A near-infrared two-zone light sheet three-dimensional in vivo imaging system

Abstract

The invention discloses a near-infrared two-zone light sheet three-dimensional in vivo imaging system, which relates to the field of biological imaging. The near-infrared two-zone light sheet three-dimensional in vivo imaging system comprises an object stage, an imaging unit and a laser unit. The laser unit comprises a laser collimator for adjusting an emitted laser to a collimated light and a light sheet generating unit for compressing the collimated light into a light sheet. The light sheet generating unit is connected to the laser collimator. Both the imaging unit and the light sheet generating unit are arranged toward the object stage. The light sheet generating unit can compress the collimated light into a light sheet, thereby compressing the laser power in a thin light sheet area, thereby improving the laser power per unit area of ​​the laser and increasing the excitation depth. The imaging unit can shoot the area where the fluorescent probe is illuminated. The shooting is subject to little laser interference and the image signal-to-noise ratio is high, so the image will be clearer. Moreover, since the thickness of the light sheet is very thin, the longitudinal resolution of the reconstructed three-dimensional model is high.

Description

Technical Field

[0001] The present invention relates to the field of biological imaging, and in particular to a near-infrared two-zone light sheet three-dimensional living body imaging system. Background Art

[0002] Traditional imaging relies primarily on visible changes in bodily, physiological, and metabolic processes during disease states, rather than understanding the specific molecular events of a disease. Molecular imaging, on the other hand, utilizes specific molecular probes to track and image targets. This shift from nonspecific to specific imaging has significantly impacted disease biology, early detection, characterization, assessment, and treatment.

[0003] Mice, rats, rabbits, and other animals are commonly used as small animal models for studying biological diseases such as tumor growth and treatment, bone repair, and vascular disease. By labeling with fluorescent probes, optical fluorescence imaging can observe changes in living small animal models with minimal or no damage to the animal model, allowing for the study of pathological mechanisms in these models. Recently developed near-infrared (NIR) region II fluorescent probes are often used for deep tissue imaging in small animals due to the deep tissue penetration of their emitted NIR fluorescence.

[0004] Existing in vivo small animal imaging systems primarily consist of lasers, imaging cameras, industrial lenses, and fluorescence filters. These systems capture flat-panel images of fluorescently labeled structures in small animals. However, the low optical power per unit area emitted by existing lasers prevents excitation of deeper fluorescent probe-labeled biological tissues, limiting the depth of in vivo fluorescence imaging in the second infrared region. Consequently, this imaging method can only capture surface distribution information, failing to capture deeper tissue layers. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the existing technology, the purpose of this application is to provide a near-infrared two-zone light sheet three-dimensional in vivo imaging system, aiming to solve the problem in the existing imaging technology that the laser excitation depth is shallow and the depth information cannot be obtained for clear three-dimensional imaging.

[0006] To solve the above technical problems, the embodiment of the present application provides a near-infrared two-zone light sheet three-dimensional in vivo imaging system, including an object stage, an imaging unit and a laser unit, the laser unit including a laser, a laser collimator for adjusting the emitted laser to collimated light, and a light sheet generation unit for compressing the collimated light into a light sheet, the laser collimator is connected to a laser for emitting Gaussian light through an optical fiber, the light sheet generation unit is connected to the laser collimator, the imaging unit and the light sheet generation unit are both arranged toward the object stage, the light sheet generation unit includes a cage plate, a telescopic lens sleeve for adjusting the distance between the focusing lens and the objective lens, a Powell prism group for uniformly distributing the light intensity in the light sheet illumination area, a focusing lens and an objective lens, through Changing the distance between the condensing lens and the objective lens can change the thickness of the light sheet and the uniform illumination range. The distance between the two main surfaces of the condensing lens and the objective lens is 27mm-32mm, the thickness of the light sheet is 100μm-190μm, and the uniform illumination range of the light sheet is 8mm-26mm. The cage plate, telescopic lens sleeve, condensing lens, objective lens and Powell prism group are arranged in sequence along the excitation optical axis, which increases the laser power per unit area. The light sheet irradiates the living body and increases the excitation depth. The imaging unit can capture the area where the fluorescent probe is illuminated. The shooting is subject to little laser interference and the image signal-to-noise ratio is high, so the image will be clearer. In addition, since the light sheet thickness is very thin, the longitudinal resolution of the reconstructed three-dimensional model is high.

