Miniaturized scanning microscopic volume imaging probe and imaging system

Abstract

The invention discloses a miniaturized scanning microscopic volume imaging probe and an imaging system. The miniature scanning microscopic volume imaging probe comprises a miniature light sheet generating unit or a miniature light needle generating unit, the miniature light sheet generating unit is used for converting an illumination light beam into a two-dimensional light sheet form, and the miniature light needle generating unit is used for converting the illumination light beam into a light needle form; the imaging light path is used for transmitting the illumination light beam from the 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; the scanning engine is used for controlling and changing the position of the illumination light beam in the imaging sample and driving the illumination light beam to perform scanning motion back to the signal light beam; and the intermediate image collector is used for receiving the intermediate image and carrying out pixelation processing on the intermediate image. According to the invention, the defect that an imaging light path is bent at a right angle at the galvanometer is avoided, and the compactness and the thinning degree of the front-end lens barrel part are greatly improved.

Description

Technical Field

[0001] The present invention particularly relates to a miniaturized scanning microscopic volume imaging probe and an imaging system with optical tomography capability, belonging to the technical fields of optical three-dimensional microscopic imaging, biomedical optics and photonics, and medical imaging equipment. Background Art

[0002] The diagnostic pathology techniques commonly used in clinical practice, including histopathology and intraoperative frozen pathology, are essentially invasive in vitro observation methods, prone to sampling bias, and unable to provide real-time dynamic information. The next generation of diagnostic pathology calls for imaging equipment that can directly implement high spatiotemporal resolution, three-dimensional structure and function visualization of in situ in vivo tissues, but the 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 image deeply under the skin; even if fluorescent signals are introduced, their spot-illumination excitation mode cannot distinguish between in-focus signals and out-of-focus backgrounds, and lacks depth tomography (i.e., optical sectioning) and three-dimensional resolution capabilities;

[0004] 2) The 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 microns, and the image contrast comes from the backscattered light of the tissue, which cannot provide effective cellular-level tissue microstructure.

[0005] 3) Confocal or two-photon fluorescence imaging modalities have micron-level spatial resolution and depth tomography capabilities, but are limited by the point-by-point scanning mode, and their two-dimensional frame rates are at most tens of frames per second. On this basis, even if axial scanning is introduced, the rate of three-dimensional volume imaging (i.e., stacking dozens to hundreds of two-dimensional images from different depths to form three-dimensional volume data) that can be achieved (hereinafter referred to as volume frame rate) is also very limited (typical values ​​are 0.1-1 volume / second); if a larger range of three-dimensional tissue is to be explored by translating the imaging probe, it will take even longer. Considering that there are inevitably irregular movements in living tissues due to breathing, heartbeat or other factors, the aforementioned limited three-dimensional volume frame rate makes the imaging results susceptible to interference from motion artifacts, resulting in irregular three-dimensional deformation of the collected volume data, and making it impossible to align two adjacent sets of volume data.

[0006] The pursuit of high spatiotemporal resolution in the field of in vivo microscopy has given rise to the emerging swept confocally-aligned planar excitation (SCAPE) microscope in recent years. SCAPE microscopy technology cleverly utilizes the ample aperture angle of high-performance microscope objectives, uses a single objective to generate an illumination beam sheet tilted relative to the main optical axis, and collects the (backward) fluorescence excited by the inclined beam sheet - thereby combining the excitation objective and detection objective of conventional light sheet microscopes into one. While inheriting the inherent optical tomography capability and low phototoxicity advantages of light sheet microscopy, it also provides an open sample space that is compatible with model animals of different sizes and various in vivo imaging scenarios, and is naturally adapted to the optical path architecture of forward-looking imaging probes. More importantly, its confocal detection mode can complete three-dimensional tomographic scanning without moving the objective or sample, avoiding the inherent speed bottleneck of mechanically moving the main objective and the disturbance to the in vivo sample. Its three-dimensional volume imaging speed can theoretically reach thousands of volumes per second.

[0007] However, the existing standard desktop confocal oblique light sheet scanning microscope is bulky and has a highly complex core optical path. To miniaturize it into a lightweight, flexible, easy-to-control slender hard tube-shaped microscopic imaging probe suitable for in situ in vivo pathology imaging applications, it faces two prominent difficulties in optical principles: (1) The standard desktop confocal oblique light sheet scanning microscope relies on a galvanometer galvanometer mirror conjugate to the rear focal plane of the main objective to drive the oblique light sheet scanning, so that the overall optical path is bent 90 degrees with the galvanometer mirror as the inflection point, which is incompatible with the compact and slender shape required for clinical applications; (2) The standard desktop confocal oblique light sheet scanning microscope needs to directly enlarge the intermediate image reconstructed in the focal area of ​​the second objective lens to a scientific research-grade CMOS camera for sampling and recording, making it difficult to physically separate the front-end imaging probe from the bulky camera, greatly restricting the portability and ease of use of the handheld probe part.

[0008] In summary, scanning oblique light sheet microscopy technology has great application potential in in vivo label-free, non-invasive, real-time in situ in vivo pathology imaging due to its advantages such as open sample space, high resolution and high-speed three-dimensional imaging (high volume rate), but its existing desktop architecture and design ideas cannot be simply transferred to the design of miniaturized, lightweight, flexible, slender and rigid tubular handheld microscope (and microendoscopy) imaging probes. How to break away from the design conventions of desktop confocal oblique light sheet scanning microscopes and achieve high-temporal and spatial resolution three-dimensional microscopic volume imaging in a compact, slender and rigid tubular imaging probe framework, so as to visualize the three-dimensional microstructure and functional dynamics of subepidermal tissues with high sensitivity and resolution without destroying living tissues and their physiological environment, and achieve an in situ pathology imaging effect of "no need for slicing, better than slicing", which is of great significance to promoting precision diagnosis and treatment and promoting the construction of a healthy China. Summary of the invention

[0009] The main purpose of the present invention is to provide a miniaturized scanning microscopic volume imaging probe and imaging system. Through innovations in principle architecture and acquisition strategy, the two core difficulties faced in miniaturizing confocal oblique light sheet scanning microscopes are solved, making it possible to achieve high temporal and spatial resolution and three-dimensional microscopic volume imaging on a miniaturized, slender, rigid tubular microscopic (or endoscopic microscopic) imaging probe, thereby overcoming the deficiencies in the prior art.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0011] In one aspect, the present invention provides a miniaturized scanning microscopic volume 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 light beam from the light source module into a two-dimensional light sheet form, and the micro light needle generating unit is used to convert the illumination light beam from the light source module into a light needle form;

[0013] An imaging optical path, used for transmitting an illumination beam from a light source module to an imaging sample, and for collecting a back-directed signal beam emitted by the imaging sample and reconstructing the back-directed signal beam into an intermediate image;

[0014] A scanning engine for controlling and changing the position of the illumination beam in the imaging sample, driving the illumination beam to perform a scanning motion, and driving the detected back signal beam to perform a scanning motion;

[0015] The intermediate image collector is used to receive the intermediate image and perform pixelation processing on the intermediate image.

[0016] In a more typical implementation case, the scanning engine includes a first scanning engine and / or a second scanning engine, the first scanning engine cooperates with the micro-light sheet generating unit and the intermediate image collector, the first scanning engine is used to drive the micro-light sheet generating unit and the intermediate image collector to synchronously perform one-dimensional scanning movement, and the second scanning engine cooperates with the imaging optical path, the second scanning engine is used to drive the illumination light beam and the back signal light beam to perform one-dimensional scanning movement.

[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 bimorphs or bending plates with deflection directions perpendicular to each other, a piezoelectric transistor with two pairs of surface electrodes perpendicular to each other, or a scanning device based on a micro-electromechanical system, a linear or rotary motor.

