lens 236 via a 4f relay system. In an alternative embodiment, any number of relay 4f systems can be introduced between the second objective lens 231 and the wedge prism scanner 21, and between the wedge prism scanner 21 and the first objective lens 236, to adjust the length of the lens barrel and adapt to the needs of the actual imaging scenario. The relay 4f system involved can be composed of a single lens, an achromatic lens, or a gradient refractive index lens (GRIN lens), or a Hopkins rod lens relay system, etc.

[0136] In this embodiment and alternative embodiments, the specific implementation schemes of the micro-light sheet generation unit and the intermediate image acquisition unit 17 are the same as those described in Embodiment 1 above, and will not be repeated here.

[0137] In this embodiment, the micro light sheet generation unit 12 is simplified to an illumination beam needle generation unit. The generated one-dimensional illumination beam needle generates a virtual illumination beam / sheet (also known as a digital scanned light sheet) under the drive of the fast axis scanner. The virtual light sheet is relayed to the front end of the probe through the imaging optical path and incident on the sample to be imaged. It is then subjected to translational scanning under the drive of the slow axis scanning engine. The generated back signal beam (fluorescence or reflected light, etc.) is scanned by the slow axis scanning engine to generate an intermediate image that is stationary relative to the intermediate image acquisition unit 17.

[0138] Example 3

[0139] Figure 4a A miniaturized virtual oblique light sheet 3D scanning microscopic volume imaging probe based on a rear-mounted scanning engine is demonstrated.

[0140] In this embodiment, the illumination beam, after being transmitted into the probe via single-mode fiber 11, is not directly converted into a sheet-like illumination beam. Instead, the micro-needle generation unit 32 generates a one-dimensional illumination "needle" that is either "long and straight" (such as a Gaussian beam or a Bessel beam) or "curved" (such as an Airy beam). Driven by the fast-axis scanner 33, this illumination beam is rapidly scanned along the x-axis direction in the figure, generating a virtual illumination beam / sheet (also called a digital light sheet). This virtual illumination beam / sheet is incident on the imaging sample via the imaging optical path. The excited backlight beam (such as fluorescence or scattered light) returns to the focal area of ​​the second objective lens 131 via the imaging optical path, forming a tilted virtual intermediate image, which is then projected onto the receiving end face of the intermediate image acquisition unit 17. Driven by the slow-axis scanner 34, the micro-needle generation unit 32, the fast-axis scanner 33, and the intermediate image acquisition unit 17 move synchronously along the y-axis direction in the figure, realizing the scanning of the virtual light sheet along the slow axis direction and keeping the virtual intermediate image and the intermediate image acquisition unit 17 relatively stationary, thus achieving the scanning reception of the intermediate image.

[0141] The micro-needle generation unit required in this embodiment can be implemented in various ways.

[0142] Figure 4bThis document demonstrates a specific implementation scheme of this embodiment (focusing on the micro-needle generation unit and scanning engine, omitting the imaging optical path). This scheme directly uses the outgoing light from the single-mode fiber 11 as the illumination beam needle. After reflection by the micro-reflector 321, the principal ray of this illumination beam needle is parallel to the z' axis of the corresponding virtual light sheet propagation direction, and the beam waist is mirrored onto the oblique receiving end face of the fiber optic image bundle 14 used as an intermediate image acquisition unit 17. The single-mode fiber 11 and the micro-reflector 321 are fixed together on the fast-axis scanner 33 via the fast-axis scanning adapter 322, and scanned along the x-axis direction under the latter's drive to generate a virtual illumination beam / sheet (i.e., a digital light sheet) parallel to the xz' plane. The fast-axis scanner 33 (along with the fiber optic cable 11, the miniature mirror 321, and the fast-axis scanning adapter 322 connected thereto) and the fiber optic image bundle 14 are fixed to the slow-axis scanner 34 via the slow-axis scanning adapter 341 and scan along the y-axis (i.e., the slow axis) under the drive of the latter, so that the virtual light sheet is scanned along the slow axis in the imaging sample after passing through the imaging optical path; the intermediate image generated by the back signal beam returned by the imaging optical path falls on the receiving end face of the fiber optic image bundle 14 and moves synchronously with the receiving end face while remaining relatively stationary, thus realizing the descanning reception of the intermediate image.

[0143] In such Figure 4c In the alternative embodiment shown (focusing on the micro-needle generation unit and scanning engine, omitting the imaging optical path), the illumination beam emitted from the single-mode fiber 11 first passes through the beam shaping lens 323 to generate an illumination beam needle with suitable morphological parameters (such as numerical aperture, beam waist diameter, Rayleigh length, etc.), and then is reflected by the micro-reflector 321 into the xz′ plane; the remaining components and working principle of this scheme are the same. Figure 4a The implementation scheme shown is the same. In this implementation scheme, the beam shaping lens 323 is preferably a miniature biconvex lens; in alternative embodiments, one or more single lenses, achromatic lenses, graded refractive index lenses, etc., with spherical or aspherical surface types such as biconcave, plano-convex, or biconvex can also be selected.

