Light sheet fluorescence microscopic imaging system

Abstract

The invention relates to the technical field of light sheet fluorescence microscopic imaging, in particular to a light sheet fluorescence microscopic imaging system. According to the system, thin-layer light beams are modulated by arranging an excitation and detection sharing module to obtain a plurality of excitation planes, fluorescence excited by the plurality of excitation planes is detected by the excitation and detection sharing module to obtain a plurality of imaging results, and the plurality of imaging results are subjected to intensity modulation and acquisition by a coding detection module to obtain a single image; and a plurality of images corresponding to the plurality of excitation planes of the to-be-tested sample are recovered by a compressed sensing algorithm. Therefore, compared with a scheme that a collected single image only contains one imaging result in the prior art, the system accumulates the multiple imaging results in the single image, so that the imaging rate of the system is greatly improved, and meanwhile, due to the fact that the single image is the accumulation of the multiple imaging results, the imaging efficiency of the system is greatly improved. The signal and the signal-to-noise ratio are higher than those of an image acquired from a single imaging image, so that the system can also break the limitation of the signal-to-noise ratio on the imaging speed to a certain extent.

Description

Technical Field

[0001] The invention relates to the technical field of light sheet fluorescence microscopy, and in particular to a light sheet fluorescence microscopy system. Background Art

[0002] Light sheet fluorescence microscopy is a new type of fluorescence microscopy technology. In light sheet fluorescence microscopy, the laser beam is compressed into a very thin light sheet by a cylindrical lens or other optical system, which illuminates the sample from the side, excites fluorescence in this thin layer, and uses a microscopic imaging system to detect this layer to achieve tomographic imaging of the sample. This surface excitation and surface imaging method can ensure a faster imaging speed and is suitable for imaging dynamic processes. In addition, since only a thin layer near the detection plane is illuminated, light sheet fluorescence microscopy can significantly reduce light damage and photobleaching in optical fluorescence tomography, and achieve long-term observation of living samples.

[0003] Traditional light sheet fluorescence microscopy usually requires two orthogonally placed objective lenses, one is the excitation objective lens, which is used to generate a thin layer of light sheet to illuminate the sample, and the other is the detection objective lens, which is used to collect the fluorescence signal emitted by the sample. Under this design, both objective lenses need to have a long working distance to ensure that they can focus on the sample at the same time. However, long working distance objective lenses usually have a low numerical aperture and it is difficult to provide sufficient resolution. In addition, the design of orthogonal placement of the two objective lenses requires that the sample be placed precisely at the intersection of the focal planes of the two objective lenses, which puts higher requirements on the placement and positioning of the sample, increases the complexity of experimental operations, and limits the application scope of light sheet fluorescence microscopy. In addition, since a camera is usually used to analyze and image the fluorescence signal collected by the detection objective lens, it is difficult to meet the needs of high-speed observation due to the limitations of the camera frame rate and signal-to-noise ratio. Summary of the invention

[0004] In view of this, an embodiment of the present invention provides a light sheet fluorescence microscopy imaging system to solve the technical problem in the prior art that light sheet fluorescence microscopy is difficult to meet the requirements of high-speed observation.

[0005] The technical solution provided by the embodiment of the present invention is as follows:

[0006] A first aspect of an embodiment of the present invention provides a light sheet fluorescence microscopy imaging system, comprising: an excitation and detection shared module, used to receive a thin layer light beam input from the outside and modulate it to sequentially form multiple thin layer light beams, the multiple thin layer light beams are irradiated on a sample to be tested to obtain multiple excitation planes, and the fluorescence excited by the multiple excitation planes passes through the excitation and detection shared module again to obtain multiple imaging results; a coding detection module, used to receive multiple imaging results, respectively perform intensity modulation on the multiple imaging results using multiple modulation patterns, and collect the modulated images to obtain a single image, and restore the single image using a compressed sensing algorithm.