[0019] Furthermore, the second scanning engine includes at least one prism pair arranged inside the imaging optical path, and the prism pair includes two coaxially arranged prisms. The two prisms can be relatively deflected around their own central axes to drive the illumination beam and the back-pointing 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 are the same or different.

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

[0023] Furthermore, when the second scanning engine drives the illumination light beam and the back-pointing signal light beam to perform translational scanning, the two prisms included in the prism pair have the same rotation angular velocity but opposite rotation directions.

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

[0025] Furthermore, the light sheet generating component includes at least one of a focusing element, a diffractive optical element with a 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 double-cemented cylindrical lens, a triple-cemented 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 assembly and the light sheet angle adjustment element are sequentially arranged on the optical path of the illumination light beam, and the light sheet angle adjustment element is used to adjust the incident angle of the illumination light beam incident on 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 body.

[0031] In a more specific implementation case, 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 light beam into a light needle shape. The third scanning engine is transmission-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 case, the light needle generating assembly includes a reflector, which is used to reflect the illumination light beam so that the main light of the reflected illumination light beam is parallel to the propagation direction of the illumination light beam in the form of a virtual light sheet, and the equivalent light waist of the reflected illumination light beam is mirrored onto the beveled receiving end surface of the intermediate image collector.

[0033] As another typical implementation case, the light needle generating component includes a beam shaping component and a reflector, the beam shaping component is used to change the morphological parameters of the illumination beam to shape the illumination beam into a light needle shape, the morphological parameters include at least one of the numerical aperture, the beam waist diameter, and the Rayleigh length, the reflector is used to reflect the illumination beam shaped by the beam shaping component, and the equivalent light waist of the reflected illumination beam is mirrored onto the beveled receiving end surface of the intermediate image collector.

[0034] Furthermore, the shape of the illumination beam in the form of a light needle includes a "long straight shape" or a "curved shape". It should be noted that the illumination beam in the form of a light needle in the present 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 component includes at least one beam shaping lens; preferably, the beam shaping lens includes at least one of a biconvex lens, a single lens with a biconcave, plano-convex or biconvex spherical or aspherical surface, an achromatic lens, and a gradient refractive index lens.

[0037] As a typical implementation case, the illumination light beam in the form of a light needle is a Bessel-Gaussian beam, and the beam shaping component includes at least one of a conical lens, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining at least one of a collimating lens, a gradient index lens, a gradient index optical fiber, a microspherical or aspherical lens with a conical lens, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining more than one diffractive optical element with an axicon holographic phase and / or a metasurface lens.

[0038] As a typical implementation case, 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, or the beam shaping component is mainly formed by combining at least one of a collimating lens, a gradient refractive index lens, a gradient refractive index optical fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask with 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 dual chips or bending plates with deflection directions perpendicular to each other, a piezoelectric transistor with two pairs of surface electrodes perpendicular to each other, or a scanning device based on a micro-electromechanical system, a linear or rotary motor.

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

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

[0043] Furthermore, the first scanning engine includes a pair of piezoelectric bimorphs or bending plates with deflection directions perpendicular to each other, a piezoelectric transistor with two pairs of surface electrodes perpendicular to each other, or a scanning device based on a micro-electromechanical system, a linear or rotary motor.

[0044] Furthermore, the second scanning engine includes at least one prism pair arranged inside the imaging optical path, and the prism pair includes two coaxially arranged prisms. The two prisms can independently deflect relative to each other around their own central axes to drive the illumination light beam and the back-pointing signal light 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 are the same or different.

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

[0048] In a typical implementation case, the micro light needle generating unit includes a reflector, which is used to reflect the illumination light beam, and the equivalent light waist of the reflected illumination light beam is mirrored onto the beveled receiving end surface of the intermediate image collector.

[0049] In another typical implementation case, the micro light needle generating unit includes a beam shaping component and a reflector, the beam shaping component is used to change the morphological parameters of the illumination beam to shape the illumination beam into a light needle shape, the morphological parameters include at least one of the numerical aperture, the beam waist diameter, and the Rayleigh length, and the reflector is used to reflect the illumination beam shaped by the beam shaping component so that the main light of the reflected illumination beam is parallel to the propagation direction of the illumination beam in the form of a virtual light sheet, and the equivalent light waist of the reflected illumination beam is mirrored onto the beveled receiving end surface of the intermediate image collector.

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

[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 with a biconcave, plano-convex or biconvex spherical or aspherical surface, an achromatic lens, and a gradient refractive index lens.

[0053] In a more typical implementation case, the illumination beam in the shape of a light needle is a Bessel-Gaussian beam, and the beam shaping component includes at least one of a conical lens, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining at least one of a collimating lens, a gradient index lens, a gradient index optical fiber, a microspherical or aspherical lens with a conical lens, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining more than one diffractive optical element with an axicon holographic phase and / or a metasurface lens.

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

[0055] Further, the imaging optical path includes a first objective lens, a relay optical system, and a second objective lens arranged in sequence, the front end of the first objective lens faces the imaging sample, the front end of the second objective lens faces the intermediate image collector, the second scanning engine is arranged 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 transmit the back signal beam collected by the first objective lens to the second objective lens, and reconstruct the intermediate image in the focal area of ​​the second objective lens;

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

[0057] Furthermore, the second scanning engine is conjugate 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 collector 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 collector itself has a photoelectric conversion function and can directly complete the pixelation and digital recording of the intermediate image.

[0060] Furthermore, the intermediate image collector includes an optical fiber image transmission bundle having an obliquely cut receiving end face, and the intermediate image formed in the imaging light path can be projected onto the obliquely cut 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 adjusted by the light sheet angle adjustment element is parallel to the beveled receiving end face of the optical fiber image transmission bundle, and the equivalent light waist of the illumination beam falls on the receiving end face of the optical fiber image transmission bundle.

[0062] Furthermore, the equivalent beam waist of the illumination light beam coincides with the focus of the second objective lens.

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

[0064] Furthermore, the intermediate image and the oblique receiving end surface of the optical fiber image transmission bundle always remain relatively still.

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

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

[0067] Furthermore, the miniaturized three-dimensional volume imaging microscope system further includes: a light source module, and the light source module is used to provide an illumination beam.

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

[0069] Furthermore, the miniaturized three-dimensional volume imaging microscope system also includes a filter element, which is arranged between the external image sensor and the intermediate image collector, and is used to filter out light signals in a specific wavelength range.

[0070] Furthermore, the filter 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, and breaks away from the design convention of standard desktop oblique light sheet scanning microscopes that rely on reflective galvanometer scanning galvanometers or micro-electromechanical systems (MEMS) scanning galvanometers, and avoids the disadvantage of the imaging light path being bent at a right angle at the galvanometer, so that the main objective lens, relay lens group, second objective lens, etc. can be placed coaxially, greatly improving the compactness and slimness of the front end barrel part.

[0073] 2) The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which has an innovative intermediate image collector design and a corresponding de-scanning detection strategy, so that the bulky camera can be separated from the imaging probe, while achieving high-speed three-dimensional microscopic volume imaging, ensuring that the imaging probe is small, light and flexible.

[0074] 3) Compared with wide-field illumination endoscopes, the miniaturized microscopic imaging probe provided by the present invention has depth tomography (i.e., optical slicing capability) and three-dimensional volume imaging capabilities, and can clearly visualize the three-dimensional microstructure of subepidermal tissue. Compared with the optical coherence tomography (OCT) imaging modality based on backscattered light, the miniaturized three-dimensional microscopic volume imaging probe provided by the present invention can be based on fluorescence signal imaging, and can provide molecular sensitivity, subcellular spatial resolution, and richer image contrast information.