[0007] The excitation optical axis is the line for laser emission. The cage plate and the laser collimator are coaxially connected by thread to form a whole. The telescopic lens sleeve is connected to the front end of the cage plate, the rear end of the focusing lens is connected to the telescopic lens sleeve, the objective lens is connected to the front end of the telescopic lens sleeve, the focusing lens is located between the telescopic lens sleeve and the objective lens, and the Powell prism group is connected to the front end of the objective lens. The Powell prism group is composed of several Powell prism arrays. The collimated laser passes through the focusing lens, objective lens and Powell prism group in sequence and is compressed to generate a thin light sheet area.

[0008] As a further improved technical solution, the laser collimator and the light sheet generating unit are arranged in sequence along the excitation light axis, and the excitation light axis intersects with the imaging light axis of the imaging unit. During actual imaging, the excitation light axis must intersect with the imaging light axis of the imaging unit to accurately capture the area actually irradiated by the light sheet.

[0009] As a further improved technical solution, the excitation optical axis is perpendicular to the imaging optical axis or the excitation optical axis and the imaging optical axis form an angle of 45 degrees, wherein when the excitation optical axis is perpendicular to the imaging optical axis, the moving direction of the single-axis translation stage is parallel to the plane of the optical system; when the moving direction of the single-axis translation stage is perpendicular to the plane of the optical system, the excitation optical axis and the imaging optical axis form an angle of 45 degrees.

[0010] As a further improved technical solution, the stage includes a fixed plate and a single-axis translation stage, wherein the fixed plate is installed on the single-axis translation stage, wherein the fixed plate is used to fix living animals to prevent the movement of the moving animals from affecting the shooting accuracy, and the single-axis translation stage is used to drive the fixed plate to move precisely.

[0011] As a further improved technical solution, the stage also includes a moving stage controller and an adjustable lifting platform. The moving stage controller is connected to the single-axis translation stage signal. The upper end surface of the adjustable lifting platform is a sloped surface. The single-axis translation stage is installed on the sloped surface. The adjustable lifting platform can adjust the overall height of the stage. By adjusting the height of the adjustable lifting platform, the center of the fixed plate is aligned with the excitation light axis.

[0012] As a further improved technical solution, the imaging unit includes a near-infrared camera, an industrial lens, and a filter. These are arranged sequentially along the imaging optical axis. A threaded tube is used to connect and mount the filter mount to the near-infrared camera. The industrial lens is threadedly connected to the filter mount and mounted below the filter mount. The filter is mounted in the filter mount. The coaxial arrangement of the near-infrared camera, industrial lens, and filter along the imaging optical axis ensures imaging accuracy.

[0013] As a further improved technical solution, the imaging unit is installed on a box body, which includes an upper partition and a lower partition. The upper partition and the lower partition are both horizontally installed in the box body, and the upper partition is located above the lower partition. The upper partition and the lower partition divide the box body into upper partition room, middle partition room and lower partition room. A circular hole is opened in the middle of the upper partition, and a circular hole is also opened on the lower partition. By opening a circular hole in the middle of the upper partition, the installation of the imaging unit is facilitated. By installing a circular hole on the lower partition, the passage of the optical fiber is facilitated. The two ends of the optical fiber are respectively connected to the laser collimator and the laser.

[0014] As a further improved technical solution, the near-infrared camera is located in the upper partition room, the near-infrared camera is installed above the upper partition room, the industrial lens is located in the middle partition room, the near-infrared camera is connected to the industrial lens through a filter mounting frame, the filter is arranged in the filter mounting frame, the near-infrared camera is installed above the upper partition room, and a threaded tube is used to pass through the middle circular hole of the upper partition room to connect and install the filter mounting frame and the near-infrared camera through the upper partition room. The industrial lens is threadedly connected to the filter mounting frame, the industrial lens can be installed under the filter mounting frame through threads, the filter is installed in the filter mounting frame, and the filter is located between the industrial lens and the filter mounting frame. After the imaging unit is fixedly connected through the upper partition room, it can be ensured that the imaging unit will not move, and at the same time, the imaging unit can be kept in a vertical state, thereby improving the shooting accuracy.