[0007] The light sheet fluorescence microscopy imaging system provided by the embodiment of the present invention modulates the thin layer light beam to obtain multiple excitation planes by setting an excitation detection common module, and the fluorescence excited by the multiple excitation planes is detected by the excitation detection common module to obtain multiple imaging results. The multiple imaging results are intensity modulated and collected by the coding detection module to obtain a single image, and the single image can be restored by a compressed sensing algorithm to obtain multiple images corresponding to the multiple excitation planes of the sample to be tested. Therefore, compared with the solution in the prior art that only one imaging result is included in the collected single image, the system accumulates multiple imaging results in a single image, thereby greatly improving the imaging rate of the system. At the same time, since a single image is an accumulation of multiple imaging results, the signal and signal-to-noise ratio are higher than the image obtained by collecting a single imaging image. Therefore, the system can also break the limitation of the signal-to-noise ratio on the imaging speed to a certain extent.

[0008] In an optional implementation, the coding detection module is further used to perform geometric transformation on the image stack restored by the compressed sensing algorithm to obtain an imaging result with the same viewing angle as the sample to be tested.

[0009] In this embodiment, accurate reconstruction of the volume image of the sample to be measured is achieved by performing geometric transformation on the restored image.

[0010] In an optional embodiment, the coding detection module includes: a detection objective lens, a tube lens, a light modulator, a first relay lens group, a first image acquisition device and a processor. The multiple imaging results received by the coding detection module form multiple magnified images on the surface of the light modulator after passing through the detection objective lens and the tube lens. The light modulator generates multiple modulation patterns to modulate the intensity of the multiple magnified images. The modulated images are projected onto the first image acquisition device through the first relay lens group. The first image acquisition device acquires the modulated images to obtain a single image. The processor uses a compressed sensing algorithm to restore the single image.

[0011] In an optional embodiment, the light modulation device includes a digital micromirror array, and the coding detection module also includes a total internal reflection prism arranged between the tube lens and the light modulation device, and the total internal reflection prism is used to reflect the magnified image output by the tube lens to the surface of the digital micromirror array, and reflect the modulated image of the digital micromirror array to the first relay lens group.

[0012] In an optional embodiment, the optical modulation device includes a digital micromirror array, the coding detection module also includes a second relay lens group and a second image acquisition device, the image modulated by the digital micromirror array is projected by the first relay lens group to the first image acquisition device, the complementary image of the image modulated by the digital micromirror array is projected by the second relay lens group to the second image acquisition device, and the processor uses a compressed sensing algorithm to restore the image captured by the first image acquisition device and the image captured by the second image acquisition device.

[0013] In an optional embodiment, the light modulation device includes a rotating disk with intensity coding, and the rotating disk is used to generate multiple modulation patterns by changing the position to modulate the intensity of the multiple magnified images.

[0014] In this embodiment, by using a digital micromirror array or a turntable as a light modulation device, modulation of multiple imaging results is achieved. At the same time, when a digital micromirror array is used, the modulated images in two directions output by the digital micromirror array can be collected separately, thereby improving the utilization rate of the light beam, and when performing image restoration, the images collected by the two image acquisition devices are combined, thereby improving the accuracy of the restored image.

[0015] In an optional embodiment, the excitation detection shared module includes: a main objective lens, a third relay lens group, a scanning galvanometer, a fourth relay lens group, a dichroic mirror, and a refocusing objective lens arranged in sequence, the main objective lens is close to the sample to be tested, and the refocusing objective lens is close to the coding detection module.

[0016] The dichroic mirror receives the thin layer light beam and reflects it. The reflected thin layer light beam passes through the fourth relay lens group, the scanning galvanometer mirror, the third relay lens group and the main objective lens in sequence, and is emitted from the edge of the main objective lens to irradiate the sample to be tested, thereby generating an excitation plane with an angle with the focal plane of the objective lens in the sample to be tested. The thin layer fluorescence generated by the excitation plane of the sample to be tested passes through the main objective lens, the third relay lens group, the scanning galvanometer mirror, the fourth relay lens group and the refocusing objective lens in sequence, and is copied to the focal plane of the refocusing objective lens to form an image identical to the excitation plane.

