CN117110267A - Light sheet microscopic imaging system and imaging method of visible light to near infrared two regions - Google Patents
Light sheet microscopic imaging system and imaging method of visible light to near infrared two regions Download PDFInfo
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Abstract
The application discloses a light sheet microscopic imaging system and an imaging method in a visible light-near infrared two-region, wherein the imaging system comprises a multi-wavelength near infrared laser light source module, a multi-wavelength visible light laser light source module, a first microscope objective, a second microscope objective, a first two-dimensional scanning galvanometer, a second two-dimensional scanning galvanometer, a long-pass dichroic mirror, a third microscope objective, a right-angle prism, a fourth microscope objective, a three-dimensional objective table, a short-pass dichroic mirror, a first photosensitive component and a second photosensitive component. Compared with the prior art, the application comprehensively applies the fluorescence microscopic imaging technology, the light sheet microscopic imaging technology and the near infrared two-region fluorescence imaging technology, gives play to the respective advantages of visible light imaging and infrared light imaging, can ensure higher imaging depth when imaging thicker samples, and can improve imaging resolution of thicker samples, thereby flexibly realizing high-quality imaging of thick samples and thin samples and solving the problem of high-resolution deep imaging of thick samples.
Description
Technical Field
The application relates to the technical field of optical microscopic imaging, in particular to a light slice microscopic imaging system and an imaging method of visible light to near infrared two regions.
Background
The appearance and development of the optical microscope have important significance for the development of key fields such as life science and the like. Heretofore, there have been various kinds of optical microscopes such as a common wide-field optical microscope, a fluorescent microscope, a confocal laser microscope, a super-resolution optical microscope, and the like. Among them, fluorescence microscope has advantages of high contrast, non-contact, almost no damage to sample, etc., and has been developed as an indispensable powerful assistant in research in life science and other fields. Intersecting a common wide-field fluorescent microscope, wherein an illumination light path and an imaging light path in a light sheet microscope are mutually orthogonal, and illumination light is in a thin 'light sheet' parallel to an imaging surface; because of the unique illumination mode, the light sheet microscope reduces the photo-bleaching and photo-toxicity to the sample, has the advantages of low background noise and imaging speed, and can be used for three-dimensional living body microscopic imaging.
Due to manufacturing level and process limitations, the detection band of early detectors was mainly centered at 400-1000nm, so that the operating band of fluorescence microscopy was mainly centered at this band: light sheet microscopes based on the visible light band greatly promote new discoveries of living cell biological mechanisms with their unique advantages. However, scattering and absorption of photons by biological tissue is severe in this band, and the image is not disturbed by autofluorescence of biological tissue in this band. Near infrared two-region (1000-1700 nm) fluorescence imaging was first proposed by university of stenford Dai Hongjie, 2009; because the scattering and absorption of the photons in the near infrared two-region wave band are relatively weak, the near infrared two-region fluorescence imaging has the advantages of large imaging depth, high spatial resolution, low image background and the like. A near infrared light sheet microscope has been proposed in the prior art (CN 113406046A, a near infrared light sheet microscope), which generates line illumination through a cylindrical lens, and generates light sheet illumination in the axial direction thereof by focusing through an illumination objective lens with the aid of a planar scanning rotating mirror, and detects fluorescent signals by means of an InGaAs camera, so that a high-quality imaging effect of a large depth can be achieved.
However, early light sheet microscopes based on visible light wave bands have limited imaging depth due to scattering and absorption of photons by tissues, are only suitable for imaging cells, and cannot be used in imaging scenes of thick samples; recently proposed near infrared band-based light sheet microscopes can obtain deeper imaging depths, but due to their longer imaging wavelength (about two to three times the wavelength of visible light), the spatial resolution of the image is not ideal for cell imaging. Therefore, the imaging technique in the prior art method has a problem that high resolution deep imaging of a thick sample cannot be achieved.
Disclosure of Invention
The embodiment of the application provides a light sheet microscopic imaging system and an imaging method in a visible light region and a near infrared region, which aim to solve the problem that the imaging technology in the prior art cannot realize high-resolution deep imaging of a thick sample.