[0015] As a further improved technical solution, the stage, adjustable lifting platform, laser collimator and light sheet generation unit are all located between the middle partitions, the lower end of the adjustable lifting platform is fixed on the lower partition, and the light sheet generation unit is installed on the lower partition through a bracket. The laser and the stage controller are both located between the lower partitions, and the control line of the stage controller passes through the circular hole of the lower partition to connect the stage controller and the single-axis displacement stage respectively. The single-axis displacement stage of the stage is installed on the slope surface, the fixed plate is installed in the center of the single-axis displacement stage, the adjustable lifting platform is fixedly installed on the lower partition of the box, the laser and the laser controller are both installed between the lower partitions of the box, the laser is connected to the laser collimator through the circular hole of the lower partition through the laser optical fiber, and the light sheet generation unit is fixedly installed on the lower partition of the box using a bracket. The position of the light sheet generation unit and the imaging unit is adjusted so that the optical system plane formed by the perpendicular intersection of the excitation optical axis and the imaging optical axis is parallel to the rear panel of the box, so that the excitation optical axis is parallel to the lower partition and the imaging optical axis is perpendicular to the lower partition, thereby ensuring imaging accuracy.

[0016] Beneficial effects: Compared with the prior art, the near-infrared two-zone light sheet three-dimensional in vivo imaging system of the present invention includes a stage, an imaging unit and a laser unit. The laser unit includes a laser collimator for adjusting the output laser to collimated light and a light sheet generating unit for compressing the collimated light into a light sheet. The light sheet generating unit is connected to the laser collimator. The imaging unit and the light sheet generating unit are both arranged toward the stage. The light sheet generating unit can compress the collimated light into a light sheet, thereby compressing the laser power in a thin light sheet area, thereby increasing the laser power per unit area of ​​the laser and increasing the excitation depth. The imaging unit can shoot the area where the fluorescent probe is illuminated. The shooting is subject to little laser interference and the image signal-to-noise ratio is high, so the image will be clearer. Moreover, since the thickness of the light sheet is very thin, the longitudinal resolution of the reconstructed three-dimensional model is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the near-infrared second-zone light sheet three-dimensional living imaging system of the present invention.

[0018] Figure 2 It is a structural diagram of the near-infrared second-zone light sheet three-dimensional living body imaging system of the present invention.

[0019] Reference numerals:

[0020] 1-Laser, 2-Laser collimator, 3-Cage plate, 4-Telescopic lens tube, 5-Converging lens, 6-Objective lens, 7-Powell prism assembly, 8-Single-axis translation stage, 9-Fixed plate, 11-Industrial lens, 12-Filter, 13-Filter mounting bracket, 14-Near-infrared camera, 15-Adjustable lifting platform, 16-Stage controller, 100-Cabinet, 200-Light sheet generation unit, 300-Imaging unit.

[0021] Among them, the near-infrared second-zone light sheet three-dimensional in vivo imaging system does not include a small animal biological model. In order to facilitate the description of the specific implementation method, it is marked as 10-mouse.

[0022] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0023] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0025] The disclosure below provides many different embodiments or examples for realizing different structures of the present invention. In order to simplify the disclosure of the present invention, the components and settings of specific examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention may repeat reference numerals and / or reference letters in different examples. Such repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides examples of various specific processes and materials, but those of ordinary skill in the art will appreciate the application of other processes and / or the use of other materials.

[0026] It should be noted that, in the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; they may refer to mechanical connections; they may refer to direct connections or indirect connections through an intermediate medium; they may refer to internal communication between two components or an interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0027] After research, the inventors found that the existing technology has the following problems:

[0028] (1) Near-infrared zone II fluorescent probes developed in recent years are often used for deep tissue imaging in small animals due to the deep tissue penetration of the near-infrared fluorescence they emit. Currently, commercially mature small animal in vivo imaging systems mainly consist of lasers, imaging cameras, industrial lenses, fluorescent filters, etc., and observe the fluorescently labeled structures of small animals by taking planar photos. This imaging method can observe the planar distribution information of tumors or blood vessels, but cannot obtain the depth information of the lesion structure, and therefore cannot accurately evaluate the lesion characteristics of biological models.

[0029] (2) In addition, the existing wide-field laser excitation of biological models results in low light power per unit area, which prevents the excitation of biological tissues labeled with fluorescent probes at deeper levels, thus limiting the depth of infrared second-zone fluorescence in vivo imaging. Current in vivo imaging systems use industrial lenses and cameras to shoot biological models from a distance, resulting in low imaging resolution and insufficient clarity for small animal biological tissue observations.