[0017] The scanning galvanometer rotates around the central axis during the imaging process to change the propagation direction of the thin-layer light beam. The thin-layer light beams with different propagation directions are irradiated on the sample to be tested to obtain multiple excitation planes. The thin-layer fluorescence generated by the multiple excitation planes of the sample to be tested passes through the main objective lens, the third relay lens group, the scanning galvanometer, the fourth relay lens group and the refocusing objective lens in sequence, and is copied to the focal plane of the refocusing objective lens to form multiple imaging results that are the same as the excitation plane.

[0018] In this embodiment, compared with the method of using two orthogonally placed objective lenses for excitation and detection in the related art, the excitation and detection shared module does not need to use an objective lens with a long working distance, thereby improving the resolution of the light sheet fluorescence microscopy imaging system. At the same time, there is no need to mechanically move the sample or detection objective lens during volume imaging, thereby improving the flexibility of use.

[0019] In an optional implementation, the back focal plane of the main objective lens, the center of the scanning galvanometer, and the back focal plane of the refocusing objective lens are conjugate.

[0020] In an optional embodiment, the front focal plane of the main objective lens, the middle plane of the third relay lens group, the middle plane of the fourth relay lens group, and the front focal plane of the refocusing objective lens are conjugate.

[0021] In an optional embodiment, the system also includes: a thin layer beam generating module, the thin layer beam generating module includes: a light source, a beam expander and a cylindrical lens, the light beam generated by the light source is expanded by the beam expander to generate a parallel light beam, and the cylindrical lens focuses the parallel light beam in one direction to form a thin layer beam. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is a structural block diagram of a light sheet fluorescence microscopy imaging system in an embodiment of the present invention;

[0024] Figure 2 is a structural block diagram of a light sheet fluorescence microscopy imaging system in another embodiment of the present invention;

[0025] Figure 3 This is a schematic diagram of the 4F system structure in an embodiment of the present invention;

[0026] Figure 4 Schematic diagram of the workflow of the light sheet fluorescence microscopy imaging system in an embodiment of the present invention;

[0027] Figure 5 Schematic diagram of the structure of the light sheet fluorescence microscopy imaging system in an embodiment of the present invention;

[0028] Figure 6 It is a schematic structural diagram of a light sheet fluorescence microscopy imaging system in another embodiment of the present invention;

[0029] Figure 7FIG. 4 is a schematic diagram of the structure of a light sheet fluorescence microscopy imaging system in another embodiment of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0032] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, it can also be the internal connection of two components, it can be a wireless connection, or it can be a wired connection. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0033] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0034] The embodiment of the present invention provides a light sheet fluorescence microscopy imaging system, such as Figure 1 As shown, the system includes: an excitation-detection shared module 1200, which is used to receive a thin layer light beam input from the outside and modulate it to form multiple thin layer light beams in sequence, the multiple thin layer light beams are irradiated on the sample to be tested to obtain multiple excitation planes, and the fluorescence excited by the multiple excitation planes passes through the excitation-detection shared module again to obtain multiple imaging results; a coding detection module 1300, which is used to receive multiple imaging results, respectively perform intensity modulation on the multiple imaging results using multiple modulation patterns, and collect the modulated images to obtain a single image, and restore the single image using a compressed sensing algorithm.

[0035] Specifically, the thin layer beam is in the shape of a thin sheet, a laser beam that is highly focused and obliquely projected onto the sample to be tested. For example, it can be obtained by compressing a laser beam, and the present embodiment does not specifically limit the specific method of generating the thin layer beam. For the thin layer beam, an excitation and detection common module can be used to modulate it. For example, the propagation direction of the thin layer beam can be changed so that the thin layer beam can irradiate different positions of the sample to be tested to form multiple excitation planes. In addition, the excitation and detection common module can not only allow the thin layer beam to irradiate the sample to be tested so that the sample to be tested excites fluorescence, but also detect and image the excited fluorescence, that is, the excitation and detection module simultaneously realizes the functions of excitation and detection, or integrates the excitation function and the detection function into the same structure.

