1. A near-infrared light sheet microscope is characterized by comprising an excitation light source system, a spatial filtering system, a directional beam expanding system, a polyhedral scanning rotating mirror, an excitation objective lens, a receiving objective lens, a sleeve lens, an optical filter and a camera, wherein the included angle between the excitation objective lens and the vertical direction is 45 degrees, and the included angle between the receiving objective lens and the vertical direction is 45 degrees; and an excitation light beam generated by the excitation light source system is filtered by the spatial filtering system, then expanded in one direction by the directional beam expanding system, and then reflected to the excitation objective lens by the polyhedral scanning rotating mirror to generate an optical sheet, the optical sheet irradiates on a sample to generate a response light beam, and the response light beam forms an image on the camera after passing through the receiving objective lens, the sleeve lens and the optical filter.

2. The near-infrared light sheet microscope of claim 1, wherein the excitation light source system comprises a laser, a fiber coupler and a fiber collimator, and the excitation light beam generated by the laser is guided into the optical fiber by the fiber coupler and then guided into the optical path by the fiber collimator.

3. The near-infrared light sheet microscope of claim 2, wherein the excitation beam wavelength comprises one or more of 660nm, 785nm, 808nm, 975nm, 1064nm, 1319nm, and 1450 nm.

4. The near-infrared light sheet microscope according to claim 2, wherein the optical fiber collimator has a wavelength ranging from 633 to 1550nm, a numerical aperture ranging from 0.15 to 0.56, a focal length ranging from 4 to 38mm, and a beam diameter ranging from 0.5 to 8 mm.

5. The near-infrared sheet microscope of claim 1, wherein the spatial filter system comprises a first achromatic lens, a precision pinhole and a second achromatic lens arranged in sequence, the precision pinhole is located at the focal length of the first achromatic lens and the second achromatic lens, the aperture of the precision pinhole is 10-1000 μm, and the focal length of the first achromatic lens and the focal length of the second achromatic lens are 30-200 mm.

6. The near-infrared ray sheet microscope of claim 1, wherein the directional beam expanding system comprises a first adjustable mechanical slit, a cylindrical lens, a third achromatic lens, a second adjustable mechanical slit and a fourth achromatic lens, which are arranged in sequence, the distance between the third achromatic lens and the fourth achromatic lens is equal to the sum of the focal lengths of the third achromatic lens and the fourth achromatic lens, the second adjustable mechanical slit is located at the focal position of the third achromatic lens and the fourth achromatic lens, and the focal lengths of the third achromatic lens and the fourth achromatic lens are in the range of 30-200 mm.

7. The near-infrared light sheet microscope of claim 6, wherein the cylindrical lens is a plano-convex or biconvex lens, is circular or rectangular, has a focal length of 50-1000 mm, is coated with an antireflection film, and has a light transmission range of 350-1620 nm.

8. The near-infrared sheet microscope of claim 1, wherein the distance between the receiving objective and the telescopic lens is equal to the interpupillary distance of the telescopic lens, and the distance between the telescopic lens and the camera is equal to the working distance of the telescopic lens; the working distance of the sleeve lens is 60-180 mm, the wavelength range is 400-2000 nm, the interpupillary distance range is 0-170 mm, and the thickness range is 25-210 mm.

9. The NIR light sheet microscope of claim 1, wherein the excitation objective and the receiving objective have a magnification of 4X, 5X, 10X or 20X, a wavelength range of 480 to 1800nm, a working distance of 15 to 40mm, and a numerical aperture of 0.13 to 0.4.