In a first aspect, an embodiment of the present application provides a light sheet microscopic imaging system from visible light to near infrared, where the system includes a multi-wavelength near infrared laser light source module, a multi-wavelength visible light laser light source module, a first microscope objective, a second microscope objective, a first two-dimensional scanning galvanometer, a second two-dimensional scanning galvanometer, a long-pass dichroic mirror, a third microscope objective, a rectangular prism, a fourth microscope objective, a three-dimensional objective table, a short-pass dichroic mirror, a first photosensitive assembly, and a second photosensitive assembly:
the multi-wavelength near infrared laser light source module emits near infrared laser and transmits the near infrared laser to the long-pass dichroic mirror through a first light path; the multi-wavelength visible light laser source module emits visible light laser and transmits the visible light laser to the long-pass dichroic mirror through a second light path;
the first light path is sequentially provided with the first microscope objective and the first two-dimensional scanning galvanometer; the second light path is sequentially provided with the second micro objective and the second two-dimensional scanning galvanometer;
the long-pass dichroic mirror integrates the light beams from the first light path and the second light path, and irradiates into a right-angle surface of the right-angle prism through the third microscope objective lens, and excites a sample to generate fluorescence at the bottom surface of the right-angle prism;
the right angle prism is fixedly arranged above the three-dimensional object stage, and the three-dimensional object stage is used for placing a sample and driving the sample to perform three-dimensional movement;
the fourth microscope objective collects the fluorescent light beam emitted from the other right angle surface of the right angle prism to the short-pass dichroic mirror; near infrared light in the fluorescent light beam is emitted into the first photosensitive assembly by the short-pass dichroic mirror, visible light in the fluorescent light beam is emitted into the second photosensitive assembly by the short-pass dichroic mirror, and the first photosensitive assembly and the second photosensitive assembly are photosensitive assemblies with a filtering function.
The light sheet microscopic imaging system from the visible light to the near infrared two regions, wherein the multi-wavelength near infrared laser source module is transmitted to the light inlet end of the first microscope objective through a near infrared single-mode polarization maintaining fiber;
the multi-wavelength visible light laser source module is transmitted to the light inlet end of the second microscope objective through the visible light single-mode polarization maintaining fiber.
And the light slice microscopic imaging system from the visible light to the near infrared two regions, wherein a near infrared scanning lens is arranged between the first two-dimensional scanning galvanometer and the long-pass dichroic mirror.
And a visible light scanning lens is arranged between the second two-dimensional scanning galvanometer and the long-pass dichroic mirror.
And the light slice microscopic imaging system from the visible light to the near infrared two regions is characterized in that a first sleeve lens is further arranged between the long-pass dichroic mirror and the third microscope objective.
The light sheet microscopic imaging system from visible light to near infrared, wherein the first photosensitive component comprises a second sleeve lens and a near infrared camera;
near infrared light in the fluorescent light beam is emitted into the near infrared camera through the second sleeve lens.
The light sheet microscopic imaging system from visible light to near infrared, wherein the first photosensitive component further comprises a first optical filter; the first filter is disposed between the second sleeve lens and the short-pass dichroic mirror.
The light sheet microscopic imaging system from visible light to near infrared, wherein the second photosensitive assembly comprises a third sleeve lens and a visible light camera;
visible light in the fluorescent light beam is emitted into the visible light camera through the third sleeve lens.
The light sheet microscopic imaging system from visible light to near infrared, wherein the second photosensitive component further comprises a second optical filter; the second filter is disposed between the third sleeve lens and the short-pass dichroic mirror.
In a second aspect, an embodiment of the present application further provides a light sheet microscopic imaging method from visible light to near infrared, where the imaging method is applied to the imaging system in the first aspect, and the imaging method includes:
starting a multi-wavelength near infrared laser light source module to emit near infrared laser, collimating and expanding beams through a first micro objective lens, then injecting the near infrared laser into an X-axis reflecting mirror and a Y-axis reflecting mirror in a first two-dimensional scanning vibrating mirror, reflecting the near infrared laser, and then injecting the near infrared laser into the rear surface of the long-pass dichroic mirror for transmission;
simultaneously starting a multi-wavelength visible light laser source module to emit visible light laser, collimating and expanding beams through a second micro objective lens, and then injecting the visible light laser into an X-axis reflecting mirror and a Y-axis reflecting mirror in a second two-dimensional scanning vibrating mirror to be reflected and then injecting the visible light laser into the front surface of the long-pass dichroic mirror to be reflected;
the near infrared laser beam from the first light path and the visible laser beam from the second light path are converged at the long-pass dichroic mirror to form a combined beam, and the combined beam is injected into a third microscope objective to be focused, and then is coupled and irradiated through a right-angle prism to excite dye in the sample on the three-dimensional object stage to generate fluorescence and emit;
the fourth microscope objective collects fluorescent light beams emitted from the other right angle surface of the right angle prism, the fluorescent light beams are emitted into the front surface of the short-pass dichroic mirror, near infrared light in the fluorescent light beams is reflected on the front surface of the short-pass dichroic mirror and is emitted into the first photosensitive assembly to obtain a first detection record, and visible light in the fluorescent light beams is transmitted on the front surface of the short-pass dichroic mirror and is emitted into the second photosensitive assembly to obtain a second detection record;
and carrying out data processing on the first detection record and the second detection record to obtain a three-dimensional reconstruction image of the sample.