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

[0076] 5) Compared with the desktop confocal oblique light sheet scanning microscope, the present invention, while maintaining its advantage of high-speed three-dimensional volume imaging, avoids the disadvantage of the former's imaging light path being bent at a right angle at the galvanometer through an innovative illumination beam (or light sheet) scanning engine and scanning strategy, and through an innovative intermediate image and de-scanning detection strategy, the bulky camera can be separated from the imaging probe, greatly improving the compactness and slimness of the imaging probe, thereby achieving high temporal and spatial resolution, in situ in vivo pathology imaging "without slicing, better than slicing" on a miniaturized, slender hard tubular microscope (or endoscopic microscope) imaging probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 It is a structural schematic diagram of a miniaturized three-dimensional volume imaging microscope system provided in a typical implementation case of the present invention;

[0078] Figure 2a It is a schematic diagram of the structure of a miniaturized volume imaging probe based on a sheet illumination beam and a rear light sheet scanning engine;

[0079] Figure 2b To explain the working principle of the micro light sheet generating unit based on negative refractive power cylindrical mirror and the overall design and structural diagram of its assembly combination with the intermediate image collector and scanning engine;

[0080] Figure 2c To explain the working principle of the micro light sheet generating unit based on the positive refractive cylindrical mirror and the overall design and structural schematic diagram of its assembly combination with the intermediate image collector and the scanning engine;

[0081] Figure 2d To explain the working principle of the micro light sheet generating unit based on negative refractive power cylindrical mirror and polygonal prism and the overall design and structural schematic diagram of its assembly combination with intermediate image collector and scanning engine;

[0082] Figure 2e To explain the working principle of the micro light sheet generating unit integrating the negative refractive power cylinder and the polygonal prism and the overall design and structural diagram of its assembly combination with the intermediate image collector and the scanning engine;

[0083] Figure 3a It is a schematic diagram of the system structure of a miniaturized volume imaging probe based on the scanning of a sheet illumination beam and a centrally placed wedge-shaped prism pair, and shows a schematic diagram of the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the negative y limit position;

[0084] Figure 3b For the corresponding Figure 3a The design in the diagram shows the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the center position of the y-axis;

[0085] Figure 3c For the corresponding Figure 3a The design in the diagram shows the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the positive y limit position;

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

[0087] Figure 4b To explain the working principle of the illumination beam needle generating unit that directly uses the output light of the single-mode optical fiber, and the overall design and structural schematic diagram of its assembly combination with the slow-axis scanner, the intermediate image collector, and the fast-axis scanning engine;

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

[0089] Figure 4d To explain the working principle of the illumination beam needle generation unit using GRIN lens and aconic lens to generate Bessel beam, and the overall design and structural diagram of its assembly combination with slow axis scanner, intermediate image collector 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 three-dimensional phase mask to generate an Airy beam, and the overall design and structural diagram of its assembly combination with a slow-axis scanner, an intermediate image collector, and a fast-axis scanning engine;

[0091] Figure 5a It is a schematic diagram of the system structure of a miniaturized volume imaging probe based on the scanning of a virtual illumination beam sheet and a centrally placed wedge-shaped prism pair, and shows a schematic diagram of the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the negative y limit position;

[0092] Figure 5b Shows the corresponding Figure 5a The design in the diagram shows the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the center position of the y-axis;

[0093] Figure 5c Shows the corresponding Figure 5a The design in the diagram shows the illumination beam path and the back signal beam path in the yz plane view (upper part) and the illumination beam path in the xy plane view (lower part) when the oblique light sheet is scanned to the positive y limit position;

[0094] Figure 6 A schematic diagram of the system structure of a miniaturized volume imaging probe based on a needle-shaped illumination beam and a rear-mounted two-dimensional independent control scanning engine, as well as an example of a possible two-dimensional scanning trajectory;

[0095] Figure 7 Schematic diagram of the system structure of a miniaturized three-dimensional microscopic volume imaging probe based on two-dimensional light needle scanning of a centrally placed asynchronous scanning wedge prism pair, and shows an example of a scanning position of the illumination beam and the corresponding back signal beam path (marked only in the yz plane) in the yz plane view (upper half) and the xy plane (lower half) view. DETAILED DESCRIPTION

[0096] In view of the deficiencies in the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution, its implementation process and principles, etc. will be further explained in conjunction with the accompanying drawings and specific implementation cases. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for the convenience of description, only the parts related to the present invention, rather than all structures, are shown in the accompanying drawings. It should be noted that the various optical components used in the present invention are known to those skilled in the art, and they can all be purchased on the market, and their specific product models are not limited here.

[0097] See also Figure 1 , Figure 1 It is a miniaturized three-dimensional volume imaging microscope system, including a light source module, a front-end imaging probe (i.e. the aforementioned miniaturized scanning microscopic volume imaging probe, the same below) and an external image sensor (optional). The arrows in the figure are the transmission paths of the illumination beam and the backlight signal / beam.

[0098] The core of the miniaturized 3D volume imaging microscope system is the front-end imaging probe, which will directly contact the imaging sample (especially living tissue) and implement volume microscopic imaging. The front-end imaging probe generally includes the following modules:

[0099] Micro light sheet generating unit: used for converting the illumination beam from the light source module into a two-dimensional light sheet shape (form), and the illumination beam in the shape of a light sheet can also be called an illumination light sheet; the micro light sheet generating unit can use cylindrical lenses such as cylindrical lenses and Powell lenses to directly generate sheet-shaped or layered illumination beams, or can use fast-scanning needle-shaped illumination beams (i.e., light needles, specifically including Gaussian beams, Bessel beams, Airy beams, etc.) to form virtual light sheets (also called digital light sheets). It should be pointed out that the specific shape of the virtual light sheet generated by the second method is related to the trajectory of the fast scanning;

[0100] The scanning engine is a non-galvanometer scanning engine, which is mainly 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-pointing signal beam; it should be noted that "non-galvanometer" means that unlike the traditional galvanometer or MEMS scanning galvanometer, which realizes beam scanning by changing the angle of a certain reflective surface, the scanning engine adopts other working principles to realize the scanning motion of the beam or light sheet in the downstream imaging optical path. The 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 conjugate coupled with the rear focal plane of the main objective lens; "rear" means that the scanning engine is located outside the imaging optical path;

[0101] Imaging optical path: used to transmit the illumination beam to the imaging sample, collect the back signal beam (fluorescence or scattered light) emitted by the imaging sample, and transmit the back signal beam back to the end away from the imaging sample for collection; the 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, the front end of the second objective lens faces the image acquisition module, and the relay optical system generally uses a 4f-system with a suitable 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 transmit the back signal beam collected by the main objective lens to the second objective lens; when a central scanning engine is used, the optical path of the central scanning engine may also become a part of the relay optical system;

[0102] It should be noted that the overall optical design of the imaging light path should minimize aberrations so that the illumination beam or light sheet can be transmitted to the imaging sample with high fidelity, and the back signal beam generated by the area illuminated by the illumination beam / light sheet in the imaging sample can be collected and transmitted back to the second objective lens with high fidelity, and an intermediate (optical) image with the smallest possible aberration can be reconstructed in the focal area of ​​the second objective lens.

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

[0104] In some embodiments, the intermediate image collector samples the intermediate image at the optical level. For example, a special optical fiber element 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 other embodiments, the intermediate image collector itself may have a photoelectric conversion function, for example, by using a miniaturized image sensor to directly complete the pixelation 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 other embodiments, the intermediate image is not stationary. In this case, the intermediate image collector moves synchronously with the intermediate image and remains relatively stationary, thereby achieving efficient "de-scanning" acquisition of the intermediate image.