10. The near-infrared light sheet microscope of claim 1, wherein the camera is an InGaAs camera, the photosensitive chip is an InGaAs array, the resolution is 320 x 256 or 640 x 512, the pixel size is 10 μm, 15 μm or 20 μm, the frame rate is greater than or equal to 50Hz, the response wavelength range is 900-1700 nm, and the dark current is less than or equal to 150e^-Read noise of 50e or less^-。

Background

Optical microscopy is the most fundamental research tool in biomedical research and is an important foundation for the establishment and development of modern biology. Currently, optical microscopes have been developed into various types, such as common optical microscopes, fluorescence microscopes, laser confocal microscopes, and the like. Among them, fluorescence microscopy has become an important imaging tool in molecular biology and biological tissue research because it can image a specific labeled target. However, the fluorescence microscope commonly used in the laboratory is used for imaging in the visible light band (400-700nm), and as is well known, many organisms are opaque, and the penetrability of visible light in biological tissues is poor, so that the fluorescence imaging in the visible light band can only be used for imaging in vitro cells, thin tissues and transparent organisms, which greatly limits the application of the fluorescence microscopy in living tissues.

Near-infrared fluorescence imaging is a fluorescence imaging technology emerging in recent years, imaging is performed in a near-infrared band (800-. However, the larger imaging depth of the near-infrared fluorescence imaging brings new problems, and in a common bright field illumination and imaging system, fluorescence images with different depths can be simultaneously shot, and the images with different depths are superposed together and cannot distinguish the depth of an acquired signal, so that researchers urgently need a near-infrared fluorescence imaging system capable of realizing tomography.

Disclosure of Invention

In view of the above, an object of the embodiments of the present invention is to provide a near-infrared sheet microscope capable of performing fast three-dimensional tomographic imaging on a stationary sample in a right position.

The embodiment of the invention provides a near-infrared light sheet microscope, which comprises an excitation light source system, a spatial filtering system, a directional beam expanding system, a polyhedron scanning rotating mirror, an excitation objective lens, a receiving objective lens, a sleeve lens, an optical filter and a camera, wherein the included angle between the excitation objective lens and the vertical direction is 45 degrees, and the included angle between the receiving objective lens and the vertical direction is 45 degrees; and an excitation light beam generated by the excitation light source system is filtered by the spatial filtering system, then expanded in one direction by the directional beam expanding system, and then reflected to the excitation objective lens by the polyhedral scanning rotating mirror to generate an optical sheet, the optical sheet irradiates on a sample to generate a response light beam, and the response light beam forms an image on the camera after passing through the receiving objective lens, the sleeve lens and the optical filter.

Optionally, the excitation light source system includes a laser, an optical fiber coupler, and an optical fiber collimator, and an excitation light beam generated by the laser is guided into an optical fiber by the optical fiber coupler and then guided into a light path by the optical fiber collimator.

Optionally, the excitation beam has a wavelength comprising one or more of 660nm, 785nm, 808nm, 975nm, 1064nm, 1319nm, and 1450 nm.

Optionally, the wavelength range of the optical fiber collimator is 633-1550 nm, the numerical aperture is 0.15-0.56, the focal length is 4-38 mm, and the beam diameter is 0.5-8 mm.

Optionally, the spatial filtering system includes first achromat, accurate pinhole and the second achromat that arrange in proper order, accurate pinhole is located first achromat and the focus department of second achromat, the aperture of accurate pinhole is between 10 ~ 1000 mu m, the focus scope of first achromat and second achromat is between 30 ~ 200 mm.

Optionally, the directional beam expanding system includes a first adjustable mechanical slit, a cylindrical lens, a third achromatic lens, a second adjustable mechanical slit, and a fourth achromatic lens, which are arranged in sequence, a distance between the third achromatic lens and the fourth achromatic lens is equal to a sum of focal lengths of the third achromatic lens and the fourth achromatic lens, the second adjustable mechanical slit is located at a focal position of the third achromatic lens and the fourth achromatic lens, and focal lengths of the third achromatic lens and the fourth achromatic lens are in a range of 30-200 mm.

Optionally, the cylindrical lens is a plano-convex or biconvex lens, is round or rectangular, has a focal length of 50-1000 mm, is coated with an antireflection film, and has a light transmission range of 350-1620 nm.