The embodiment of the application provides a light sheet microscopic imaging system and an imaging method in a visible light-near infrared two region, wherein the imaging system comprises a multi-wavelength near infrared laser light source module, a multi-wavelength visible light laser light source module, a first microscope objective, a second microscope objective, a first two-dimensional scanning galvanometer, a second two-dimensional scanning galvanometer, a long-pass dichroic mirror, a third microscope objective, a right-angle prism, a fourth microscope objective, a three-dimensional objective table, a short-pass dichroic mirror, a first photosensitive assembly and a second photosensitive assembly. Compared with the prior art, the application comprehensively applies the fluorescence microscopic imaging technology, the light sheet microscopic imaging technology and the near infrared two-region fluorescence imaging technology, gives play to the respective advantages of visible light imaging and infrared light imaging, can ensure higher imaging depth when imaging thicker samples, and can improve imaging resolution of thicker samples, thereby flexibly realizing high-quality imaging of thick samples and thin samples and solving the problem of high-resolution deep imaging of thick samples.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a light sheet microscopic imaging system in the visible to near infrared region according to an embodiment of the present application;
fig. 2 is a flow chart of a light sheet microscopic imaging method from visible light to near infrared according to an embodiment of the present application.
Reference numerals: 1. a multi-wavelength near infrared laser light source module; 2. near infrared single-mode polarization maintaining optical fiber; 3. a first microobjective; 4. a first two-dimensional scanning galvanometer; 5. a near infrared scanning lens; 6. a first mirror; 7. a long-pass dichroic mirror; 8. a multi-wavelength visible light laser source module; 9. visible light single-mode polarization maintaining optical fiber; 10. a second microobjective; 11. a second two-dimensional scanning galvanometer; 12. a visible light scanning lens; 13. a first sleeve lens; 14. a third microobjective; 15. a right angle prism; 16. a fourth microobjective; 17. a three-dimensional stage; 18. a short-pass dichroic mirror; 19. a first optical filter; 20. a second sleeve lens; 21. a near infrared camera; 22. a second mirror; 23. a second optical filter; 24. a third sleeve lens; 25. a visible light camera.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It should be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
It should be further understood that the term "and/or" as used in the present specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations.
In this embodiment, please refer to fig. 1. As shown in the drawing, the embodiment of the application provides a light sheet microscopic imaging system from visible light to near infrared, wherein the system comprises a multi-wavelength near infrared laser light source module 1, a multi-wavelength visible light laser light source module 8, a first microscope objective 3, a second microscope objective 10, a first two-dimensional scanning galvanometer 4, a second two-dimensional scanning galvanometer 11, a long-pass dichroic mirror 7, a third microscope objective 14, a right angle prism 15, a fourth microscope objective 16, a three-dimensional objective table 17, a short-pass dichroic mirror 18, a first photosensitive component and a second photosensitive component: the multi-wavelength near infrared laser light source module 1 emits near infrared laser light and transmits the near infrared laser light to the long-pass dichroic mirror 7 through a first light path; the multi-wavelength visible light laser source module 8 emits visible light laser and transmits the visible light laser to the long-pass dichroic mirror 7 through a second optical path; the first light path is sequentially provided with the first microscope objective 3 and the first two-dimensional scanning galvanometer 4; the second light path is sequentially provided with the second microscope objective 10 and the second two-dimensional scanning galvanometer 11; the long-pass dichroic mirror 7 integrates the light beams from the first light path and the second light path, and irradiates into one side surface of the right-angle prism 15 through the third micro objective lens 14, and excites a sample at the bottom surface of the right-angle prism 15 to generate fluorescence; the rectangular prism 15 is fixedly arranged above the three-dimensional objective table 17, a certain gap is reserved between the rectangular prism 15 and the three-dimensional objective table 17, and the three-dimensional objective table 17 can only drive a sample on the rectangular prism to move and can not drive the rectangular prism 15 to move together; the three-dimensional object stage 17 is used for placing a sample and driving the sample to perform three-dimensional movement; the fourth microscope 16 collects the fluorescent light beam emitted through the other right angle surface of the right angle prism 15 to the short-pass dichroic mirror 18; near infrared light in the fluorescent light beam is reflected by the short-pass dichroic mirror and is emitted into the first photosensitive assembly, visible light in the fluorescent light beam is transmitted by the short-pass dichroic mirror and is emitted into the second photosensitive assembly, and the first photosensitive assembly and the second photosensitive assembly are photosensitive assemblies with a filtering function.