[0030] Example 1:

[0031] like Figure 1-2 As shown, the embodiment of the present application provides a near-infrared two-zone light sheet three-dimensional in vivo imaging system to solve the above technical problems, including an object stage, an imaging unit 300 and a laser unit, the laser unit including a laser 1, a laser collimator 2 for adjusting the emitted laser into collimated light, and a light sheet generation unit 200 for compressing the collimated light into a light sheet, the laser collimator 2 is connected to the laser 1 through an optical fiber, the light sheet generation unit 200 is connected to the laser collimator 2, the imaging unit 300 and the light sheet generation unit 200 are both arranged toward the object stage, the light sheet generation unit 200 includes a cage plate 3, a A telescopic lens sleeve 4 for adjusting the distance between the condensing lens 5 and the objective lens 6, a Powell prism group 7 for uniformly distributing the light intensity in the light sheet illumination area, the condensing lens 5 and the objective lens 6. By changing the distance between the condensing lens 5 and the objective lens 6, the thickness of the light sheet and the uniform illumination range can be changed. The distance between the two main surfaces of the condensing lens and the objective lens is 27mm-32mm, the thickness of the light sheet is 100μm-190μm, and the uniform illumination range of the light sheet is 8mm-26mm. The cage plate 3, telescopic lens sleeve 4, condensing lens 5, objective lens 6 and Powell prism group 7 are arranged in sequence along the excitation optical axis.

[0032] Specifically, the stage is used to fix the living organism to be imaged to prevent it from moving. The living organism needs to be fixed on the stage before imaging. The laser unit is used to irradiate the living organism and excite the fluorescent probe in the living organism to make the fluorescent probe emit light. The imaging unit 300 is used to shoot the area of ​​the living organism irradiated by the laser unit. The imaging unit 300 is specifically used to obtain multiple frames of the image when the single-axis translation stage 8 drives the small animal to move. The multiple frames of the image are used to form a three-dimensional imaging model, wherein the fluorescent probe is inserted into the living organism in advance. The fluorescent probe uses a fluorescent substance as an indicator and produces fluorescence under the excitation of a certain wavelength of light. The qualitative or quantitative analysis of the substance to be detected is achieved by detecting the generated fluorescence. The fluorescent probe is affected by the surrounding environment, causing its fluorescence emission to change, thereby allowing people to know the characteristics of the surrounding environment or certain specific information existing in the environment.

[0033] The laser unit includes a laser collimator 2 for adjusting the output laser into collimated light and a light sheet generation unit 200 for compressing the collimated light into a light sheet. The laser collimator 2 can adjust the laser emitted by the laser 1 through the optical fiber into a collimated laser. The light sheet generation unit 200 can compress the collimated laser emitted by the laser collimator 2 to generate a thin light sheet area, thereby increasing the laser power per unit area. The light sheet irradiates the living body and increases the excitation depth. The imaging unit 300 can shoot the area where the fluorescent probe is illuminated. The shooting is subject to little laser interference and the image signal-to-noise ratio is high, so the image will be clearer. Moreover, since the thickness of the light sheet is very thin, the longitudinal resolution of the reconstructed three-dimensional model is high.

[0034] The excitation optical axis is the line of laser emission. The cage plate 3 and the laser collimator 2 are coaxially connected by threads to form a whole. The telescopic lens sleeve 4 is connected to the front end of the cage plate 3. The rear end of the condensing lens 5 is connected to the telescopic lens sleeve 4. The objective lens 6 is connected to the front end of the telescopic lens sleeve 4. The condensing lens 5 is located between the telescopic lens sleeve 4 and the objective lens 6. The Powell prism group 7 is connected to the front end of the objective lens 6. The Powell prism group 7 is composed of a plurality of Powell prism arrays. Among them, the cage plate 3 is an SM1 threaded cage plate 3. The front and rear ends of the SM1 threaded cage plate 3 are respectively used to thread the telescopic lens sleeve 4 and the laser collimator 2. The telescopic lens sleeve 4 is used to connect the condensing lens 5 and the objective lens 6, and the distance between the condensing lens 5 and the objective lens 6 can be adjusted through the telescopic lens sleeve 4. Among them, in order to achieve better results, the condensing lens 5 adopts a 0.79NA aspheric condensing lens 5, and the objective lens 6 adopts an objective lens 6 with an objective lens 6NA of 0.3 and a magnification of 10. The telescopic lens sleeve 4 adopts the base optical CSM-Z5 inch telescopic lens tube, the collimated laser passes through the focusing lens 5, objective lens 6 and Will prism group in sequence and is compressed to form a thin light sheet area.

[0035] Furthermore, the laser collimator 2 and the light sheet generating unit 200 are sequentially arranged along the excitation optical axis, and the excitation optical axis intersects with the imaging optical axis of the imaging unit 300 .