Optionally, the distance between the receiving objective lens and the sleeve lens is equal to the interpupillary distance of the sleeve lens, and the distance between the sleeve lens and the camera is equal to the working distance of the sleeve lens; the working distance of the sleeve lens is 60-180 mm, the wavelength range is 400-2000 nm, the interpupillary distance range is 0-170 mm, and the thickness range is 25-210 mm.

Optionally, the magnification of the exciting objective lens and the receiving objective lens is 4X, 5X, 10X or 20X, the wavelength range is 480-1800 nm, the working distance is 15-40 mm, and the numerical aperture is 0.13-0.4.

Optionally, the camera is an InGaAs camera, the photosensitive chip is an InGaAs array, the resolution is 320 × 256 or 640 × 512, the pixel size is 10 μm, 15 μm or 20 μm, the frame rate is greater than or equal to 50Hz, the response wavelength range is 900-1700 nm, and the dark current is less than or equal to 150e^-Read noise of 50e or less^-。

The implementation of the embodiment of the invention has the following beneficial effects: the embodiment of the invention generates an excitation beam through an excitation light source system, filters the excitation beam through a spatial filtering system, expands the filtered laser beam through a directional beam expanding system, and changes the incident angle of the laser beam through a polygon turning mirror, thereby realizing the rapid three-dimensional tomography imaging of a sample under the condition that the sample and an objective lens do not move; in addition, the included angles between the exciting objective lens and the receiving objective lens and the vertical direction are both 45 degrees, and the included angles are mutually perpendicular, so that the tomography scanning of the placed sample is realized.

Drawings

FIG. 1 is a schematic structural diagram of a near-infrared light sheet microscope according to an embodiment of the present invention;

FIG. 2 is a diagram showing the result of the intracerebral vascular imaging of a sample using a near-infrared light sheet microscope according to an embodiment of the present invention;

fig. 3 is a diagram showing the result of brain imaging of a sample using a near-infrared light sheet microscope according to an embodiment of the present invention.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific embodiments. The step numbers in the following embodiments are provided only for convenience of illustration, the order between the steps is not limited at all, and the execution order of each step in the embodiments can be adapted according to the understanding of those skilled in the art.

The embodiment of the invention provides a near-infrared light sheet microscope, which comprises an excitation light source system, a spatial filtering system, a directional beam expanding system, a polyhedron scanning rotating mirror, an excitation objective lens, a receiving objective lens, a sleeve lens, an optical filter and a camera, wherein the included angle between the excitation objective lens and the vertical direction is 45 degrees, and the included angle between the receiving objective lens and the vertical direction is 45 degrees; and an excitation light beam generated by the excitation light source system is filtered by the spatial filtering system, then expanded in one direction by the directional beam expanding system, and then reflected to the excitation objective lens by the polyhedral scanning rotating mirror to generate an optical sheet, the optical sheet irradiates on a sample to generate a response light beam, and the response light beam forms an image on the camera after passing through the receiving objective lens, the sleeve lens and the optical filter.

Specifically, as shown in fig. 1, an excitation light beam generated by an excitation light source system 1 is reflected by a reflector M1 and enters a spatial filter system for filtering, and the spatial filter system sequentially includes a first achromatic lens 2-1, a precision pinhole 2-2, and a second achromatic lens 2-3 along an optical axis direction; the excitation light beam enters a directional beam expanding system for expansion after being filtered by a spatial filtering system, and the directional beam expanding system sequentially comprises a first adjustable mechanical slit 3-1, a cylindrical lens 3-2, a third achromatic lens 3-3, a second adjustable mechanical slit 3-4 and a fourth achromatic lens 3-5 along the direction of an optical axis; the expanded excitation light beam is reflected to the polyhedral scanning rotating mirror 4 by the reflecting mirror M2, and is reflected to the excitation objective 5 by the polyhedral scanning rotating mirror 4 to generate light sheets with different angles, the light sheets irradiate on a sample laboratory mouse to generate response light beams, and the response light beams are imaged by the camera 9 after passing through the receiving objective 6, the sleeve lens 7 and the optical filter 8.