[0144] Figure 4dAn alternative embodiment based on a Bessel beam is illustrated (focusing on the micro-needle generation unit and scanning engine, omitting the imaging optical path). The illumination beam emanating from single-mode fiber 11 is first collimated by collimating lens 324, and then passes through a micro-axicon 325 (also called a cone lens) to generate an illumination beam needle with a Bessel beam (strictly speaking, a Bessel-Gaussian beam) shape. Compared to a Gaussian beam with the same waist diameter, a Bessel beam has a longer equivalent beam waist length, enabling it to maintain uniform lateral resolution over a longer axial range. In some alternative embodiments, the micro-axicon 325 can also be replaced with diffractive optical elements or metasurface lenses with axial pyramidal holographic phase; other methods well-known to those in the art for generating (quasi-)Bessel beams, including annular pupils, can also be employed.

[0145] Figure 4e An alternative embodiment based on an Airy beam is shown (focusing on the micro-needle generation unit and scanning engine, omitting the imaging optical path). The illumination beam emitted from the single-mode fiber 11 is first collimated by the collimating lens 324, and then passes through a phase mask 326 with a cubic phase structure to generate an Airy beam with self-accelerating properties. It should be noted that the Airy beam has two sets of mutually perpendicular side lobes, extending along the x and y axes of the cubic phase mask itself; an optimized implementation here is to rotate the cubic phase mask so that the two sets of side lobes align with... Figure 4e The x-axis (i.e., fast axis) is at 45 degrees, so that the side lobes of the virtual Airy sheet generated under the action of the fast axis scanner 33 are distributed as symmetrically as possible, so as to obtain better spatial resolution.

[0146] In such Figures 4d-4e In the specific (alternative) embodiment shown, the collimating lens 324 preferably uses a graded refractive index lens (i.e., a GRiN lens); in alternative embodiments, the collimating lens may also use graded refractive index optical fiber, micro spherical or aspherical lens, diffractive optical element, metasurface lens, etc. for beam collimation.

[0147] In such Figures 4a-4eIn the (alternative) embodiment shown, the slow-axis scanner 34 and the fast-axis scanner 33 are preferably elongated piezoelectric bicrystalline wafers, with their deflection directions perpendicular to each other. The slow-axis scanner 34 can drive the obliquely shaped fiber image bundle 14 used for intermediate image acquisition, the fast-axis scanner 33, and the micro-needle generation unit 32 (driven by the fast-axis scanner 33) to move synchronously via a slow-axis scanning adapter 341. In an alternative embodiment, the fast-axis and slow-axis scanners can also be piezoelectric transistors or microelectromechanical systems (MEMS) drivers, micro linear motors, or other driving devices based on various transduction principles capable of performing one-dimensional linear scanning motion.

[0148] exist Figures 4b-4e In the (alternative) embodiment shown, the intermediate image acquisition unit 17 preferably uses an optical fiber image bundle 14 with a suitable end face bevel angle; other optional specific implementation schemes of the intermediate image acquisition unit 17 are the same as those described in embodiments 1-2 above, and will not be repeated here.

[0149] Example 4

[0150] Figures 5a-5c A three-dimensional microscopic volumetric imaging probe based on a virtual oblique light plate and a centrally positioned wedge prism for the scanning engine is demonstrated.

[0151] In this embodiment, the illumination beam emitted from the light source module is input to the rear end of the probe via a single-mode fiber 11 and enters the micro-light needle generation unit 32. The one-dimensional illumination "light needles" emitted from the micro-light needle generation unit 32, which are either "long straight" (such as a Gaussian beam or a Bessel beam) or "curved" (such as an Airy beam), are scanned along the fast axis direction (x-axis direction in the figure) under the drive of the fast axis scanner 33 to generate a virtual illumination beam / sheet (also called a digital light sheet). This virtual light sheet enters the imaging optical path composed of the second objective lens 231, the second barrel lens 232, the second scanning lens 233, the wedge prism to the scanner 21, the first scanning lens 234, the first barrel lens 235, and the first objective lens (i.e., the main objective lens) 236 at a certain tilt angle relative to the main optical axis. The wedge prism scanner 21 includes two coaxially arranged first wedge prisms (also called optical wedges or Risley prisms) 211 and second wedge prisms (also called optical wedges or Risley prisms) 212 with the same wedge angle; by controlling the relative rotation of the two about the central axis, the deflection of the light beam can be controlled, thereby controlling the virtual light sheet to scan along the slow axis (y-axis in the figure).