The multi-wavelength near infrared laser light source module 1, the first microscope objective 3, the first two-dimensional scanning galvanometer 4 and the first reflecting mirror 6 are combined to form a first light path, and the multi-wavelength near infrared laser light source module 1 is used for emitting near infrared laser and transmitting the near infrared laser to the long-pass dichroic mirror 7 through the first light path. The multi-wavelength visible light laser source module 8, the second micro objective lens 10 and the second two-dimensional scanning galvanometer 11 are combined to form a second light path, and the multi-wavelength visible light laser source module 8 is used for emitting visible light laser and transmitting the visible light laser to the long-pass dichroic mirror 7 through the second light path. Wherein, the multi-wavelength near-infrared laser light source module 1 and the multi-wavelength visible light laser light source module 8 are adopted as light sources, near-infrared laser is collimated and expanded by the first micro objective lens 3 and then is injected into the first two-dimensional scanning galvanometer 4, and the first two-dimensional scanning galvanometer 4 has high reflectivity to the wave band of the near-infrared laser; after the visible light laser is collimated and expanded by the second micro objective lens 10, the visible light laser is injected into the second two-dimensional scanning galvanometer 11, and the second two-dimensional scanning galvanometer 11 has high reflectivity for the wave band of the visible light laser. The right-angle prism is used for refracting light rays, so that the light rays irradiate objects on the three-dimensional object stage at a proper angle, and total reflection is avoided. The two-dimensional scanning galvanometer consists of two reflecting mirrors, and X, Y axis scanning is controlled respectively. When the scanning galvanometer does not work, the generated laser is converged on the focal plane of the third microscope objective, and the converged laser carries out fluorescence excitation on a straight line segment of the focal plane of the imaging objective in a line mode. When the X-axis is operated at high frequency (> 1000 hz), the linear scanning laser at the focal plane of the imaging objective lens is expanded to a planar scanning laser. On the basis of the X-axis maintenance work, when the Y-axis scanning work, the excitation area is expanded from planar scanning to three-dimensional scanning, and then the application effect of three-dimensional scanning imaging is realized.
The light beam of the first optical path and the light beam of the second optical path are integrated at the long-pass dichroic mirror 7 and then enter the right-angle prism 15. The sample to be imaged is placed on the three-dimensional stage 17, and the three-dimensional stage 17 can be driven to drive the sample to move in three dimensions, so that the position where the light beam irradiates the sample can be adjusted. The light beam excites the sample at the bottom surface of the right angle prism 15 to generate fluorescence; the fourth microscope objective 16 collects the fluorescent light beam emitted from the other right-angle surface of the right-angle prism 15 to the short-pass dichroic mirror 18, and the short-pass dichroic mirror 18 splits the fluorescent light beam into two beams, that is, separates the visible light and the near-infrared light in the fluorescent light beam by the short-pass dichroic mirror 18; near-infrared light in the fluorescent light beam is incident on the first photosensitive member by the short-pass dichroic mirror 18, and visible light in the fluorescent light beam is incident on the second photosensitive member by the short-pass dichroic mirror 18. That is, the fluorescence signals in the visible light and near infrared regions are collected as the fluorescence beams by the fourth microscope objective 16, and the fourth microscope objective 16 is an imaging objective having a high transmittance in the 400-1700nm band. The controller can be used for respectively and independently controlling the multi-wavelength near infrared laser light source module 1 and the multi-wavelength visible light laser light source module 8, wherein the wavelength range of near infrared laser generated by the multi-wavelength near infrared laser light source module 1 is 700nm-1100nm, and the wavelength range of visible light laser generated by the multi-wavelength visible light laser light source module 8 is 400nm-700nm. The fourth micro objective 16 collects the light signal formed by reflecting the incident light contained in the fluorescent light beam emitted from the other right-angle surface of the right-angle prism 15, and the first photosensitive assembly and the second photosensitive assembly are both photosensitive assemblies with a filtering function, and the first photosensitive assembly filters the received light beam, so as to remove the light signal formed by reflecting the incident light contained therein; also, the received light beam is filtered by the second photosensitive assembly, thereby removing the optical signal formed by reflection of the incident light contained therein.