[0036] Among them, the laser collimator 2 and the light sheet generating unit 200 are coaxially connected by a thread to form a whole, and the laser collimator 2 and the light sheet generating unit 200 are both on the excitation optical axis. The imaging optical axis of the imaging unit 300 is the center line of the shooting light when the imaging unit 300 is shooting, and the excitation optical axis is the center line of the laser beam. During actual imaging, the excitation optical axis must intersect with the imaging optical axis of the imaging unit 300 to accurately capture the area actually irradiated by the light sheet. The excitation optical axis and the imaging optical axis intersect to form an optical system plane, and the intersection of the excitation optical axis and the imaging optical axis is located at the center of the light sheet waist generated by the light sheet generating unit 200.

[0037] Furthermore, the excitation optical axis is perpendicular to the imaging optical axis or the excitation optical axis forms an angle of 45 degrees with the imaging optical axis. The stage includes a fixed plate 9 and a single-axis translation stage 8. The fixed plate 9 is installed on the single-axis translation stage 8. When the excitation optical axis is perpendicular to the imaging optical axis, the moving direction of the single-axis translation stage 8 is parallel to the plane of the optical system. When the moving direction of the single-axis translation stage 8 is perpendicular to the plane of the optical system, the excitation optical axis and the imaging optical axis form an angle of 45 degrees. The fixed plate 9 is provided with a structure for fixing living animals. The upper end surface of the fixed plate 9 is used to fix living animals. The lower end surface of the fixed plate 9 is installed on the single-axis translation stage 8. The single-axis translation stage 8 can drive the fixed plate 9 and the living animals to move. The single-axis translation stage 8 is a device that can drive the fixed plate 9 to move in a certain direction with a single axis. The single-axis translation stage 8 can be a device with a displacement function such as a single-axis flexible translation stage.

[0038] Furthermore, the loading platform also includes a platform controller 16 and an adjustable lifting platform 15. The platform controller 16 is connected to the single-axis translation platform 8 by signal. The upper end surface of the adjustable lifting platform 15 is a sloped surface, and the single-axis translation platform 8 is installed on the sloped surface.

[0039] Among them, the stage controller 16 is used to control the operation of the single-axis displacement stage 8. The stage controller 16 can send instructions to the single-axis displacement stage 8 to move the single-axis displacement stage 8 in a certain direction, so that the single-axis displacement stage 8 drives the fixed plate 9 to move in a certain direction. The adjustable lifting platform 15 can be a cylinder lifting platform or a threaded lifting platform or other devices with lifting and adjusting functions. The upper end surface of the adjustable lifting platform 15 is a sloped surface, and the inclination angle of the sloped surface is 45°. The sloped surface can ensure that the single-axis displacement stage 8 and the fixed plate 9 are tilted at a certain angle, so that the living animal remains in a tilted state, so that the excitation optical axis and the imaging optical axis can intersect on the living animal. When in use, the position of the adjustable lifting platform 15 is adjusted so that the center of the fixed plate 9 is facing the industrial lens 11.

[0040] Furthermore, the imaging unit 300 includes a near-infrared camera 14, an industrial lens 11 and a filter 12, and the near-infrared camera 14, the industrial lens 11 and the filter 12 are arranged in sequence along the imaging optical axis.

[0041] The near-infrared camera 14 can be an indium gallium arsenide cooled camera. A threaded tube is used to connect the filter mount 13 and the near-infrared camera 14. The industrial lens 11 is threadedly connected to the filter mount 13 and mounted below the filter mount 13. The filter 12 is mounted in the filter mount 13. The near-infrared camera 14, the industrial lens 11, and the filter 12 are coaxially arranged along the imaging optical axis to ensure accurate imaging.

[0042] Furthermore, the imaging unit 300 is installed on the box 100, and the box 100 includes an upper partition and a lower partition. The upper partition and the lower partition are both horizontally installed in the box 100, and the upper partition is located above the lower partition. The upper partition and the lower partition divide the box 100 into an upper partition room, a middle partition room, and a lower partition room.

[0043] The imaging unit 300 is mounted on the housing 100, which can be made of 2mm thick aluminum sheet metal. The housing 100 is 97cm high, 60cm long, and 50cm wide. It is divided into three layers: upper, middle, and lower. Each layer has an upper, middle, and lower partition, respectively. The upper partition is 17cm high, and the lower partition is 20cm high. The upper partition has a circular hole in the middle, and the lower partition also has a circular hole.

[0044] Furthermore, the near-infrared camera 14 is located in the upper partition room, the near-infrared camera 14 is installed above the upper partition room, the industrial lens 11 is located in the middle partition room, the near-infrared camera 14 is connected to the industrial lens 11 through the filter mounting frame 13, and the filter 12 is arranged in the filter mounting frame 13.