[0152] The backscattered signal beam (fluorescence or backscattered photons) emitted from the illumination beam / sheet passes through the first objective lens 236, the first barrel lens 235, and the first scanning lens 234 before entering the wedge prism scanner 21. It is scanned by the wedge prism scanner 21, then passes through the second scanning lens 233 and the second barrel lens 232, forming a static intermediate image in the focal region of the second objective lens 231, which is then received by the intermediate image acquisition unit 17. It should be noted that in this embodiment, both the intermediate image and the intermediate image acquisition unit 17 are stationary.

[0153] In this embodiment and alternative embodiments, the principle of generating the illumination beam needle 32 and the corresponding virtual light sheet is the same as that described in Embodiment 3, and will not be repeated here.

[0154] In this embodiment and alternative embodiments, the optional specific implementation schemes of the intermediate image acquisition device 17 are the same as those described in embodiments 1-3, and will not be repeated here.

[0155] In this embodiment, the first wedge prism 211 and the second wedge prism 212 are mounted in a plane-to-plane manner. In an alternative embodiment, they can also be mounted in a plane-to-wedge or wedge-to-wedge manner to achieve the same beam deflection and light sheet scanning effect.

[0156] In this embodiment and alternative embodiments, the control logic of the wedge prism on the scanner 21 is the same as that described in Embodiment 2, that is, their angular velocities are the same, their rotation directions are opposite, and according to specific scanning requirements, they can undergo... Figures 5a to 5b Again Figure 5c All states can also be derived from, for example Figure 5a The state shown evolves to the point where... Figure 5b The state shown will then return to the state shown. Figure 5a The states shown can also be customized to other steering and turning angle ranges according to application requirements.

[0157] exist Figures 5a-5c In the illustrated embodiment, the second objective lens 231 is coupled to the wedge prism scanner 21, and the wedge prism scanner 21 is coupled to the first objective lens 236 via a set of 4f relay systems. In an alternative embodiment, the second objective lens 231 can be coupled to the wedge prism scanner 21, and the wedge prism scanner 21 can be coupled to the first objective lens 236 via any number of relay 4f systems to adjust the length of the lens barrel and adapt to the needs of the actual imaging scenario. The relay 4f system can be composed of a single lens, an achromatic lens, or a gradient refractive index lens (GRiN lens), or a Hopkins rod lens relay system, etc.

[0158] In a third embodiment of the invention, the scanning trajectory of the miniaturized illumination beam needle does not need to follow a common raster scan trajectory (in other words, it does not need to generate a digital scan virtual light sheet first), but can be designed arbitrarily according to application requirements; correspondingly, the scanning engine does not need to explicitly distinguish between fast and slow axis scanners, but can use any two-dimensional scanning engine; the two-dimensional scanning engine will also scan the generated back signal beam directly in the xy direction simultaneously to generate an intermediate image that is stationary (essentially one-dimensional) relative to the intermediate image acquisition unit.

[0159] Example 5

[0160] Figure 6 A three-dimensional microscopic volumetric imaging probe based on a needle-shaped illumination beam and a rear-mounted independently controlled two-dimensional scanning actuator is demonstrated.

[0161] In this embodiment, the micro light needle generation unit 32 is bound to the intermediate image acquisition unit 18 and scans synchronously under the drive of the two-dimensional scanning engine 51. The optical path design of the system ensures that the one-dimensional intermediate image corresponding to the illumination beam needle can be accurately projected onto the intermediate image acquisition unit 18. During the scanning process, the one-dimensional intermediate image moves synchronously with the intermediate image acquisition unit 18, thereby maintaining relative stillness and realizing the de-scanning detection of the intermediate image.

[0162] In this embodiment, the two-dimensional scan driver 51 preferably employs a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions. In an alternative embodiment, the two-dimensional scan driver 51 may also be a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes. By applying a suitable dual-channel voltage waveform, the two-dimensional scan driver 51 can drive the scanning cantilever, composed of the micro-needle generation unit 32 and the intermediate image acquisition unit 18, to deflect along the x-axis and y-axis directions, thereby acquiring the desired scan trajectory. It should be noted that the two-dimensional piezoelectric driver can output sufficient oscillation amplitude to directly drive the scanning cantilever to deflect and scan, or it can operate near the resonant frequency of the scanning cantilever, thereby using resonant amplification to drive the scanning cantilever to generate sufficient deflection amplitude and scanning range. By reasonably designing the mechanical structure of the scanner and the amplitude, frequency, and phase of the driving waveform, scanning trajectories suitable for full-field dense sampling, such as spiral scanning and Lissajous scanning, can be generated, as well as scanning trajectories suitable for covering several regions of interest, such as random jump scanning of sub-fields (see...). Figure 6 (See the schematic diagram in the included figures). In an alternative embodiment, the two-dimensional scan driver 51 may also be a scanning device based on other transduction mechanisms such as microelectromechanical systems (MEMS), linear or rotary motors.