The first two-dimensional scanning galvanometer 4 and the second two-dimensional scanning galvanometer 11 adopted in the embodiment of the application can be replaced by a Spatial Light Modulator (SLM), an acousto-optic deflector (AODF), a digital micromirror array (DMD) and a Micro Electro Mechanical System (MEMS) with high frame rate, and can be used for replacing the components of the two-dimensional scanning galvanometer in the embodiment of the application to realize the generation and the layer-by-layer scanning of the light sheet.
In a more specific embodiment, the multi-wavelength near-infrared laser light source module 1 is transmitted to the light inlet end of the first micro objective lens 3 through a near-infrared single-mode polarization maintaining fiber 2; the multi-wavelength visible light laser source module 8 transmits the light to the light inlet end of the second microscope objective 10 through the visible light single-mode polarization maintaining fiber 9.
The multi-wavelength near infrared laser light source module 1 and the multi-wavelength visible light laser light source module 8 are respectively connected with a single-mode polarization maintaining optical fiber, and the port of the single-mode polarization maintaining optical fiber can be used for outputting visible light laser or near infrared laser and reducing laser transmission loss, so that the quality of fluorescence imaging of a sample is further improved.
In a more specific embodiment, a near infrared scanning lens 5 is further arranged between the first two-dimensional scanning galvanometer 4 and the long-pass dichroic mirror 7. Wherein, a visible light scanning lens 12 is also arranged between the second two-dimensional scanning galvanometer 11 and the long-pass dichroic mirror 7.
A near-infrared scanning lens 5 may be provided between the first two-dimensional scanning galvanometer 4 and the long-pass dichroic mirror 7, and a visible light scanning lens 12 may be provided between the second two-dimensional scanning galvanometer 11 and the long-pass dichroic mirror 7; the effect of the scanning process of the near infrared laser beam by the first two-dimensional scanning galvanometer 4 is improved by providing the near infrared scanning lens 5, and similarly, the effect of the scanning process of the visible laser beam by the second two-dimensional scanning galvanometer 11 is improved by providing the visible light scanning lens 12.
In a more specific embodiment, the first photosensitive assembly includes a second sleeve lens 20 and a near infrared camera 21; near infrared light in the fluorescent light beam is incident into the near infrared camera 21 through the second sleeve lens 20. Further, the first photosensitive assembly further comprises a first optical filter 19; the first filter 19 is disposed between the second sleeve lens 20 and the short-pass dichroic mirror 18. Specifically, the second photosensitive assembly includes a third sleeve lens 24 and a visible light camera 25; visible light in the fluorescent light beam is incident into the visible light camera 25 through the third sleeve lens 24. Wherein the second photosensitive assembly further comprises a second optical filter 23; the second filter 23 is disposed between the third sleeve lens 24 and the short-pass dichroic mirror 18.
The first photosensitive assembly can be arranged to be composed of a second sleeve lens 20 and a near infrared camera 21, wherein the second sleeve lens 20 transmits near infrared light in the fluorescent light beam and irradiates the near infrared camera 21, so that the quality of a near infrared fluorescent signal acquired by the near infrared camera 21 is improved; similarly, the second photosensitive assembly may be configured to be composed of a third sleeve lens 24 and a visible light camera 25, where the third sleeve lens 24 transmits visible light in the fluorescent light beam and irradiates the visible light camera 25, so as to improve quality of the visible light fluorescent signal obtained by the visible light camera 25, and a second reflecting mirror 22 may be disposed between the short-pass dichroic mirror 18 and the third sleeve lens 24, so that the visible light emitted by the short-pass dichroic mirror 18 is reflected by the second reflecting mirror 22 and then irradiates the third sleeve lens 24.