[0107] Specifically, the light source module preferably uses a laser light source, and may also use light-emitting diodes, lamps, and various light sources emitted after being transmitted through optical fibers; the light source module may include multiple groups of physically separated light source devices, may include 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 may 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 collector of the aforementioned front-end imaging probe does not have the photoelectric conversion function, it is necessary to use an external image sensor separated from the imaging probe. Specifically, the external image sensor includes but is not limited to a charge-coupled device (CCD), a complementary metal oxide semiconductor (CMOS), a single photon avalanche diode (SPAD) or a SPAD array, a photomultiplier tube (PMT) or a PMT array, a silicon photomultiplier tube (SiPM) or a SiPM array. 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 the first type of embodiments of the present invention, the micro-light sheet generating unit directly generates a sheet-shaped illumination light beam, which is relayed to the front end of the probe via the imaging optical path and incident on the sample to be imaged, and is scanned translationally under the drive of the scanning engine. The excited back-signal light 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 light sheet three-dimensional microscopic volume 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 light beam is transmitted to the micro-light sheet generating unit 12 at the rear end of the probe through the single-mode optical fiber 11. The thin light sheet emitted from the micro-light sheet generating unit 12 enters the imaging light 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 inclination angle relative to the main optical axis, and generates an inclined illumination light beam / sheet in the focal area of ​​the first objective lens 136. The excited back-signal light beam (fluorescence or scattered light, etc.) is returned via the imaging light path, forming an inclined intermediate image in the focal area of ​​the second objective lens 131, and is received by the intermediate image collector.

[0113] In this embodiment, the intermediate image collector uses a fiber optic imaging bundle 14 with a beveled receiving end face. By adjusting the placement orientation of the fiber optic imaging 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 fiber optic imaging bundle 14. The intermediate image is received and optically sampled by the fiber optic array and then transmitted back to the camera and host at the remote end for image recording, display, and processing.

[0114] In order to realize light sheet scanning and descanning detection, the present embodiment binds the micro light sheet generating unit 12 and the optical fiber image transmission bundle 14 together through the scanning cantilever 15, and uses the piezoelectric driver (i.e., scanning engine) 16 to drive the two to move synchronously; on the one hand, the illumination beam / sheet is moved along the y-axis direction (see Figure 2a Secondly, since the optical fiber imaging bundle 14 and the micro-light sheet generating unit 12 are scanned synchronously, according to the principle of optical path reversibility, the intermediate image formed in the focal area of ​​the second objective lens 131 always remains relatively still with the oblique receiving end face of the optical fiber imaging bundle 14 during the scanning process, thereby cleverly ensuring the de-scanning, collection and reception of the intermediate image.

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

[0116] Figure 2b A specific implementation scheme of the micro-light sheet generating unit 12 is shown. The illumination light beam emitted from the single-mode optical fiber 11 is first collimated by a gradient refractive index (GRIN) lens 121, then focused into a light sheet form by a micro cylindrical mirror 122, and finally reflected by a micro reflector 123 to achieve a suitable inclination angle relative to the optical axis of the imaging light path. Figure 2b The micro cylindrical mirror 122 shown has a negative refractive power and can diverge the collimated illumination beam emitted from the gradient refractive index lens 121 along the y-axis direction shown in the figure to form an equivalent thin light sheet with a width direction along the x-axis; the installation orientation of the micro 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 transmission bundle 14, and the equivalent light waist ( Figure 2b (2) the equivalent light waist coincides with the focus of the second objective lens 131 to optimize the spatial resolution of the system.

[0117] Figure 2c Another specific implementation scheme of the micro light sheet generating unit 12 is shown, which adopts a cylindrical mirror 124 with positive refractive power, that is, focusing along the y-axis direction to generate a thin light sheet. By designing and optimizing the relative positions of the positive refractive 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 optical fiber imaging bundle 14, and the equivalent light waist can overlap with the focus (or front focal plane) of the second objective lens 131.

[0118] In an alternative implementation, the micro light sheet generating unit 12 may also use spherical cylindrical mirrors of plano-concave, plano-convex, bi-concave, bi-convex, meniscus or other surface types, or double-cemented, triple-cemented or other cylindrical mirrors (to better eliminate chromatic aberration), aspherical cylindrical mirrors including Powell lenses, flat top light shapers, etc., or a combination of the above various types of cylindrical mirrors, to generate an illumination beam / sheet and optimize its uniformity. In an alternative implementation, the micro light sheet generating unit 12 may also use a combination of a pyramid cylindrical mirror and a slit mask, or other design solutions familiar to professionals in the field, to generate a Bessel light sheet with an extended depth of field.

[0119] In an alternative implementation, the micro-light sheet generating unit 12 may also introduce a diffractive optical element or a metasurface element / lens with a beam shaping function, and may combine the diffractive element or metasurface lens with the aforementioned various types of cylindrical mirrors or axicon mirrors, etc., to optimize and control the geometric shape of the generated thin light sheet to meet the needs of actual imaging applications.

[0120] Figure 2b and Figure 2c The implementation schemes of the micro light sheet generating unit 12 shown all use a micro reflector 123 to control the output 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 to adapt to the assembly and scanning load requirements.

[0121] Figure 2dAnother specific implementation scheme of the micro light sheet generating unit 12 is shown, which adopts a polygonal prism 125 to adjust the inclination angle of the illumination beam / sheet (relative to the main optical axis); its reflective surface can be based on the total internal reflection mechanism, or it can be plated with metal or dielectric film on the surface; in the corresponding alternative implementation scheme, the polygonal prism can have any shape, the light sheet can be reflected once or multiple times inside the prism, and can be refracted when it is emitted, and the specific design is based on the orientation of the output light sheet required by the imaging system.

[0122] Figure 2e Another specific implementation scheme of the micro-light sheet generating unit 12 is shown. Figure 2d The micro cylindrical mirror 122 for forming the light sheet and the polygonal prism 125 for adjusting the tilt angle of the light sheet are integrated into a single device 126. In a corresponding alternative implementation scheme, the refractive cylindrical surface and other reflective prism surfaces of the device 126 can also be designed to have a conic curve cylindrical surface or a generalized aspherical surface type, so as to further adjust the emission direction, divergence degree and position of the equivalent light waist of the illumination beam / sheet.

[0123] In such Figure 2b-Figure 2e In all the implementation schemes and alternative implementation schemes of the micro-light sheet generating unit shown, the gradient refractive index lens 121 used for collimation can be replaced by a gradient refractive index optical fiber, which can be directly fused with the single-mode optical fiber 11; it can also be replaced by a micro-spherical or aspherical lens or a diffractive optical element or a super-surface lens, etc., to achieve the purpose of beam shaping. It is particularly important to point out that in the alternative embodiments, after the light emitted from the single-mode optical fiber passes through the gradient refractive index lens 121 or the gradient refractive index optical fiber or the micro-spherical or aspherical lens or the diffractive optical element or the super-surface lens, etc., it may not be completely collimated, but may have a certain degree of convergence or divergence, as long as it is guaranteed that it can generate an illumination beam / sheet that meets the application requirements after being used in combination with subsequent optical elements.

[0124] In such Figure 2a-2e In the embodiment or implementation of the micro-light sheet generating unit shown, the scanning engine preferably uses a piezoelectric driver 16, and the specific implementation can be a bar-shaped piezoelectric bimorph, or a ring-shaped or cylindrical piezoelectric transistor or any type of piezoelectric bender, etc., which can drive the scanning cantilever, thereby driving the micro-light sheet generating unit and the optical fiber image transmission bundle to synchronize the scanning motion of the piezoelectric driving device. In alternative embodiments, the scanning engine can also use a micro-electromechanical system (MEMS) scanner, a micro-motor, or other driving devices based on various other transducer principles to meet the one-dimensional linear motion required for light sheet scanning.

[0125] In such Figure 2a-2eIn the illustrated embodiment or the implementation scheme of the micro-light sheet generating unit, the intermediate image collector preferably uses a fiber optic image bundle 14 with a beveled receiving end face, and the inclination angle of the beveled receiving end face relative to the optical axis of the imaging light path (hereinafter referred to as the main optical axis) should be consistent with the inclination angle of the illumination beam / sheet relative to the main optical axis. It is particularly important to point out that the inclination angle of the beveled receiving end face of the fiber optic image bundle relative to the main optical axis and the bevel angle of the beveled receiving end face itself (i.e., the inclination angle of the beveled receiving end face relative to the central axis of the fiber optic image bundle or the bevel angle of the end face of each component optical fiber in the fiber optic image bundle) can be independently controlled. In view of the fact that the end face bevel angle of a single-mode or multi-mode optical fiber waveguide will affect the direction of its emission and receiving light cones, in an alternative embodiment, the end face bevel angle of the fiber optic image bundle can be optimized to maximize the collection efficiency of the back-directed signal beam while ensuring that the entire receiving end face coincides with the intermediate image.