[0163] Unlike the previous embodiments, the intermediate image acquisition unit 18 in this embodiment only needs to acquire a one-dimensional intermediate image. In its specific implementation, a slanted one-dimensional or two-dimensional fiber optic image transmission bundle is preferred, and its refractive index and end face slant angle can be optimized to maximize the collection efficiency of the back-facing signal beam. After the received one-dimensional intermediate image is transmitted back to the remote end along the fiber optic image transmission bundle, it can be directly mapped onto a one-dimensional linear array detector to complete photoelectric conversion and digital recording. Alternatively, a rescanning unit can be introduced to tile the one-dimensional intermediate images corresponding to different time points (thus originating from different scanning positions) onto a two-dimensional area array detector, thereby fully utilizing the speed advantage of commercial two-dimensional area array detectors to complete photoelectric conversion and digital recording. One-dimensional linear array detectors can be selected from linear CCD, ICCD, EMCCD, or CMOS cameras, or linear multi-anode photomultiplier tubes (PMTs), silicon photomultiplier tubes (SiPMs), avalanche photodiodes (APDs), etc.; two-dimensional area array detectors can be selected from area CCD, ICCD, EMCCD, or CMOS cameras, or area PMTs, silicon photomultiplier tubes (SiPMs), avalanche photodiodes (APDs), etc.

[0164] In some alternative embodiments, the intermediate image acquisition unit 18 can be a fiber optic array or a multi-core fiber optic cable. After the received one-dimensional intermediate image is transmitted back to the remote end, the optical signals corresponding to different "pixels" can be separated and transmitted to multiple discrete photodiodes, photomultiplier tubes (PMTs), silicon photomultiplier tubes (SiPMs), or avalanche photodiodes (APDs) to complete photoelectric conversion and digital recording in parallel. The above-mentioned optical signal separation and transmission process is natural for fiber optic arrays; for multi-core fibers, it can be accomplished with the help of fiber optic splitters.

[0165] In some alternative embodiments, the intermediate image acquisition unit 18 may also be an array photodetector with a suitable structure and size (i.e., small enough), including linear or area CCD, ICCD, EMCCD or CMOS camera, as well as linear or area multi-anode photomultiplier tubes (PMT), silicon photomultiplier tubes (SiPM), avalanche photodiodes (APD), etc., to directly complete the photoelectric conversion and digitization of the intermediate image inside the probe.

[0166] Example 6

[0167] Figure 7 A two-dimensional optical needle scanning three-dimensional microscopic volume imaging probe based on an asynchronous scanning wedge prism pair is demonstrated.

[0168] Unlike Embodiments 2 and 4, where the first wedge prism 211 and the second wedge prism 212 constituting the scanner 21 require synchronous rotation (at the same speed and in opposite directions), in this embodiment, the rotation control of the first wedge prism 211 and the second wedge prism 212 is independent of each other, thereby generating arbitrary two-dimensional scanning trajectories. By reasonably designing the rotation speed and phase of the two wedge prisms, scanning trajectories suitable for full field-of-view coverage and dense sampling, such as helical scanning and Lissajous scanning, can be generated. Scanning trajectories suitable for local field-of-interest coverage, such as random jump scanning between several defined sub-fields of view, can also be generated.

[0169] In this embodiment, both the micro-needle generation unit 32 and the intermediate image acquisition unit 18 remain stationary. The principle of optical path reversibility ensures that the one-dimensional intermediate image reconstructed by the generated back signal beam in the focal area of ​​the second objective lens 231 can be accurately projected onto the receiving end face of the (one-dimensional) intermediate image acquisition unit 18, thereby realizing the descan detection of the intermediate image.

[0170] In this embodiment, the principle and optional alternative embodiments of the illumination beam needle generating unit 32 are the same as those described in Embodiment 3. Figures 4b-4e The same applies as shown in Example 5, and will not be repeated here. Optional implementations for the (one-dimensional) intermediate image acquisition unit 18 are the same as those described in Example 5, and will not be repeated here.

[0171] In this embodiment, the first wedge prism 211 and the second wedge prism 212 are mounted in a plane-to-plane manner. In an alternative embodiment, they can also be mounted in a plane-to-wedge or wedge-to-wedge manner to achieve the same beam deflection and light sheet scanning effect.

[0172] In this embodiment, the second objective lens 231 is coupled to the wedge prism scanner 21, and the wedge prism scanner 21 is coupled to the first objective lens 236 via a set of 4f relay systems. In an alternative embodiment, the second objective lens 231 can be coupled to the wedge prism scanner 21, and the wedge prism scanner 21 can be coupled to the first objective lens 236 via any number of relay 4f systems to adjust the length of the lens barrel and adapt to the needs of the actual imaging scenario. The relay 4f system involved can be composed of a single lens, an achromatic lens, or a gradient refractive index lens (GRIN lens), or a Hopkins rod lens relay system, etc.