Further, to improve the signal acquisition quality of the visible light camera 25 and the near infrared camera 21, a first filter 19 may be disposed between the second sleeve lens 20 and the short-pass dichroic mirror 18, where the first filter 19 may be used to filter out the light signal formed by reflection of the incident light contained in the near infrared light in the fluorescent light beam, so as to improve the purity of the light beam entering the second sleeve lens 20, thereby further improving the quality of the near infrared fluorescent signal acquired by the near infrared camera 21. Similarly, a second optical filter 23 may be disposed between the third sleeve lens 24 and the short-pass dichroic mirror 18, where the second optical filter 23 may be used to filter out an optical signal formed by reflection of incident light included in the visible light in the fluorescent light beam, so as to improve the purity of the light beam incident on the third second sleeve lens 20, that is, further improve the quality of the visible light fluorescent signal obtained by the visible light camera 25.
In a specific application process, the short-pass dichroic mirror 18 can be replaced according to needs, so that a two-region light sheet conversion technology of visible light and near infrared light can be combined, and a visible-near infrared two-region light sheet microscopic imaging technology based on the conversion technology can be realized, so that an imaging effect is further improved.
In a more specific embodiment, a first sleeve lens 13 is further arranged between the long-pass dichroic mirror 7 and the third microscope objective 14. The first sleeve lens 13 is arranged to transmit the combined light beam emitted by the long-pass dichroic mirror 7, so that the quality of the light beam entering the third microscope objective lens 14 is improved, and the combined light beam can be more accurately emitted into the third microscope objective lens 14 for focusing.
The embodiment of the application also provides a light sheet microscopic imaging method from visible light to near infrared, referring to fig. 2, and the imaging method comprises steps S110 to S150.
S110, starting the multi-wavelength near infrared laser light source module to emit near infrared laser, collimating and expanding the beam through the first micro objective lens, then injecting the near infrared laser into the X-axis reflecting mirror and the Y-axis reflecting mirror in the first two-dimensional scanning vibrating mirror, reflecting the near infrared laser, and then injecting the near infrared laser into the rear surface of the long-pass dichroic mirror to transmit the near infrared laser.
When a near infrared band light sheet is imaged on a sample, near infrared laser is emitted through a multi-wavelength near infrared laser source module, and a dye in the sample is excited to emit fluorescent signals, and a near infrared camera records the near infrared fluorescent signals; and then scanning the vibrating mirror layer by layer on the Y axis to acquire the three-dimensional polished section data of the sample.
S120, simultaneously starting the multi-wavelength visible light laser source module to emit visible light laser, collimating and expanding the beam through the second micro objective lens, and then injecting the visible light laser into an X-axis reflecting mirror and a Y-axis reflecting mirror in the second two-dimensional scanning vibrating mirror for reflection, and then injecting the visible light laser into the front surface of the long-pass dichroic mirror for reflection.
When a light sheet of a visible light wave band is imaged on a sample, a multi-wavelength visible light laser source module is used for emitting visible light laser and exciting dye in the sample to emit fluorescent signals, and a visible light camera is used for recording the visible light fluorescent signals; and then scanning the vibrating mirror layer by layer on the Y axis to acquire the three-dimensional polished section data of the sample.
S130, the near infrared laser beam from the first light path and the visible laser beam from the second light path are converged at the long-pass dichroic mirror to form a combined beam, and the combined beam is injected into a third microscope objective lens to be focused, and then is coupled and irradiated through a right-angle prism to excite dyes in a sample arranged on the three-dimensional object stage to generate fluorescence and emit.
And S140, collecting fluorescent light beams emitted from the other right angle surface of the right angle prism by the fourth micro objective lens, and enabling near infrared light in the fluorescent light beams to be reflected on the front surface of the short-pass dichroic mirror and enter the first photosensitive assembly to obtain a first detection record, and enabling visible light in the fluorescent light beams to be transmitted on the front surface of the short-pass dichroic mirror and enter the second photosensitive assembly to obtain a second detection record.
The first detection record is that the obtained light sheet information of the near infrared fluorescence signal is recorded by a near infrared camera; the second detection record is the light sheet information of the obtained visible light fluorescence signal recorded by the visible light camera.