Optionally, the excitation light source system includes a laser, an optical fiber coupler, and an optical fiber collimator, and an excitation light beam generated by the laser is guided into an optical fiber by the optical fiber coupler and then guided into a light path by the optical fiber collimator.

Specifically, the interface with the optical fiber coupler is SMA or FC/PC; the optical fiber interface is SMA or FC/PC, the core diameter is 50-200 μm, the numerical aperture is 0.1-0.5, and the transmission wavelength range is 400-2200 nm.

It should be noted that, excitation light generated by the laser is guided into the optical fiber by the optical fiber coupler and then guided into the optical path by the optical fiber collimator, and the optical fiber can be connected with a plurality of lasers to realize fast switching of the wavelength of the excitation light.

Optionally, the excitation beam has a wavelength comprising one or more of 660nm, 785nm, 808nm, 975nm, 1064nm, 1319nm, and 1450 nm.

Specifically, the power range of the laser is 1-12000 mW, the wavelength of the excitation beam is determined by the wavelength of the laser used, the laser power density can also be adjusted by the excitation current of the laser, the adjustable range is determined by the selected laser wavelength, for example, the power density of the 660nm laser is adjusted in the range of 0-0.2W cm^-2The power density of 785nm laser is adjusted within the range of 0-0.3W cm^-2The power density of 808nm laser is adjusted within the range of 0-0.33W cm^-2The power density of 975nm laser is adjusted within the range of 0-0.71W cm^-2The power density adjusting range of 1064nm and 1319nm lasers is 0-1W cm^-2The power density of 1450nm laser is adjusted within 0-0.1W cm^-2。

Optionally, the wavelength range of the optical fiber collimator is 633-1550 nm, the numerical aperture is 0.15-0.56, the focal length is 4-38 mm, and the beam diameter is 0.5-8 mm.

Specifically, the interface of the optical fiber collimator is SMA or FC/PC.

Optionally, the spatial filtering system includes first achromat, accurate pinhole and the second achromat that arrange in proper order, accurate pinhole is located first achromat and the focus department of second achromat, the aperture of accurate pinhole is between 10 ~ 1000 mu m, the focus scope of first achromat and second achromat is between 30 ~ 200 mm.

Specifically, the precise pinhole is made of any one of stainless steel, tungsten or gold-plated copper, the diameter of the shell is 0.5 or 1 inch, and the aperture range is 10-1000 μm. The aperture of the precision pinhole is 1.3 lambdaf/r, wherein lambdaf is the wavelength of the excitation light, f is the focal length of the first achromatic lens, and r is the radius of the incident light spot.

Specifically, the diameters of the first achromatic lens and the second achromatic lens are within 0.5-2 inches, and the first achromatic lens and the second achromatic lens are coated with antireflection films, and the light transmission range is within 400-1700 nm.

It should be noted that the spatial filtering system can filter out stray light and multi-order diffracted light, so as to obtain a pure gaussian beam.

Optionally, the directional beam expanding system includes a first adjustable mechanical slit, a cylindrical lens, a third achromatic lens, a second adjustable mechanical slit, and a fourth achromatic lens, which are arranged in sequence, a distance between the third achromatic lens and the fourth achromatic lens is equal to a sum of focal lengths of the third achromatic lens and the fourth achromatic lens, the second adjustable mechanical slit is located at a focal position of the third achromatic lens and the fourth achromatic lens, and focal lengths of the third achromatic lens and the fourth achromatic lens are in a range of 30-200 mm.

Specifically, the diameters of the third achromatic lens and the fourth achromatic lens are in the range of 0.5-2 inches, the third achromatic lens and the fourth achromatic lens are coated with antireflection films, and the light transmission range is 400-1700 nm.

Optionally, the cylindrical lens is a plano-convex or biconvex lens, is round or rectangular, has a focal length of 50-1000 mm, is coated with an antireflection film, and has a light transmission range of 350-1620 nm.