[0173] It should be particularly noted that in Embodiments 5 and 6, the illumination beam needle is preferably a straight beam such as a Gaussian beam or a Bessel beam, so that the resulting one-dimensional intermediate image is also straight, consistent with the pixel arrangement of common linear or area array detectors. In corresponding alternative embodiments, if an illumination beam needle based on a curved shape, such as an Airy beam, is selected—resulting in a curved intermediate image—then the (one-dimensional) intermediate image acquisition unit 18 can preferably use a (customized) fiber array, multi-core fiber, or linear array photodetector with a pixel arrangement consistent with the curved intermediate image, or use a fiber image bundle or area array detector with a sufficiently large receiving end face, etc.

[0174] This invention provides a miniaturized scanning microscopic volumetric imaging probe and imaging system, featuring an innovative illumination beam (or light sheet) scanning engine and scanning strategy. It breaks away from the design convention of standard tabletop oblique light sheet scanning microscopes that rely on reflective galvanometer scanning mirrors or microelectromechanical systems (MEMS) scanning mirrors, avoiding the drawback of the imaging optical path being bent at a right angle at the mirror. This allows the main objective, relay lens group, and second objective to be placed coaxially, greatly improving the compactness and slimness of the front end tube.

[0175] The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which features an innovative intermediate image acquisition device design and a corresponding descan detection strategy, enabling the bulky camera to be separated from the imaging probe. This achieves high-speed three-dimensional microscopic volume imaging while ensuring the small size and lightweight flexibility of the imaging probe.

[0176] Compared to wide-field illumination endoscopes, the miniaturized microscopic imaging probe provided by this invention possesses depth tomography (i.e., optical slicing capability) and three-dimensional volumetric imaging capabilities, enabling clear visualization of the three-dimensional microstructure of subepidermal tissues. Compared to backscattered light-based optical coherence tomography (OCT) imaging modality, the miniaturized three-dimensional microscopic volumetric imaging probe provided by this invention can utilize fluorescence signals for imaging, providing molecular sensitivity, subcellular spatial resolution, and richer image contrast information.

[0177] Compared with endoscopic imaging probes based on point scanning imaging modes that rely on tightly focused excitation light, such as confocal fluorescence, two-photon fluorescence, or coherent Raman, the miniaturized volumetric imaging probe provided by this invention adopts a light sheet illumination and parallel detection imaging strategy. It can perform three-dimensional microscopic imaging without mechanically moving the imaging probe or the imaging sample, resulting in an order-of-magnitude improvement in the three-dimensional volume ratio. Even if there is irregular axial relative movement between the living tissue and the probe, there is still sufficient three-dimensional overlap between the adjacent sets of volumetric data blocks acquired by the imaging probe provided by this invention. Therefore, the three-dimensional misalignment between adjacent volumetric data blocks can be intuitively and accurately estimated from the obtained high-speed "three-dimensional volumetric data stream". Then, after registration and fusion, a panoramic three-dimensional image covering a range of several millimeters is generated, providing richer and more accurate in vivo in situ pathological feedback for clinical applications.

[0178] Compared with desktop confocal oblique light sheet scanning microscopes, this invention maintains the advantages of high-speed three-dimensional volumetric imaging while avoiding the drawback of the former's imaging optical path being bent at a right angle at the galvanometer through an innovative illumination beam (or light sheet) scanning engine and scanning strategy. Furthermore, through innovative intermediate image and descan detection strategies, the bulky camera can be separated from the imaging probe, greatly improving the compactness and slimness of the imaging probe. Thus, it achieves high spatiotemporal resolution and in situ in vivo pathological imaging that is "better than a slice" without the need for a slice on a miniaturized, slender rigid tube-shaped microscopic (or endoscopic) imaging probe.

[0179] It should be understood that the above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A miniaturized scanning microscopic volumetric imaging probe, characterized in that, include: A micro light sheet generating unit is used to convert an illumination beam originating from a light source module into a two-dimensional light sheet shape. An imaging optical path is used to transmit an illumination beam from a light source module to an imaging sample, and to collect a back signal beam emitted by the imaging sample and reconstruct the back signal beam to form an intermediate image. A scanning engine is used to control and change the position of the illumination beam in the imaging sample, drive the illumination beam to perform scanning motion, and drive the detected back signal beam to perform scanning motion. An intermediate image acquisition unit is used to receive the intermediate image and perform pixelation processing on the intermediate image. The intermediate image acquisition unit includes an optical fiber image transmission bundle with a beveled receiving end face. The intermediate image formed in the imaging optical path can be projected onto the beveled receiving end face of the optical fiber image transmission bundle, thereby realizing the reception and optical sampling of the intermediate image. The scanning engine includes a first scanning engine and / or a second scanning engine. The first scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine. The first scanning engine cooperates with the micro-light sheet generating unit and the intermediate image acquisition unit. The first scanning engine is used to drive the micro-light sheet generating unit and the intermediate image acquisition unit to perform one-dimensional scanning motion synchronously. The second scanning engine cooperates with the imaging optical path. The second scanning engine is used to drive the illumination beam and the back signal beam to perform one-dimensional scanning motion. The second scanning engine includes at least one set of prism pairs disposed inside the imaging optical path. The prism pair includes two coaxially arranged prisms. The two prisms can be relatively deflected around their own central axis to drive the illumination beam and the back signal beam to perform one-dimensional scanning motion.

2. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: The first scanning engine includes a piezoelectric actuator or a scanning device based on a microelectromechanical system, a linear or rotary motor.

3. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: The prism is a wedge-shaped prism.

4. The miniaturized scanning microscopic volumetric imaging probe according to claim 3, characterized in that: The wedge angles of the two prisms contained in the prism pair may be the same or different.

5. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: The prism pair contains two prisms made of the same material.

6. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: When the second scanning engine drives the illumination beam and the back signal beam to perform translational scanning, the prism has the same rotational angular velocity but opposite rotational directions for the two prisms it contains.

7. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: The micro light sheet generating unit includes a light sheet generating component, which is used to directly shape the illumination beam into a light sheet shape.

8. The miniaturized scanning microscopic volumetric imaging probe according to claim 7, characterized in that: The light-generating assembly includes at least one of a focusing element, a diffractive optical element with beam-shaping function, and a metasurface element.

9. The miniaturized scanning microscopic volumetric imaging probe according to claim 8, characterized in that: The focusing element includes a cylindrical lens.

10. The miniaturized scanning microscopic volumetric imaging probe according to claim 9, characterized in that: The cylindrical lens includes a spherical cylindrical lens, a cemented doublet cylindrical lens, a cemented triplicate cylindrical lens, or an aspherical cylindrical lens.

11. The miniaturized scanning microscopic volumetric imaging probe according to claim 8, characterized in that: The micro light sheet generating unit also includes a light sheet angle adjustment element. The light sheet generating component and the light sheet angle adjustment element are sequentially arranged in the optical path of the illumination beam. The light sheet angle adjustment element is used to adjust the incident angle of the illumination beam onto the imaging optical path.

12. The miniaturized scanning microscopic volumetric imaging probe according to claim 11, characterized in that: The light sheet angle adjustment element includes a reflector and / or a polygonal prism.

13. The miniaturized scanning microscopic volumetric imaging probe according to claim 11, characterized in that: The focusing element and the light sheet angle adjustment element are integrated into one unit.

14. The miniaturized scanning microscopic volumetric imaging probe according to claim 1, characterized in that: The micro light sheet generating unit includes a light needle generating component and a third scanning engine. The light needle generating component is used to shape the illumination beam into a light needle shape. The third scanning engine is connected to the light needle generating component and is used to drive the light needle generating component to scan in a selected direction and form a virtual light sheet shape.

15. The miniaturized scanning microscopic volumetric imaging probe according to claim 14, characterized in that: The light needle generating component includes a reflector for reflecting the illumination beam, and the equivalent optical waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

16. The miniaturized scanning microscopic volumetric imaging probe according to claim 14, characterized in that: The light needle generation component includes a beam shaping component and a reflector. The beam shaping component is used to change the shape parameters of the illumination beam to shape the illumination beam into a light needle shape. The shape parameters include at least one of numerical aperture, beam waist diameter, and Rayleigh length. The reflector is used to reflect the illumination beam after it has been shaped by the beam shaping component. The equivalent beam waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

17. The miniaturized scanning microscopic volumetric imaging probe according to claim 14, characterized in that: The illumination beam shape of the light needle includes "long straight" or "curved".

18. The miniaturized scanning microscopic volumetric imaging probe according to claim 14, characterized in that: Illumination beams in the form of light needles include Gaussian beams, Bessel beams, Bessel-Gaussian beams, or Airy beams.

19. The miniaturized scanning microscopic volumetric imaging probe according to claim 16, characterized in that: The beam shaping assembly includes at least one beam shaping lens.

20. The miniaturized scanning microscopic volumetric imaging probe according to claim 19, characterized in that: The beam-shaping lens includes at least one of the following: a biconvex lens, a single lens having a biconcave, plano-convex, or biconvex spherical or aspherical surface, an achromatic lens, and a graduated refractive index lens.

21. The miniaturized scanning microscopic volumetric imaging probe according to claim 16, characterized in that: The illumination beam in the form of a light needle is a Bessel-Gaussian beam. The beam shaping assembly includes at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens; or, the beam shaping assembly is formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens with at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens; or, the beam shaping assembly is formed by combining one or more diffractive optical elements with an axial pyramidal holographic phase and / or metasurface lenses.

22. The miniaturized scanning microscopic volumetric imaging probe according to claim 16, characterized in that: The illumination beam in the form of a light needle is an Airy beam. The beam shaping component has a phase mask with a cubic phase structure. Alternatively, the beam shaping component is mainly formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask having a cubic phase structure. The third scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine.