And S150, performing data processing on the first detection record and the second detection record to obtain a three-dimensional reconstruction image of the sample.
The acquired first detection record and second detection record can be subjected to data processing, so that three-dimensional image reconstruction of the sample is realized, and a three-dimensional reconstruction image of the sample is obtained. When the two-dimensional scanning galvanometer works, the three-dimensional area excitation is carried out on the sample, and the Z axis of the fourth microscope objective is matched with the Y axis of the scanning galvanometer to move, so that the focal plane of the fourth microscope objective is always matched with the excitation plane. The Z axis of the fourth microscope objective lens can be moved each time, a first detection record and a second detection record can be obtained through a near infrared camera and a visible light camera, then three-dimensional reconstruction is carried out on images contained in the first detection record and images contained in the second detection record, and then three-dimensional images of the sample in an illumination area can be restored, so that three-dimensional reconstructed image images of the sample are obtained.
In a specific application process, the embodiment of the application adopts the visible light and near infrared light sources in the illumination light path, so that the generation of the visible light and near infrared illumination light sheets can be realized, and the imaging light path comprises the detection light path aiming at the visible light fluorescence signal and the near infrared two-region fluorescence signal, so that the full-band imaging target from the visible light to the near infrared two-region can be realized. Compared with the traditional mode of generating the light sheet by adopting the cylindrical lens, the imaging field of view and the light beam type are relatively fixed, but the application adopts the galvanometer scanning mode, the imaging field of view can be adjusted by controlling the voltage of the galvanometer, and the light beam type can be adjusted by adding an element in a light path (such as adding a conical lens to generate Bessel light beam), so that the imaging field of view range selection and the light beam type adjustment are more flexible. The application adopts the multi-wavelength module and can cover the spectrum range from visible to near infrared, so the application has more flexible selection of dye in the implementation process.
The embodiment of the application provides a light sheet microscopic imaging system and an imaging method in a visible light-near infrared two region, wherein the imaging system comprises a multi-wavelength near infrared laser light source module, a multi-wavelength visible light laser light source module, a first microscope objective, a second microscope objective, a first two-dimensional scanning galvanometer, a second two-dimensional scanning galvanometer, a long-pass dichroic mirror, a third microscope objective, a right-angle prism, a fourth microscope objective, a three-dimensional objective table, a short-pass dichroic mirror, a first photosensitive assembly and a second photosensitive assembly. Compared with the prior art, the application comprehensively applies the fluorescence microscopic imaging technology, the light sheet microscopic imaging technology and the near infrared two-region fluorescence imaging technology, gives play to the respective advantages of visible light imaging and infrared light imaging, can ensure higher imaging depth when imaging thicker samples, and can improve imaging resolution of thicker samples, thereby flexibly realizing high-quality imaging of thick samples and thin samples and solving the problem of high-resolution deep imaging of thick samples.
While the application has been described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions of equivalents may be made and equivalents will be apparent to those skilled in the art without departing from the scope of the application. Therefore, the protection scope of the application is subject to the protection scope of the claims.
Claims (10)
1. The system is characterized by comprising a multi-wavelength near infrared laser light source module, a multi-wavelength visible light laser light source module, a first microscope objective, a second microscope objective, a first two-dimensional scanning galvanometer, a second two-dimensional scanning galvanometer, a long-pass dichroic mirror, a third microscope objective, a right angle prism, a fourth microscope objective, a three-dimensional object stage, a short-pass dichroic mirror, a first photosensitive component and a second photosensitive component:
the multi-wavelength near infrared laser light source module emits near infrared laser and transmits the near infrared laser to the long-pass dichroic mirror through a first light path; the multi-wavelength visible light laser source module emits visible light laser and transmits the visible light laser to the long-pass dichroic mirror through a second light path;
the first light path is sequentially provided with the first microscope objective and the first two-dimensional scanning galvanometer; the second light path is sequentially provided with the second micro objective and the second two-dimensional scanning galvanometer;
the long-pass dichroic mirror integrates the light beams from the first light path and the second light path, and irradiates into a right-angle surface of the right-angle prism through the third microscope objective lens, and excites a sample to generate fluorescence at the bottom surface of the right-angle prism;
the right angle prism is fixedly arranged above the three-dimensional object stage, and the three-dimensional object stage is used for placing a sample and driving the sample to perform three-dimensional movement;
the fourth microscope objective collects the fluorescent light beam emitted from the other right angle surface of the right angle prism to the short-pass dichroic mirror; near infrared light in the fluorescent light beam is emitted into the first photosensitive assembly by the short-pass dichroic mirror, visible light in the fluorescent light beam is emitted into the second photosensitive assembly by the short-pass dichroic mirror, and the first photosensitive assembly and the second photosensitive assembly are photosensitive assemblies with a filtering function.