[0126] In alternative embodiments, the intermediate image collector may also use any other type of array detector with a suitable size (generally small enough), including charge-coupled device (CCD), intensified CCD (ICCD), electron magnified CCD (EMCCD) or complementary metal-oxide-semiconductor (CMOS) image sensor, as well as multi-anode photomultiplier tube (PMT), multi-anode silicon photomultiplier (Si-PMT) or avalanche photodiode (APD) array, etc. In addition, microlens arrays may be provided in front of these array detectors for focusing light or analyzing light field information.

[0127] In such Figure 2a In the embodiment shown, the second objective lens 131 and the first objective lens 136 are coupled by two groups of 4f system relays; in an alternative embodiment, any multiple groups of relay 4f systems may be included between the second objective lens 131 and the first objective lens 136 to flexibly adjust the length of the lens barrel to meet the needs of the actual imaging scene. The relay 4f system involved may be composed of a single lens, an achromatic lens or a gradient refractive index lens (GRIN lens), or a Hopkins rod lens (Hopkins rodlens) relay system, etc.

[0128] Example 2

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

[0130] In this embodiment, the illumination light beam emitted from the light source module is input from the single-mode optical fiber 11 to the rear end of the probe, and enters the micro-light sheet generating unit 12 from the end of the single-mode optical fiber. The thin light sheet emitted from the micro-light sheet generating unit 12 enters the imaging light 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 inclination angle relative to the main optical axis, wherein the wedge prism pair scanner 21 (i.e., the optical wedge) is composed of two coaxially arranged first wedge prisms (also called optical wedge, Risley prism) 211 and second wedge prisms (also called optical wedge, Risley prism) 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 light beam can be controlled.

[0131] In this embodiment, the two wedge-shaped prisms rotate at the same (angular) speed and in opposite directions, so that the light beam can be controlled Figure 3a-3c Specifically, when the two wedge-shaped prisms are rotated until their thickest and thinnest parts are aligned (as shown in FIG. Figure 3a and Figure 3c As shown), the entire wedge-shaped prism is equivalent to an isosceles prism with a vertex angle equal to the sum of the two wedge angles for the scanner 21. At this time, the deflection angle of the illumination light beam after passing through the prism reaches the positive direction of the y-axis ( Figure 3c ) or negative ( Figure 3a ); when the two are rotated until the thickest (thinnest) part of the first wedge-shaped prism 211 is aligned with the thinnest (thickest) part of the second wedge-shaped prism 212, the wedge surfaces of the two wedge-shaped prisms are parallel to each other, and the entire wedge-shaped prism pair scanner 21 can be equivalent to a parallel glass plate, and the light beam does not deflect at an angle 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 deflection degree of the light beam 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 light beam and the illumination light beam / sheet in the focal area of ​​the first objective lens 236 can be controlled.

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

[0133] It should be noted that, depending on the specific scanning requirements, the wedge-shaped prism can be driven to rotate synchronously and continuously with the scanner 21, that is, after Figure 3a to Figure 3b To Figure 3c can also drive the wedge-shaped prism to the scanner 21 from such Figure 3a The state shown evolves to Figure 3b After the state shown, reverse the rotation direction of the two wedge prisms, from Figure 3b The status shown returns to Figure 3a The state shown; any desired wedge prism steering and angle range can also be formulated according to application requirements.

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

[0135] exist Figure 3a-3c In the embodiment, the second objective lens 231 and the wedge prism pair scanner 21, and the wedge prism pair scanner 21 and the first objective lens 236 are coupled through a set of 4f relay systems; in alternative embodiments, any multiple sets of relay 4f systems can be introduced between the second objective lens 231 and the wedge prism pair scanner 21, and the wedge prism pair scanner 21 and the first objective lens 236 to adjust the length of the lens barrel part to meet the needs of the actual imaging scene. 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 the alternative embodiments, the specific implementation schemes that can be adopted by the micro light sheet generation unit and the intermediate image collector 17 are the same as those described in the above embodiment 1, and will not be repeated here.

[0137] In this embodiment, the micro light sheet generating unit 12 is simplified to an illumination beam needle generating unit, and the generated one-dimensional illumination beam needle generates a virtual illumination beam / sheet (also called 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, and is 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 collector 17.

[0138] Example 3

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

[0140] In this embodiment, after the illumination beam is transmitted into the probe through the single-mode optical fiber 11, it is not directly converted into a sheet-shaped illumination beam, but a one-dimensional illumination "light needle" of "long straight shape" (such as Gaussian beam or Bessel beam) or "bent shape" (such as Airy beam) is generated by the micro light needle generating unit 32. The illumination beam is rapidly scanned along the x-axis direction in the figure under the drive of the fast axis scanner 33 to generate a virtual illumination beam / sheet (also called digital light sheet); the virtual illumination beam / sheet is incident on the imaging sample through the imaging optical path, and the excited back signal beam (such as fluorescence or scattered light) is returned to the focal area of ​​the second objective lens 131 through the imaging optical path to form an inclined virtual intermediate image, which is projected onto the receiving end surface of the intermediate image collector 17. The micro light needle generating unit 32, the fast axis scanner 33, and the intermediate image collector 17 are synchronously moved along the y-axis direction in the figure under the drive of the slow axis scanner 34, realizing the scanning of the virtual light sheet along the slow axis direction, and making the virtual intermediate image and the intermediate image collector 17 relatively stationary, realizing the de-scanning reception of the intermediate image.

[0141] The micro optical needle generating unit required by this embodiment may have a variety of specific implementation schemes.

[0142] Figure 4bA specific implementation scheme of this embodiment is shown (focusing on the micro light needle generation unit and the scanning engine, omitting the imaging optical path). This scheme directly uses the output light of the single-mode optical fiber 11 as the illumination beam needle. After being reflected by the micro-reflector 321, the main light of the illumination beam needle is parallel to the z' axis of the corresponding virtual light sheet propagation direction, and the light waist is mirrored to the beveled receiving end surface of the optical fiber image transmission bundle 14 used as the intermediate image collector 17. The single-mode optical fiber 11 and the micro-reflector 321 are fixed to the fast-axis scanner 33 through the fast-axis scanning adapter 322, and are scanned along the x-axis direction under the drive of the latter to generate a virtual illumination beam / sheet (i.e., a digital light sheet) parallel to the xz' plane. The fast axis scanner 33 (and the optical fiber 11, micro-reflector 321 and fast axis scanning adapter 322 connected thereto) and the optical fiber imaging bundle 14 are fixed on the slow axis scanner 34 through the slow axis scanning adapter 341, and are driven by the latter to scan along the y-axis (i.e., slow axis) direction, so that the virtual light sheet is scanned along the slow axis direction in the imaging sample after passing through the imaging optical path; the intermediate image generated by the back-pointing signal light beam returned by the imaging optical path falls on the receiving end face of the optical fiber imaging bundle 14, and moves synchronously with the receiving end face and is relatively stationary, thereby realizing the de-scanning reception of the intermediate image.

[0143] In such Figure 4c In the alternative embodiment shown (focusing on the micro-light needle generation unit and the scanning engine, omitting the imaging optical path), the illumination light beam emitted from the single-mode optical fiber 11 first passes through the beam shaping lens 323 to generate an illumination light 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 principles of this solution are similar to Figure 4a In this implementation, the beam shaping lens 323 preferably uses a micro biconvex lens; in an alternative embodiment, one or more single lenses, achromatic lenses, gradient refractive index lenses, etc. with spherical or aspherical surfaces such as biconcave, plano-convex, or biconvex can also be used.