It should be noted that, the first adjustable mechanical slit is used to control the width of the light beam in one direction, and then the cylindrical lens is used to expand the width of the light beam in the direction, so the first adjustable mechanical slit can be used to adjust the width of the light sheet. In addition, two achromatic lenses are used for expanding the excitation beam, the distance between the two achromatic lenses is equal to the sum of the focal lengths of the two achromatic lenses, and a second adjustable mechanical slit is arranged between the two achromatic lenses and at the focal positions of the two achromatic lenses and is perpendicular to the beam expansion direction in the direction, so that the second adjustable mechanical slit can be used for adjusting the thickness of the optical sheet and the effective numerical aperture of the excitation light path.

Specifically, the mirror surface of the polyhedral scanning rotating mirror is 8-12 surfaces, the polyhedral scanning rotating mirror is provided with a rotating motor, the rotating speed is 0-500 Hz, and an air bearing or a ball bearing is used.

It should be noted that the angular scanning rate is determined by the number of facets of the polygon scanning rotating mirror and the rotating speed of the rotating mirror, and the angular scanning rate can be 0 to 160rad s^-1And internal adjustment, the more the number of the surfaces of the rotating mirror is, the faster the rotating speed is, and the higher the angular scanning speed is. The angular scanning range is determined by the number of the surfaces of the polyhedron scanning rotating mirror, the angular scanning range of the 8-surface rotating mirror is 45 degrees, and the angular scanning range of the 12-surface rotating mirror is 30 degrees. The angular scanning rate and the scanning range need to be matched with the acquisition rate of the InGaAs camera, namely the polyhedral scanning rotating mirror needs to be synchronously controlled with the InGaAs camera.

Optionally, the magnification of the exciting objective lens and the receiving objective lens is 4X, 5X, 10X or 20X, the wavelength range is 480-1800 nm, the working distance is 15-40 mm, and the numerical aperture is 0.13-0.4.

Note that the magnification of the receiving objective lens is generally 2 times that of the exciting objective lens.

It should be noted that an angle between the excitation objective lens and the vertical direction is 45 °, and an angle between the receiving objective lens and the vertical direction is 45 °, that is, the excitation objective lens and the receiving objective lens are perpendicular to each other.

It should be noted that the thickness and shape of the excitation lens are determined by the magnification of the used excitation objective lens and the laser wavelength, the thickness (i.e. waist width) of the excitation lens can be adjusted within the range of 9-30 μm, the length (i.e. 2 times of rayleigh range) of the optical sheet can be adjusted within the range of 0.2-2.5 mm, the smaller the magnification of the excitation objective lens is, the longer the laser wavelength is, the larger the thickness of the optical sheet is, and the longer the length is. The effective numerical aperture of the excitation light path is determined by the focal length and the light sheet width of the used objective lens and can be adjusted between 0.035 and 0.2, and the shorter the focal length of the objective lens is, the wider the light sheet is, the larger the effective numerical aperture is.

It should be noted that the imaging resolution depends on the magnification of the receiving objective lens and the wavelength of the received light, the resolution perpendicular to the optical sheet direction can be adjusted within a range of 2-10 μm, the resolution parallel to the optical sheet direction can be adjusted within a range of 0.8-4 μm, and the higher the magnification of the receiving objective lens, the shorter the wavelength, the higher the resolution.

Optionally, the distance between the receiving objective lens and the sleeve lens is equal to the interpupillary distance of the sleeve lens, and the distance between the sleeve lens and the camera is equal to the working distance of the sleeve lens; the working distance of the sleeve lens is 60-180 mm, the wavelength range is 400-2000 nm, the interpupillary distance range is 0-170 mm, and the thickness range is 25-210 mm.

Specifically, the filter is a long-wave pass filter with a diameter of 25mm and cut-off wavelengths of 1000nm, 1100nm, 1200nm, 1300nm, 1400nm and 1500 nm.