2. The optical chip microscopic imaging system from visible light to near infrared according to claim 1, wherein the multi-wavelength near infrared laser source module is transmitted to the light inlet end of the first microscope objective through a near infrared single-mode polarization maintaining fiber;
the multi-wavelength visible light laser source module is transmitted to the light inlet end of the second microscope objective through the visible light single-mode polarization maintaining fiber.
3. The optical sheet microscopic imaging system of two regions from visible light to near infrared according to claim 1 or 2, wherein a near infrared scanning lens is further provided between the first two-dimensional scanning galvanometer and the long-pass dichroic mirror.
4. The light sheet microscopic imaging system of two areas from visible light to near infrared according to claim 1 or 2, wherein a visible light scanning lens is further arranged between the second two-dimensional scanning galvanometer and the long-pass dichroic mirror.
5. The optical sheet microscopic imaging system of two regions from visible light to near infrared according to claim 1 or 2, wherein a first sleeve lens is further provided between the long-pass dichroic mirror and the third microscope objective.
6. The optical sheet microscopy imaging system of claim 1 or 2, wherein the first photosensitive assembly comprises a second sleeve lens and a near infrared camera;
near infrared light in the fluorescent light beam is emitted into the near infrared camera through the second sleeve lens.
7. The optical sheet microscopy imaging system of two visible to near infrared regions of claim 6, wherein the first photosensitive assembly further comprises a first optical filter; the first filter is disposed between the second sleeve lens and the short-pass dichroic mirror.
8. The optical sheet microscopic imaging system of two regions from visible light to near infrared according to claim 1 or 2, wherein the second photosensitive assembly comprises a third sleeve lens and a visible light camera;
visible light in the fluorescent light beam is emitted into the visible light camera through the third sleeve lens.
9. The light sheet microimaging system of claim 8, wherein the second photosensitive assembly further comprises a second filter; the second filter is disposed between the third sleeve lens and the short-pass dichroic mirror.
10. A method for microscopic imaging of light sheet in the visible to near infrared range, wherein the imaging method is applied to the imaging system of any one of claims 1 to 9, and the imaging method comprises:
starting a multi-wavelength near infrared laser light source module to emit near infrared laser, collimating and expanding beams through a first micro objective lens, then injecting the near infrared laser into an X-axis reflecting mirror and a Y-axis reflecting mirror in a first two-dimensional scanning vibrating mirror, reflecting the near infrared laser, and then injecting the near infrared laser into the rear surface of the long-pass dichroic mirror for transmission;
simultaneously starting a multi-wavelength visible light laser source module to emit visible light laser, collimating and expanding beams through a second micro objective lens, and then injecting the visible light laser into an X-axis reflecting mirror and a Y-axis reflecting mirror in a second two-dimensional scanning vibrating mirror to be reflected and then injecting the visible light laser into the front surface of the long-pass dichroic mirror to be reflected;
the near infrared laser beam from the first light path and the visible laser beam from the second light path are converged at the long-pass dichroic mirror to form a combined beam, the combined beam is injected into a third microscope objective to be focused, and then is coupled and irradiated to a sample arranged on the three-dimensional object stage through a right-angle prism, and dyes in the sample are excited to generate fluorescence and are emitted;
the fourth microscope objective collects fluorescent light beams emitted from the other right angle surface of the right angle prism, the fluorescent light beams are emitted into the front surface of the short-pass dichroic mirror, near infrared light in the fluorescent light beams is reflected on the front surface of the short-pass dichroic mirror and is emitted into the first photosensitive assembly to obtain a first detection record, and visible light in the fluorescent light beams is transmitted on the front surface of the short-pass dichroic mirror and is emitted into the second photosensitive assembly to obtain a second detection record;
and carrying out data processing on the first detection record and the second detection record to obtain a three-dimensional reconstruction image of the sample.
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