[0144] Figure 4dAn alternative embodiment based on a Bessel beam is shown (focusing on the micro-light needle generation unit and the scanning engine, and omitting the imaging optical path). The illumination light beam emitted from the single-mode optical fiber 11 is first collimated by the collimating lens 324, and then passes through the micro-axicon 325 (also called axicon) to generate an illumination light beam needle in the form of a Bessel beam (strictly speaking, a Bessel-Gaussian beam). Compared with a Gaussian beam with the same waist diameter, a Bessel beam has a longer equivalent waist length and can maintain uniform lateral resolution over a longer axial range. In some alternative embodiments, the micro-axicon 325 can also be replaced by a device such as a diffractive optical element or a metasurface lens with an axicon holographic phase; other methods of generating (quasi) Bessel beams, including annular pupils, that are well known to researchers in the field can also be used.

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

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

[0147] In such Figure 4a-4eIn the (alternative) embodiment shown, the slow-axis scanner 34 and the fast-axis scanner 33 preferably use long strip-shaped piezoelectric bimorphs, and the deflection directions of the two are perpendicular to each other; the slow-axis scanner 34 can drive the beveled optical fiber image transmission bundle 14 for intermediate image acquisition, the fast-axis scanner 33, and the micro-light needle generating unit 32 (driven by the fast-axis scanner 33) to move synchronously with the help of the slow-axis scanning adapter 341. In alternative embodiments, the fast-axis and slow-axis scanners can also use piezoelectric transistors or micro-electromechanical systems (MEMS) drivers, micro linear motors, and other driving devices based on various transducer principles that can implement one-dimensional linear scanning motion.

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

[0149] Example 4

[0150] Figure 5a-5c A three-dimensional microscopic volume imaging probe based on a virtual oblique light sheet and a centrally placed wedge prism 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 through the single-mode optical fiber 11 and enters the micro light needle generating unit 32. The one-dimensional illumination "light needle" of "long straight shape" (such as Gaussian beam or Bessel beam) or "bent shape" (such as Airy beam) emitted from the micro light needle generating unit 32 is 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 digital light sheet). The 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 pair 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 inclination angle relative to the main optical axis. The wedge prism pair scanner 21 includes a first wedge prism (also called optical wedge, Risley prism) 211 and a second wedge prism (also called optical wedge, Risley prism) 212 coaxially arranged with the same wedge angle; by controlling the relative rotation of the two around the central axis, the deflection of the light beam can be controlled, thereby controlling the virtual light sheet to scan along the slow axis (the y-axis shown in the figure).

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

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

[0154] In this embodiment and alternative embodiments, the optional specific implementation scheme of the intermediate image collector 17 is the same as that described in Embodiments 1-3 and will not be repeated here.

[0155] In this embodiment, the first wedge-shaped prism 211 and the second wedge-shaped prism 212 are installed in a plane-to-plane manner. In an alternative embodiment, the two may also be installed 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 the alternative embodiment, the control logic of the wedge-shaped prism on the scanner 21 is the same as that described in Embodiment 2, that is, the angular velocities of the two are the same, the rotation directions are opposite, and according to the specific scanning requirements, the wedge-shaped prism can go through Figure 5a to Figure 5b To Figure 5c The full status of Figure 5a The state shown evolves to Figure 5b The status shown returns to Figure 5a Other steering and turning angle ranges can also be formulated according to application requirements.

[0157] exist Figure 5a-5c In the embodiment shown, the second objective lens 231 and the wedge prism pair scanner 21, and the wedge prism pair scanner 21 and the first objective lens 236 are coupled through a set of 4f relay systems. In alternative embodiments, there can be any number of relay 4f systems between the second objective lens 231 and the wedge prism pair scanner 21, and between the wedge prism pair scanner 21 and the first objective lens 236 to adjust the length of the lens barrel to meet the needs of the actual imaging scene. The relay 4f system involved can be composed of a single lens, an achromatic lens or a gradient refractive index lens (GRiN lens), and a Hopkins rod lens (Hopkins rodlens) relay system can also be used.

[0158] In the third type of embodiments of the present invention, the scanning trajectory of the miniaturized illumination beam needle does not need to follow the common raster scan trajectory (in other words, there is no need to first generate a digital scanning virtual light sheet), but an arbitrary scanning trajectory can be optimized and designed according to application requirements; accordingly, the scanning engine does not need to explicitly distinguish between fast-axis and slow-axis scanners, but can use an arbitrary two-dimensional scanning engine; the two-dimensional scanning engine also scans the generated back-signal light beam directly in the xy direction simultaneously to generate a stationary (essentially one-dimensional) intermediate image relative to the intermediate image collector.

[0159] Example 5

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

[0161] In this embodiment, the micro light needle generating unit 32 is bound to the intermediate image collector 18, and is scanned 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 collector 18, and during the scanning process, the one-dimensional intermediate image moves synchronously with the intermediate image collector 18, thereby maintaining relative stillness, thereby realizing de-scanning detection of the intermediate image.

[0162] In the present embodiment, the two-dimensional scanning driver 51 preferably uses a pair of piezoelectric dual chips or bending plates whose deflection directions are perpendicular to each other. In an alternative embodiment, the two-dimensional scanning driver 51 may also use a piezoelectric transistor having two pairs of surface electrodes perpendicular to each other. By applying a suitable dual-path voltage waveform, the two-dimensional scanning driver 51 can drive the scanning cantilever composed of the micro-light needle generating unit 32 and the intermediate image collector 18 to deflect along the x-axis and y-axis directions, thereby obtaining the required scanning trajectory. It should be pointed out that the two-dimensional piezoelectric driver can output sufficient swing amplitude to directly drive the scanning cantilever to deflect and scan, and can also work near the resonant frequency of the scanning cantilever, thereby using resonance 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, and scanning trajectories suitable for covering several areas of interest such as sub-field random jump scanning can also be generated (see Figure 6 In alternative embodiments, the two-dimensional scanning driver 51 may also be a scanning device based on other transducer mechanisms such as a micro-electromechanical system (MEMS), a linear or rotary motor, etc.

[0163] Different from the above-mentioned embodiments, the intermediate image collector 18 of this embodiment only needs to collect a one-dimensional intermediate image. In the specific implementation, a beveled one-dimensional or two-dimensional optical fiber image transmission bundle is preferably used, and the efficiency of collecting the back signal beam can be maximized by optimizing the design of its refractive index, end bevel angle, etc. After the received one-dimensional intermediate image is transmitted back to the far end along the optical fiber image transmission bundle, it can be directly mapped to a one-dimensional linear array detector to complete photoelectric conversion and digital recording. A rescanning unit can also be introduced to tile the one-dimensional intermediate images corresponding to different time points (thus originating from different scanning positions) onto a two-dimensional array detector, thereby making full use of the speed advantage of commercial two-dimensional array detectors to complete photoelectric conversion and digital recording. Here, the one-dimensional linear array detector can be a linear array CCD, ICCD, EMCCD or CMOS camera, and can also be a linear array multi-anode photomultiplier tube (PMT), a linear array silicon photomultiplier tube (SiPM), a linear array avalanche photodiode (APD), etc.; the two-dimensional area array detector can be an area array CCD, ICCD, EMCCD or CMOS camera, and can also be an area array PMT, an area array silicon photomultiplier tube (SiPM), an area array avalanche photodiode (APD) and other sensors.

[0164] In some alternative embodiments, the intermediate image collector 18 can use an optical fiber array or a multi-core optical fiber. 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 (photodiodes), photomultiplier tubes (PMTs), silicon photomultiplier tubes (SiPMs), or avalanche photodiodes (APDs), and photoelectric conversion and digital recording are completed in parallel. The above optical signal separation and transmission process is very natural for optical fiber arrays; for multi-core optical fibers, it can be completed with the help of optical fiber splitters.