Optionally, the camera is an InGaAs camera, the photosensitive chip is an InGaAs array, the resolution is 320 × 256 or 640 × 512, the pixel size is 10 μm, 15 μm or 20 μm, the frame rate is greater than or equal to 50Hz, the response wavelength range is 900-1700 nm, and the dark current is less than or equal to 150e^-Read noise of 50e or less^-。

The implementation of the embodiment of the invention has the following beneficial effects: the embodiment of the invention generates an excitation beam through an excitation light source system, filters the excitation beam through a spatial filtering system, expands the filtered laser beam through a directional beam expanding system, and changes the incident angle of the laser beam through a polygon turning mirror, thereby realizing the rapid three-dimensional tomography imaging of a sample under the condition that the sample and an objective lens do not move; in addition, the included angles between the exciting objective lens and the receiving objective lens and the vertical direction are both 45 degrees, and the included angles are mutually perpendicular, so that the tomography scanning of the placed sample is realized.

It is noted that the embodiments of the present invention can be used for deep optical imaging of ex vivo tissues, organs or small living animals, including but not limited to untreated ex vivo tumor tissues, brain tissues or cardiac muscle tissues and the like derived from experimental animals,derived from the isolated heart or brain of a small animal, derived from the isolated brain of a small animal subjected to tissue clearing treatment, derived from the tumor tissue, heart or brain of a living small animal, or the like. The embodiment of the invention can perform near-infrared fluorescence imaging on the sample subjected to near-infrared fluorescence labeling, and also can perform near-infrared imaging on blood vessels in a living body sample by utilizing the absorption of water in a near-infrared band. The imaging depth can reach 2mm under micron resolution, and the single scanning volume can reach 1mm³。

The following two specific examples are provided to illustrate the imaging of a living body sample using the near-infrared light sheet microscope of the present application.

Example one

The PbS @ CdS-PEG quantum dots are dispersed in PBS buffer solution to obtain 0.2mL of dispersion system with OD808 ═ 4, the dispersion system is injected into a C57 mouse with the age of 4 weeks through a tail vein, hairs on the head of the mouse are shaved off, a cover glass and a prism are placed at the top of the head of the mouse, and gaps among the PbS @ CdS-PEG quantum dots, the cover glass and the prism are filled with 80% glycerol solution to improve refractive index matching. After anaesthetizing, the mouse is placed under an exciting objective lens and a receiving objective lens, and blood vessels in the skull of the mouse are imaged through the skin and the skull. The used excitation wavelength is 808nm, the used receiving filter is 1500nm, namely, the receiving wavelength is longer than the fluorescence signal of 1500nm, the used excitation objective lens is 5X, the used receiving objective lens is 10X, the exposure time is 5ms, the thickness of the optical sheet is 11.9 μm, the length of the optical sheet is 522.3 μm, and the effective numerical aperture is 0.051. As shown in figure 2, under the micron resolution, the imaging can penetrate through the skin and the skull and penetrate into the brain of a mouse by 0.7mm, the imaging quality is high, the distribution of capillary vessels in the brain can be clearly observed, and the signal-to-noise ratio is over 10.

Example two

An IRFEP-Hoechst probe (nuclear targeting) was dissolved in PBS buffer to give 0.02mL of dispersion with OD808 ═ 20, injected intracranially into C57 mice of 4 weeks of age by brain stereotactic, the head of the mice was shaved, a cover glass and a triangular prism were placed on top of the mouse head, and the space between the three was filled with 80% glycerol solution to improve refractive index matching. After being anesthetized, the mouse is placed under an exciting objective lens and a receiving objective lens, and the brain of the mouse is imaged through the skin and the skull. The used excitation wavelength is 808nm, the used receiving filter is 1000nm, namely, the receiving wavelength is longer than 1000nm, the used excitation objective lens is 5X, the used receiving objective lens is 10X, the exposure time is 5ms, the thickness of the optical sheet is 11.9 μm, the length of the optical sheet is 522.3 μm, and the effective numerical aperture is 0.051. As shown in fig. 3, at micron resolution, the imaging can penetrate the skin and skull and penetrate 0.36mm deep into the brain of the mouse with a signal-to-noise ratio above 3.

While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.