[0045] The near-infrared camera 14 is installed above the upper partition. A threaded pipe is used to pass through the middle round hole of the upper partition to connect and install the filter mounting frame 13 and the near-infrared camera 14 through the upper partition. The industrial lens 11 is threadedly connected to the filter mounting frame 13. The industrial lens 11 can be installed below the filter mounting frame 13 through threads. The filter 12 is installed in the filter mounting frame 13. The filter 12 is located between the industrial lens 11 and the filter mounting frame 13.

[0046] Furthermore, the stage, adjustable lifting platform 15, laser collimator 2 and light sheet generating unit 200 are all located in the middle partition room, the lower end of the adjustable lifting platform 15 is fixed on the lower partition room, the light sheet generating unit 200 is installed on the lower partition room through a bracket, and the laser 1 and the stage controller 16 are both located in the lower partition room.

[0047] The optical fiber is a single-mode optical fiber. The light output by the single-mode optical fiber is Gaussian light. After passing through the focusing lens 5 and the objective lens 6, it is still Gaussian distributed. After passing through the Powell prism group 7, the light intensity in the light sheet illumination area can be evenly distributed.

[0048] The loading platform is installed between the middle partitions of the box 100, and the moving stage controller 16 is installed between the lower partitions of the box 100. The control line of the moving stage controller 16 passes through the circular hole of the lower partition to connect the moving stage controller 16 and the single-axis displacement stage 8 respectively. The upper end surface of the adjustable lifting platform 15 is a 45-degree slope surface. The single-axis displacement stage 8 of the loading platform is installed on the slope surface, and the fixed plate 9 is installed in the center of the single-axis displacement stage 8. By adjusting the position of the adjustable lifting platform 15 so that the center of the fixed plate 9 faces the industrial lens 11, the adjustable lifting platform 15 is fixedly installed on the lower partition of the box 100. The laser collimator 2 can use an integrated laser collimator 2 for fiber laser alignment. The laser 1 and the controller of the laser 1 are both installed between the lower partitions of the box 100. The laser 1 is connected to the laser collimator 2 through the circular hole of the lower partition via the laser 1 optical fiber. The laser collimator 2 is coaxially connected with the light sheet generating unit 200 to form a whole. The light sheet generating unit 200 is fixedly installed on the lower partition of the box 100 using a bracket. The position of the light sheet generating unit 200 and the imaging unit 300 is adjusted so that the optical system plane formed by the perpendicular intersection of the excitation optical axis and the imaging optical axis is parallel to the rear panel of the box 100, so that the excitation optical axis is parallel to the lower partition and the imaging optical axis is perpendicular to the lower partition.

[0049] The working principle of the present invention is:

[0050] This embodiment of the present invention uses a mouse 10 as a living animal, a near-infrared camera 14 that is an indium gallium arsenide cooled camera, a fiber laser 1, and a liquid-core fiber as the transmission fiber. The laser wavelength can be selected from 808 nm, 980 nm, 1064 nm, and the specific laser wavelength is selected based on the near-infrared region II fluorescent material. For example, when using rare earth erbium ion-doped nanoparticles to prepare a biological probe, a 980 nm laser 1 is used for excitation. The focusing lens 5 is a 0.79 NA aspheric focusing lens 5, the objective lens 66 has an NA of 0.3 and a magnification of 10, and the industrial lens 11 is a near-infrared lens with a focal length of 12 mm. The filter is selected according to the emission spectrum of the fluorescent probe. For example, if a biological probe is prepared using nanoparticles doped with rare earth erbium ions and its emission peak is around 1530nm, a bandpass filter with a central wavelength of 1500nm and a passband of 50nm can be selected; if a single fluorescent probe is emitting light, a 1450nm longpass filter can also be selected as the imaging filter. The laser collimator 2 can be a single convex lens, a fiber optic collimator, or an objective lens 6.

[0051] During use, the laser collimator 2 adjusts the laser emitted by the laser 1 through the optical fiber into collimated light, and the light sheet generation unit 200 compresses the collimated light emitted by the laser collimator 2 into a thin light sheet. The light sheet can be irradiated deeper into the tissue of the mouse 10. The light sheet is irradiated on the mouse 10 and reacts with the fluorescent probe on the mouse 10. The fluorescent probe emits fluorescence. The area irradiated by the light sheet is the light sheet area. The near-infrared camera 14 is aimed at the light sheet area to take a frame of image. Then the translation stage controller 16 controls the movement of the single-axis translation stage 8. The moving table drives the fixed plate 9 and the mouse 10 to move. The displacement step is 120um, where the movement step range is 0.8-1.42 times the light sheet waist thickness. Every time the single-axis translation stage 8 moves one step, the near-infrared camera 14 takes a frame of image. When the shooting of this mouse 10 is completed, all the captured photos are imported into the three-dimensional model generator to generate a three-dimensional small animal sample imaging model.