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

[0166] Example 6

[0167] Figure 7 A 3D microscopic volume imaging probe based on 2D optical needle scanning and asynchronous scanning wedge prism pairs is demonstrated.

[0168] Unlike the first wedge prism 211 and the second wedge prism 212 constituting the wedge prism pair scanner 21 in Examples 2 and 4, which need to rotate synchronously (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, so that any two-dimensional scanning trajectory can be generated. 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 spiral scanning and Lissajous scanning can be generated, and scanning trajectories suitable for local field of view coverage such as random jump scanning between a number of delimited sub-fields of view can also be generated.

[0169] In this embodiment, the micro light needle generating unit 32 and the intermediate image collector 18 remain stationary. The principle of optical path reversibility ensures that the one-dimensional intermediate image generated by the generated back-pointing signal light beam in the focal area of ​​the second objective lens 231 can be accurately projected onto the receiving end surface of the (one-dimensional) intermediate image collector 18, thereby realizing the de-scanning 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 and Figure 4b-4e The optional implementation scheme for the (one-dimensional) intermediate image collector 18 is the same as that described in Embodiment 5, and will not be repeated here.

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

[0172] In this embodiment, the second objective lens 231 and the wedge prism pair scanner 21, and the wedge prism pair scanner 21 and the first objective lens 236 are coupled through a set of 4f relay systems. In alternative embodiments, there can be any multiple sets of relay 4f systems between the second objective lens 231 and the wedge prism pair scanner 21, and between the wedge prism pair scanner 21 and the first objective lens 236 to adjust the length of the barrel part to meet the needs of the actual imaging scene. 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.

[0173] It should be particularly pointed out that in Examples 5 and 6, the illumination beam needle preferably uses a straight beam such as a Gaussian beam or a Bessel beam, so that the obtained one-dimensional intermediate image is also in a straight line shape, which is consistent with the pixel arrangement habits of common linear array or planar array detectors. In corresponding alternative embodiments, if an illumination beam needle based on a curved shape such as an Airy beam is selected - thereby obtaining a curved intermediate image - the (one-dimensional) intermediate image collector 18 may preferably use a (customized) optical fiber array, a multi-core optical fiber or a linear array photoelectric detector whose pixel arrangement is consistent with the curved intermediate image, or a fiber optic image transmission bundle or a planar array detector with a sufficiently large receiving end surface.

[0174] The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which have an innovative illumination beam (or light sheet) scanning engine and scanning strategy, and break away from the design convention of standard desktop oblique light sheet scanning microscopes that rely on reflective galvanometer scanning galvanometers or micro-electromechanical systems (MEMS) scanning galvanometers, and avoid the disadvantage that the imaging light path is bent at a right angle at the galvanometer, so that the main objective lens, relay lens group, second objective lens, etc. can be placed coaxially, greatly improving the compactness and slenderness of the front end barrel part.

[0175] The present invention provides a miniaturized scanning microscopic volume imaging probe and imaging system, which have an innovative intermediate image collector design and a corresponding de-scanning detection strategy, so that a bulky camera can be separated from the imaging probe, while achieving high-speed three-dimensional microscopic volume imaging and ensuring that the imaging probe is small, light and flexible.

[0176] Compared with wide-field illumination endoscopes, the miniaturized microscopic imaging probe provided by the present invention has depth tomography (i.e., optical slicing capability) and three-dimensional volume imaging capabilities, and can clearly visualize the three-dimensional microscopic structure of subepidermal tissue. Compared with the optical coherence tomography (OCT) imaging modality based on backscattered light, the miniaturized three-dimensional microscopic volume imaging probe provided by the present invention can be based on fluorescence signal imaging, and can provide molecular sensitivity, subcellular spatial resolution, and richer image contrast information.

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

[0178] Compared with the desktop confocal oblique light sheet scanning microscope, the present invention, while maintaining its advantage of high-speed three-dimensional volume imaging, avoids the disadvantage of the former that the imaging light path is bent at a right angle at the galvanometer through an innovative illumination beam (or light sheet) scanning engine and scanning strategy. The innovative intermediate image and de-scanning detection strategy allows the bulky camera to be separated from the imaging probe, greatly improving the compactness and slenderness of the imaging probe, thereby achieving high temporal and spatial resolution, in situ in vivo pathology imaging that "does not require slicing, but is better than slicing" on a miniaturized, slender, rigid tubular microscope (or endoscopic microscope) imaging probe.

[0179] It should be understood that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A miniaturized scanning microscopic volume imaging probe, characterized in that: include: 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 light beam from the light source module into a two-dimensional light sheet form, and the micro light needle generating unit is used to convert the illumination light beam from the light source module into a light needle form; An imaging optical path, used for transmitting an illumination beam from a light source module to an imaging sample, and for collecting a back-directed signal beam emitted by the imaging sample and reconstructing the back-directed signal beam into an intermediate image; A scanning engine for controlling and changing the position of the illumination beam in the imaging sample, driving the illumination beam to perform a scanning motion, and driving the detected back signal beam to perform a scanning motion; The intermediate image collector is used to receive the intermediate image and perform pixelation processing on the intermediate image.

2. The miniaturized scanning micro-volume imaging probe according to claim 1, characterized in that: The scanning engine includes a first scanning engine and / or a second scanning engine. The first scanning engine cooperates with the micro-light sheet generating unit and the intermediate image collector, and is used to drive the micro-light sheet generating unit and the intermediate image collector to synchronously perform one-dimensional scanning motion. The second scanning engine cooperates with the imaging optical path, and is used to drive the illumination light beam and the back-pointing signal light beam to perform one-dimensional scanning motion.

3. The miniaturized scanning micro-volume imaging probe according to claim 2, characterized in that: The first scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine; Preferably, the first scanning engine comprises a piezoelectric drive or a scanning device based on a micro-electromechanical system, a linear or a rotary motor; And / or, the second scanning engine includes at least one prism pair arranged 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 light beam and the back-pointing signal light beam to perform one-dimensional scanning motion; Preferably, the prism is a wedge-shaped prism; Preferably, the wedge angles of the two prisms included in the prism pair are the same or different; Preferably, the two prisms included in the prism pair are made of the same material; Preferably, when the second scanning engine drives the illumination light beam and the back-pointing signal light beam to perform translational scanning, the two prisms included in the prism pair have the same rotation angular velocity but opposite rotation directions.

4. The miniaturized scanning micro-volume imaging probe according to claim 2, characterized in that: The micro light sheet generating unit comprises a light sheet generating component, and the light sheet generating component is used to directly shape the illumination light beam into a light sheet form; Preferably, the light sheet generating assembly comprises at least one of a focusing element, a diffractive optical element with a beam shaping function, and a metasurface element; Preferably, the focusing element comprises a cylindrical lens; Preferably, the cylindrical lens comprises a spherical cylindrical lens, a double-cemented cylindrical lens, a triple-cemented cylindrical lens or an aspherical cylindrical lens; And / or, the micro light sheet generating unit further comprises a light sheet angle adjusting element, the light sheet generating assembly and the light sheet angle adjusting element are sequentially arranged on the optical path of the illumination light beam, and the light sheet angle adjusting element is used to adjust the incident angle of the illumination light beam incident on the imaging optical path; Preferably, the light sheet angle adjustment element comprises a reflector and / or a polygonal prism; Preferably, the focusing element and the light sheet angle adjustment element are integrated into one body.