[0036] When the excitation detection common module is working, it can first make a thin layer of light beam in one direction irradiate on the sample to be tested to form an excitation plane, and the fluorescence excited by the excitation plane on the sample to be tested returns to the excitation detection common module, and the module realizes the detection imaging of the excited fluorescence. Then, the direction of the thin layer of light beam is changed by the excitation detection common module, so that the thin layer of light beam is irradiated on the sample to be tested from another direction, forming an excitation plane again, and the fluorescence excited by the excitation plane is detected and imaged by the excitation detection common module again. Repeat the above process, so as to obtain multiple imaging results.

[0037] In addition, since the coding detection module performs one exposure acquisition within one exposure time, when the coding detection module receives multiple imaging results within one exposure time, the multiple imaging results can be exposed and acquired to obtain a single image. The number of multiple imaging results can be determined according to actual conditions, for example, according to the exposure time of the coding detection module and the number of images that can be restored by the compressed sensing algorithm, etc. This embodiment does not specifically limit the number of multiple imaging results.

[0038] At the same time, before collecting multiple imaging results, the coding detection module may first perform intensity modulation, i.e., intensity coding, on them, and then collect the modulated imaging. The imaging collection may be implemented by a camera. For example, within one exposure time of the camera, multiple modulated images are collected by the camera, and the camera may accumulate and collect these multiple modulated images to obtain a single image. The obtained single image may be restored using a compressed sensing algorithm, that is, a single image is restored to obtain multiple images. For example, a single image is obtained by accumulating and collecting K modulated images, and K images are restored by a compressed sensing algorithm. Therefore, if K imaging images are obtained within one exposure time, the maximum volume imaging speed will be increased by K times, that is, when the maximum frame rate of the camera is M and the total number of volume imaging layers is N, the maximum volume imaging rate of the existing solution is M / N, and the maximum volume imaging rate of the present invention is K*M / N.

[0039] The compressed sensing algorithm used may be an algorithm in related technologies, such as an iterative optimization algorithm, dictionary learning, or a deep neural network, etc. This embodiment does not specifically limit the specific algorithm used and the specific recovery process.

[0040] The light sheet fluorescence microscopy imaging system provided by the embodiment of the present invention modulates the thin layer light beam to obtain multiple excitation planes by setting an excitation detection common module, and the fluorescence excited by the multiple excitation planes is detected by the excitation detection common module to obtain multiple imaging results. The multiple imaging results are intensity modulated and collected by the coding detection module to obtain a single image, and the single image can be restored by a compressed sensing algorithm to obtain multiple images corresponding to the multiple excitation planes of the sample to be tested. Therefore, compared with the solution in the prior art that only one imaging result is included in the collected single image, the system accumulates multiple imaging results in a single image, thereby greatly improving the imaging rate of the system. At the same time, since a single image is an accumulation of multiple imaging results, the signal and signal-to-noise ratio are higher than the image obtained by collecting a single imaging image. Therefore, the system can also break the limitation of the signal-to-noise ratio on the imaging speed to a certain extent.

[0041] In an optional embodiment, the coding detection module is also used to perform geometric transformation on the image stack restored by the compressed sensing algorithm to obtain an imaging result with the same viewing angle as the sample to be tested. Specifically, since the excitation plane is usually an inclined plane, the image restored by the compressed sensing algorithm cannot be used to reconstruct the volume image of the sample to be tested by simple image stacking, and it is necessary to perform geometric transformation on it to convert it to the correct viewing angle. For example, to reconstruct the volume image of the sample to be tested, an affine transformation can be performed on the stacked image, and the transformation matrix is ​​the product of the scaling matrix and the shearing matrix.

[0042] In an optional embodiment, if Figure 2 As shown, the system also includes: a thin layer light beam generating module 1100, and the thin layer light beam generating module 1100 includes: a light source, a beam expander and a cylindrical lens. The light beam generated by the light source is expanded by the beam expander to generate a parallel light beam, and the cylindrical lens focuses the parallel light beam in one direction to form a thin layer light beam. Specifically, the light source can be a laser for outputting a laser beam. The beam expander can be a lens group composed of two convex lenses, which can expand the laser beam output by the laser to generate a parallel light beam, and the parallel light beam is compressed by the cylindrical lens to form a thin layer light beam in one direction. Wherein, when setting the thin layer light beam generating module, the back focal plane of the cylindrical lens is conjugate with the central plane of the sample to be tested, so as to ensure that the thin layer light beam can be projected onto the sample to be tested.