23. The miniaturized scanning microscopic volumetric imaging probe according to claim 14, characterized in that: The third scanning engine includes a piezoelectric driver, a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions, a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes, or a scanning device based on a microelectromechanical system, a linear or rotary motor.

24. The miniaturized scanning microscopic volumetric imaging probe according to any one of claims 7-23, characterized in that: The imaging optical path includes a first objective lens, a relay optical system, and a second objective lens arranged sequentially. The front end of the first objective lens faces the imaging sample, and the front end of the second objective lens faces the intermediate image acquisition device. The second scanning engine is disposed between the first objective lens and the second objective lens. The relay optical system is used to relay the illumination beam and the scanning motion of the second scanning engine to the imaging sample located at the front end of the first objective lens and to transmit the back signal beam collected by the first objective lens to the second objective lens, and to reconstruct the intermediate image in the focal area of ​​the second objective lens.

25. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The imaging optical path is coaxially arranged with the prism contained in the second scanning engine.

26. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The second scanning engine is conjugately coupled to the back focal plane of the first objective lens.

27. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The relay optical system includes a 4F system.

28. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The intermediate image acquisition device is used to sample the intermediate image at the optical level and then transmit it to an external image sensor to complete the digital recording of the intermediate image. Alternatively, the intermediate image acquisition device itself has photoelectric conversion function and can directly complete the pixelation and digital recording of the intermediate image.

29. The miniaturized scanning microscopic volumetric imaging probe according to claim 11, characterized in that: The propagation direction of the illumination beam / sheet, after being adjusted by the light sheet angle adjustment element, is parallel to the oblique receiving end face of the optical fiber image bundle, and the equivalent optical waist of the illumination beam falls on the receiving end face of the optical fiber image bundle.

30. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The equivalent optical waist of the illumination beam coincides with the focal point of the second objective lens.

31. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The first scanning engine is located outside the imaging optical path. The micro-light sheet generating unit is fixedly coupled with the intermediate image acquisition unit. The micro-light sheet generating unit and the intermediate image acquisition unit can be driven by the first scanning engine to move synchronously. The intermediate image and the intermediate image acquisition unit move synchronously and remain relatively stationary.

32. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: The intermediate image remains relatively stationary with respect to the oblique receiving end face of the optical fiber image bundle.

33. The miniaturized scanning microscopic volumetric imaging probe according to claim 24, characterized in that: Both the intermediate image and the intermediate image acquisition device are stationary.

34. A miniaturized scanning microscopic volumetric imaging probe, characterized in that, include: A micro light needle generating unit is used to convert an illumination beam originating from a light source module into a light needle shape. An imaging optical path is used to transmit an illumination beam from a light source module to an imaging sample, and to collect a back signal beam emitted by the imaging sample and reconstruct the back signal beam to form an intermediate image. A scanning engine is used to control and change the position of the illumination beam in the imaging sample, drive the illumination beam to perform scanning motion, and drive the detected back signal beam to perform scanning motion. An intermediate image acquisition unit is used to receive the intermediate image and perform pixelation processing on the intermediate image. The intermediate image acquisition unit includes an optical fiber image transmission bundle with a beveled receiving end face. The intermediate image formed in the imaging optical path can be projected onto the beveled receiving end face of the optical fiber image transmission bundle, thereby realizing the reception and optical sampling of the intermediate image. The scanning engine includes a first scanning engine and / or a second scanning engine. Both the first and second scanning engines are two-dimensional scanning engines. The first scanning engine is driven and cooperates with the micro-light needle generating unit and the intermediate image acquisition unit. The first scanning engine is used to drive the micro-light needle generating unit and the intermediate image acquisition unit to perform two-dimensional scanning motion synchronously. The second scanning engine cooperates with the imaging optical path. The second scanning engine is used to drive the illumination beam and the back signal beam to perform two-dimensional scanning motion. The second scanning engine includes at least one set of prism pairs disposed inside the imaging optical path. The prism pair includes two coaxially arranged prisms. The two prisms can independently deflect relative to each other around their own central axis to drive the illumination beam and the back signal beam to perform two-dimensional scanning motion.

35. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The first scanning engine includes a pair of piezoelectric bicrystalline wafers or curved wafers with mutually perpendicular deflection directions, a piezoelectric transistor with two pairs of mutually perpendicular surface electrodes, or a scanning device based on a microelectromechanical system, a linear or rotary motor.

36. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The prism is a wedge-shaped prism.

37. The miniaturized scanning microscopic volumetric imaging probe according to claim 36, characterized in that: The wedge angles of the two prisms contained in the prism pair may be the same or different.

38. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The prism pair contains two prisms made of the same material.

39. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The micro-light needle generating unit includes a reflector for reflecting an illumination beam. The equivalent optical waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

40. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The micro-needle generating unit includes a beam shaping component and a reflector. The beam shaping component is used to change the shape parameters of the illumination beam to shape the illumination beam into a needle shape. The shape parameters include at least one of numerical aperture, beam waist diameter, and Rayleigh length. The reflector is used to reflect the illumination beam after it has been shaped by the beam shaping component. The equivalent beam waist of the reflected illumination beam is mirrored onto the oblique receiving end face of the intermediate image acquisition unit.

41. The miniaturized scanning microscopic volumetric imaging probe according to claim 40, characterized in that: The illumination beam shape of the light needle includes "long straight" or "curved".

42. The miniaturized scanning microscopic volumetric imaging probe according to claim 40, characterized in that: Illumination beams in the form of light needles include Gaussian beams, Bessel beams, Bessel-Gaussian beams, or Airy beams.

43. The miniaturized scanning microscopic volumetric imaging probe according to claim 40, characterized in that: The beam shaping assembly includes at least one beam shaping lens.

44. The miniaturized scanning microscopic volumetric imaging probe according to claim 43, characterized in that: The beam-shaping lens includes at least one of the following: a biconvex lens, a single lens having a biconcave, plano-convex, or biconvex spherical or aspherical surface, an achromatic lens, and a graduated refractive index lens.

45. The miniaturized scanning microscopic volumetric imaging probe according to claim 40, characterized in that: The illumination beam in the form of a light needle is a Bessel-Gaussian beam. The beam shaping assembly includes at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Alternatively, the beam shaping assembly is formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, and a micro spherical or aspherical lens with at least one of a conical lens, a diffractive optical element with an axial pyramidal holographic phase, and a metasurface lens. Or, the beam shaping assembly is formed by combining one or more diffractive optical elements with an axial pyramidal holographic phase and / or metasurface lenses.

46. ​​The miniaturized scanning microscopic volumetric imaging probe according to claim 40, characterized in that: The illumination beam in the form of a light needle is an Airy beam. The beam shaping component has a phase mask with a cubic phase structure. Alternatively, the beam shaping component is mainly formed by combining at least one of a collimating lens, a graded refractive index lens, a graded refractive index fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask having a cubic phase structure.

47. The miniaturized scanning microscopic volumetric imaging probe according to any one of claims 34-46, characterized in that: The imaging optical path includes a first objective lens, a relay optical system, and a second objective lens arranged sequentially. The front end of the first objective lens faces the imaging sample, and the front end of the second objective lens faces the intermediate image acquisition device. The second scanning engine is disposed between the first objective lens and the second objective lens. The relay optical system is used to relay the illumination beam and the scanning motion of the second scanning engine to the imaging sample located at the front end of the first objective lens and to transmit the back signal beam collected by the first objective lens to the second objective lens, and to reconstruct the intermediate image in the focal area of ​​the second objective lens.

48. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: The imaging optical path is coaxially arranged with the prism contained in the second scanning engine.

49. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: The second scanning engine is conjugately coupled to the back focal plane of the first objective lens.

50. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: The relay optical system includes a 4F system.

51. The miniaturized scanning microscopic volumetric imaging probe according to claim 34, characterized in that: The intermediate image acquisition device is used to sample the intermediate image at the optical level and then transmit it to an external image sensor to complete the digital recording of the intermediate image. Alternatively, the intermediate image acquisition device itself has photoelectric conversion function and can directly complete the pixelation and digital recording of the intermediate image.

52. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: The first scanning engine is located outside the imaging optical path. The micro light needle generating unit is fixedly coupled with the intermediate image acquisition unit. The micro light needle generating unit and the intermediate image acquisition unit can be driven by the first scanning engine to move synchronously. The intermediate image and the intermediate image acquisition unit move synchronously and remain relatively stationary.

53. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: The intermediate image remains relatively stationary with respect to the oblique receiving end face of the optical fiber image bundle.

54. The miniaturized scanning microscopic volumetric imaging probe according to claim 47, characterized in that: Both the intermediate image and the intermediate image acquisition device are stationary.

55. A miniaturized three-dimensional volumetric imaging microscope system, characterized in that, Includes the miniaturized scanning microscopic volumetric imaging probe according to any one of claims 1-33 or 34-54.

56. The miniaturized three-dimensional volumetric imaging microscope system according to claim 55, characterized in that, Also includes: A light source module, which provides an illumination beam.

57. The miniaturized three-dimensional volumetric imaging microscope system according to claim 56, characterized in that: The miniaturized three-dimensional volumetric imaging microscope system also includes an external image sensor, which is used to receive intermediate images obtained by the intermediate image acquisition unit.

58. The miniaturized three-dimensional volumetric imaging microscope system according to claim 57, characterized in that: The miniaturized three-dimensional volumetric imaging microscope system also includes a filter element, which is disposed between the external image sensor and the intermediate image acquisition unit. The filter element is used to filter out light signals within a specific wavelength range.

59. The miniaturized three-dimensional volumetric imaging microscope system according to claim 58, characterized in that: The filtering element includes at least one filter.

Images