5. The miniaturized scanning micro-volume imaging probe according to claim 2, characterized in that: The micro light sheet generating unit comprises a light needle generating component and a third scanning engine, wherein the light needle generating component is used to shape the illumination light beam into a light needle form, and the third scanning engine is connected to the light needle generating component in a transmission manner and is used to drive the light needle generating component to scan in a selected direction and form a virtual light sheet form; Preferably, the light needle generating assembly comprises a reflector, and the reflector is used to reflect the illumination light beam, and the equivalent light waist of the reflected illumination light beam is mirrored onto the beveled receiving end surface of the intermediate image collector; And / or, the light needle generating component includes a beam shaping component and a reflector, the beam shaping component is used to change the morphological parameters of the illumination beam to shape the illumination beam into a light needle shape, the morphological parameters include at least one of the numerical aperture, the beam waist diameter, and the Rayleigh length, the reflector is used to reflect the illumination beam shaped by the beam shaping component, and the equivalent beam waist of the reflected illumination beam is mirrored onto the beveled receiving end surface of the intermediate image collector; Preferably, the shape of the illumination beam of the light needle shape includes "long straight shape" or "curved shape"; Preferably, the illumination beam in the form of a light needle comprises a Gaussian beam, a Bessel beam, a Bessel-Gaussian beam or an Airy beam; Preferably, the beam shaping assembly comprises at least one beam shaping lens; preferably, the beam shaping lens comprises 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 gradient refractive index lens; Preferably, the illumination beam in the form of a light needle is a Bessel-Gaussian beam, and the beam shaping component includes at least one of an axicon, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining at least one of a collimating lens, a gradient index lens, a gradient index optical fiber, a micro spherical or aspherical lens with an axicon, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining more than one diffractive optical element with an axicon holographic phase and / or a metasurface lens; Preferably, 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, or the beam shaping component is mainly formed by combining at least one of a collimating lens, a gradient refractive index lens, a gradient refractive index optical fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask with a cubic phase structure; preferably, the third scanning engine is a one-dimensional scanning engine or a two-dimensional scanning engine; Preferably, the third scanning engine includes a piezoelectric driver, a pair of piezoelectric bimorphs or bending plates with deflection directions perpendicular to each other, a piezoelectric transistor with two pairs of surface electrodes perpendicular to each other, or a scanning device based on a micro-electromechanical system, a linear or rotary motor.

6. The miniaturized scanning micro-volume imaging probe according to claim 1, characterized in that: The scanning engine includes a first scanning engine and / or a second scanning engine. The first scanning engine is in transmission cooperation with the micro-light needle generating unit and the intermediate image collector. The first scanning engine is used to drive the micro-light needle generating unit and the intermediate image collector to synchronously perform two-dimensional scanning motion. The second scanning engine is in cooperation with the imaging optical path. The second scanning engine is used to drive the illumination light beam and the back-pointing signal light beam to perform two-dimensional scanning motion. Preferably, the first scanning engine and the second scanning engine are both two-dimensional scanning engines; Preferably, the first scanning engine comprises a pair of piezoelectric bimorphs or flexures with mutually perpendicular deflection directions, a piezoelectric transistor with two mutually perpendicular pairs of surface electrodes, or a scanning device based on a micro-electromechanical system, a linear or rotary motor; And / or, the second scanning engine includes at least one prism pair arranged inside the imaging optical path, the prism pair includes two coaxially arranged prisms, and the two prisms can independently deflect relative to each other around their own central axis to drive the illumination light beam and the back-pointing signal light beam to perform two-dimensional scanning motion; And / or, the prism is a wedge prism; Preferably, the wedge angles of the two prisms included in the prism pair are the same or different; Preferably, the two prisms included in the prism pair are made of the same material; Preferably, the micro light needle generating unit comprises a reflector, and the reflector is used to reflect the illumination light beam, and the equivalent light waist of the reflected illumination light beam is mirrored onto the beveled receiving end surface of the intermediate image collector; And / or, the micro light needle generating unit comprises a beam shaping component and a reflector, the beam shaping component is used to change the morphological parameters of the illumination beam to shape the illumination beam into a light needle shape, the morphological parameters include at least one of the numerical aperture, the beam waist diameter, and the Rayleigh length, the reflector is used to reflect the illumination beam shaped by the beam shaping component, and the equivalent beam waist of the reflected illumination beam is mirrored onto the beveled receiving end surface of the intermediate image collector; Preferably, the shape of the illumination beam in the form of a light needle includes "long straight shape" or "curved shape"; preferably, the illumination beam in the form of a light needle includes a Gaussian beam, a Bessel beam or a Bessel-Gaussian beam or an Airy beam; Preferably, the beam shaping assembly comprises at least one beam shaping lens; preferably, the beam shaping lens comprises 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 gradient refractive index lens; Preferably, the illumination beam in the form of a light needle is a Bessel-Gaussian beam, and the beam shaping component includes at least one of an axicon, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining at least one of a collimating lens, a gradient index lens, a gradient index optical fiber, a micro spherical or aspherical lens with an axicon, a diffractive optical element with an axicon holographic phase, and a metasurface lens; or, the beam shaping component is formed by combining more than one diffractive optical element with an axicon holographic phase and / or a metasurface lens; Preferably, 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, or the beam shaping component is mainly formed by combining at least one of a collimating lens, a gradient refractive index lens, a gradient refractive index optical fiber, a micro spherical or aspherical lens, a diffractive optical element, and a metasurface lens with a phase mask with a cubic phase structure.

7. The miniaturized scanning micro-volume imaging probe according to any one of claims 2 to 6, characterized in that: The imaging optical path comprises a first objective lens, a relay optical system and a second objective lens which are arranged in sequence, the front end of the first objective lens faces the imaging sample, the front end of the second objective lens faces the intermediate image collector, the second scanning engine is arranged 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; Preferably, the imaging optical path is coaxially arranged with a prism included in the second scanning engine; Preferably, the second scanning engine is conjugate coupled to the back focal plane of the first objective lens; Preferably, the relay optical system comprises a 4F system; And / or, the intermediate image collector is used to sample the intermediate image at the optical level, and then transmit it to the external image sensor to complete the digital recording of the intermediate image, or the intermediate image collector itself has a photoelectric conversion function and can directly complete the pixelation and digital recording of the intermediate image; Preferably, the intermediate image collector comprises an optical fiber image transmission bundle having an oblique receiving end face, and the intermediate image formed in the imaging optical path can be projected onto the oblique receiving end face of the optical fiber image transmission bundle, thereby achieving reception and optical sampling of the intermediate image; Preferably, the propagation direction of the illumination beam / sheet adjusted by the light sheet angle adjustment element is parallel to the beveled receiving end face of the optical fiber image transmission bundle, and the equivalent light waist of the illumination beam falls on the receiving end face of the optical fiber image transmission bundle; Preferably, the equivalent light waist of the illumination light beam coincides with the focus of the second objective lens.

8. The miniaturized scanning micro-volume imaging probe according to claim 7, characterized in that: The first scanning engine is arranged outside the imaging optical path, the micro light sheet generating unit or the micro light needle generating unit is fixedly matched with the intermediate image collector, the micro light sheet generating unit or the micro light needle generating unit and the intermediate image collector can be driven by the first scanning engine to move synchronously, and the intermediate image and the intermediate image collector move synchronously and are relatively stationary; Preferably, the intermediate image and the oblique receiving end surface of the optical fiber image transmission bundle always remain relatively stationary; Preferably, the intermediate image and the intermediate image collector are both stationary.

9. A miniaturized three-dimensional volume imaging microscope system, characterized in that: A miniaturized scanning micro-volume imaging probe comprising any one of claims 1-8.

10. The miniaturized three-dimensional volume imaging microscope system according to claim 9, characterized in that: Also includes: A light source module, the light source module is used to provide an illumination beam; Preferably, the miniaturized three-dimensional volume imaging microscope system further comprises an external image sensor, and the external image sensor is used to receive the intermediate image obtained by the intermediate image collector; Preferably, the miniaturized three-dimensional volume imaging microscope system further comprises a filter element, which is disposed between the external image sensor and the intermediate image collector, and is used to filter out light signals within a specific wavelength range; Preferably, the filter element comprises at least one filter.

Images