[0052] In response to the current problem of shallow depth of in vivo imaging in the second near-infrared zone, the present invention compresses the laser beam into a light sheet and compresses the laser power into a thin light sheet area, thereby increasing the laser power per unit area and increasing the excitation depth. Using light sheet illumination, the area illuminated by the fluorescent probe is almost the same width as the light sheet, and the area is photographed and imaged. Then, the displacement stage carries the small animal biological model and moves the distance of the light sheet thickness, and then takes another image, until all areas that need to be imaged are photographed to form a series of images, and the image is reconstructed into a three-dimensional biological model. Since each image is taken of an area illuminated by a light sheet, the laser interference is small and the image signal-to-noise ratio is high, so the image will be clearer; similarly, due to the excitation of light sheet illumination, the thickness of the light sheet is very thin, and the longitudinal resolution of the reconstructed three-dimensional model is the thickness of the light sheet divided by 0.707. This resolution is much higher than the resolution of the plane imaging taken by the industrial camera lens 11. The system can obtain three-dimensional information of the small animal in vivo model and has the advantages of deep imaging and high resolution three-dimensional in vivo imaging.

[0053] The light sheet is generated by using a focusing lens, combined with an objective lens (10×, NA = 0.3, NexscopeplanF Apo), to focus the Gaussian parallel beam emitted by the collimator. This Gaussian beam is then reshaped through the light sheet using a Powell prism array (PMMA-Powell lens array-30). The light sheet thickness can be calculated using the following formula:

[0054]

[0055] In the formula, 2ω0 is the thickness of the light sheet, λ is the wavelength, and NA is the numerical aperture of the lens group. Using the Gaussian beam model, we get the diameter and length of the beam waist. The Gaussian beam function is shown below:

[0056]

[0057]

[0058]

[0059] Where E is the amplitude of the beam, E0 is the amplitude at the focal plane, z is the distance from the focal plane, r is the distance from the optical axis, ω(z) is the spot radius, and ω0 is the spot radius at the focal plane. z0 is the Rayleigh length, η is the refractive index of the medium, and λ is the wavelength. The Rayleigh range is the range over which the spot area becomes twice its minimum value. For a Gaussian beam, the Rayleigh length is the length from the waist to the Rayleigh range.

[0060] To achieve sufficient light-sheet illumination depth, the Rayleigh length of the light sheet can be selected to be 8mm. When the Rayleigh length is approximately 8mm, a focal point with a diameter of 100.5μm can be obtained. To achieve deep illumination with a Rayleigh length of up to 26mm, the thickness of the light sheet can be adjusted to 183.1μm by adjusting the distance between the focusing lens and the objective lens. Compared to conventional light-sheet lasers, which can only produce light sheets with a thickness of approximately 500μm, the present invention has stronger tomographic capabilities and more flexible adjustments. Compared to light sheets generated by conventional objective lenses, the present invention has a wider illumination range and is suitable for in vivo imaging at the mouse scale.

[0061] The calculation method of the thickness of the light sheet and the uniform illumination range is as follows:

[0062] The focal length of the condenser lens is 16.7mm, the equivalent focal length of the objective lens is 20mm, and the maximum travel of the telescopic lens tube is 4.1mm.

[0063] The distance between the condenser lens and the two main surfaces of the objective lens is 27-32mm. The focal length formula of the combined lens is: 1 / f=1 / f1+1 / f2-d / f1f2

[0064] The focal length of the system is calculated to be 35mm by the formula of the focal length of the combined lens, where f1 is the focal length of the focusing lens, f2 is the focal length of the objective lens, and d is the distance between the principal surfaces.

[0065] When the light sheet thickness is 100 μm, the equivalent NA can be calculated as 0.0062. The formula NA = D / 2f shows that the effective aperture is 0.434 mm and the uniform illumination range is 8.01 mm. The specific calculation formula is:

[0066] z0=(50^2)*3.14*1 / 0.98=8010μm=8.01mm

[0067] When the telescopic tube advances Δd, the distance between the two main surfaces is (d + Δd), and the equivalent focal length is 1 / f = 1 / f1 + 1 / f2 - (d - Δd) / f1f2. Substituting NA = D / 2f, the formula for the light sheet thickness is:

[0068]

[0069] The thickness of the available light sheet is (μm):

[0070]

[0071] The uniform illumination range is:

[0072] Compared with the prior art, the near-infrared two-zone light sheet three-dimensional in vivo imaging system of the present invention includes a stage, an imaging unit 300 and a laser unit. The laser unit includes a laser collimator 2 for adjusting the output laser into collimated light and a light sheet generation unit 200 for compressing the collimated light into a light sheet. The light sheet generation unit 200 is connected to the laser collimator 2. The imaging unit 300 and the light sheet generation unit 200 are both arranged towards the stage. The light sheet generation unit 200 can compress the collimated light into a light sheet, thereby compressing the laser power into a thin light sheet area, improving the laser power per unit area and increasing the excitation depth. The imaging unit 300 can shoot the area where the fluorescent probe is illuminated. The shooting is subject to little laser interference and the image signal-to-noise ratio is high, so the image will be clearer. Moreover, since the thickness of the light sheet is very thin, the longitudinal resolution of the reconstructed three-dimensional model is high.

[0073] It should be pointed out that in the description of the present invention, it should be understood that the terms "thickness", "up", "down", "inside" and "outside" indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0074] Throughout this specification, reference to terms such as "certain embodiments," "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the exemplary descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0075] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A near-infrared two-zone light sheet three-dimensional in vivo imaging system, characterized in that: The invention comprises an objective stage, an imaging unit and a laser unit, wherein the laser unit comprises a laser, a laser collimator for adjusting the emitted laser to collimated light, and a light sheet generating unit for compressing the collimated light into a light sheet, the laser collimator is connected to the laser for emitting Gaussian light through an optical fiber, the light sheet generating unit is connected to the laser collimator, the imaging unit and the light sheet generating unit are both arranged toward the objective stage, the light sheet generating unit comprises a cage plate, a telescopic lens sleeve for adjusting the distance between a condensing lens and an objective lens, a Powell prism group for making the light intensity of the light sheet illumination area uniformly distributed, a condensing lens and an objective lens, the thickness of the light sheet and the uniform illumination range can be changed by changing the distance between the condensing lens and the objective lens, the distance between the two main surfaces of the condensing lens and the objective lens is 27mm-32mm, the thickness of the light sheet is 100μm-190μm, the uniform illumination range of the light sheet is 8mm-26mm, and the cage plate, the telescopic lens sleeve, the condensing lens, the objective lens and the Powell prism group are arranged in sequence along the excitation optical axis.

2. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 1, characterized in that: The laser collimator and the light sheet generating unit are sequentially arranged along an excitation optical axis, and the excitation optical axis intersects with an imaging optical axis of the imaging unit.

3. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 2, characterized in that: The excitation light axis is perpendicular to the imaging light axis or the excitation light axis forms an angle of 45 degrees with the imaging light axis.

4. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 3, characterized in that: The stage comprises a fixed plate and a uniaxial translation stage, wherein the fixed plate is mounted on the uniaxial translation stage.

5. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 4, characterized in that: The loading platform also includes a platform controller and an adjustable lifting platform. The platform controller is connected to the single-axis displacement platform by signal. The upper end surface of the adjustable lifting platform is a slope surface, and the single-axis displacement platform is installed on the slope surface.

6. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 5, characterized in that: The imaging unit comprises a near-infrared camera, an industrial lens and a filter, which are arranged in sequence along an imaging optical axis.

7. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 6, characterized in that: The imaging unit is installed on a box body, which includes an upper partition and a lower partition. The upper partition and the lower partition are both horizontally installed in the box body, the upper partition is located above the lower partition, and the upper partition and the lower partition divide the box body into an upper partition room, a middle partition room, and a lower partition room.

8. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 7, characterized in that: The near-infrared camera is located in the upper partition room, the near-infrared camera is installed above the upper partition, the industrial lens is located in the middle partition room, the near-infrared camera is connected to the industrial lens through a filter mounting frame, and the filter is arranged in the filter mounting frame.

9. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 8, characterized in that: The stage, adjustable lifting platform, laser collimator and light sheet generating unit are all located in the middle partition, the lower end of the adjustable lifting platform is fixed on the lower partition, and the light sheet generating unit is installed on the lower partition through a bracket.

10. The near-infrared two-zone light sheet three-dimensional in vivo imaging system according to claim 9, characterized in that: The laser and the stage controller are both located in the lower partition.

Images