[0043] In an optional embodiment, the excitation detection shared module includes: a main objective lens, a third relay lens group, a scanning galvanometer, a fourth relay lens group, a dichroic mirror, and a refocusing objective lens arranged in sequence, the main objective lens is close to the sample to be tested, the refocusing objective lens is close to the encoding detection module, the dichroic mirror receives the thin layer light beam and reflects it, the reflected thin layer light beam passes through the fourth relay lens group, the scanning galvanometer, the third relay lens group and the main objective lens in sequence, is emitted from the edge of the main objective lens, and is irradiated on the sample to be tested, an excitation plane with an angle with the focal plane of the objective lens is generated in the sample to be tested, and the thin layer fluorescence generated by the excitation plane of the sample to be tested is sequentially After passing through the main objective lens, the third relay lens group, the scanning galvanometer, the fourth relay lens group and the refocusing objective lens, it is copied to the focal plane of the refocusing objective lens to form an image identical to the excitation plane; the scanning galvanometer rotates around the central axis during the imaging process to change the propagation direction of the thin-layer light beam, and the thin-layer light beams with different propagation directions are irradiated on the sample to be tested to obtain multiple excitation planes. The thin-layer fluorescence generated by the multiple excitation planes of the sample to be tested passes through the main objective lens, the third relay lens group, the scanning galvanometer, the fourth relay lens group and the refocusing objective lens in sequence, and is copied to the focal plane of the refocusing objective lens to form multiple imaging results identical to the excitation plane.

[0044] Specifically, the third relay lens group and the fourth relay lens group can be a 4F system consisting of two convex lenses or microscope tube lenses. Figure 3 As shown, the 4F system usually represents an optical system consisting of two lenses, with the focal length of the first lens being f. 1 , the focal length of the second lens is f 2 , the distance between the two lenses is f 1 +f 2 The sample is placed in front of the first lens at a distance f 1 At (front focal plane), then at the distance f behind the second lens 2 The image of the sample is formed at the back focal plane, and the imaging magnification is -f 2 / f 1 , that is u 3 (x,y)=-f 2 / f 1 ·u 1 (x, y), in the present invention, u 2 The (x,y) plane is called the middle plane of the 4F system. The back focal plane of the main objective lens, the center of the scanning galvanometer, and the back focal plane of the refocusing objective lens are set to be conjugate. The front focal plane of the main objective lens, the middle plane of the third relay lens group, the middle plane of the fourth relay lens group, and the front focal plane of the refocusing objective lens are conjugate. It is ensured that the thin layer light beam can be transmitted through the excitation detection common module and irradiated on the sample to be tested, and at the same time, the excited fluorescence is imaged on the front focal plane of the refocusing objective lens through the excitation detection common module.

[0045] In addition, when the thin layer of light beam is irradiated on the dichroic mirror, it is necessary to deviate from the center of the dichroic mirror, so that the thin layer of light beam that is convenient to transmit is incident from the edge of the main objective lens, forming an inclined light sheet that is irradiated on the sample to be tested. In addition, through the excitation and detection of the excitation detection common module, the excitation plane and the scanning galvanometer plane are in a Fourier transform relationship. When the sample to be tested is imaged, the scanning galvanometer rotates around the center. According to the principle of Fourier transform, the excitation plane will be translated in the sample to be tested. Similarly, the rotation of the scanning galvanometer offsets the translation of the excited fluorescence, that is, the scanning galvanometer has the function of descanning, so the image of the excitation plane after the refocusing objective lens does not move.

[0046] In addition, compared with the method of using two orthogonally placed objective lenses for excitation and detection in related technologies, the excitation and detection shared module does not need to use an objective lens with a long working distance, thereby improving the resolution of the light sheet fluorescence microscopy system. At the same time, there is no need to mechanically move the sample or detection objective lens during volume imaging, thereby improving the flexibility of use.

[0047] In an optional embodiment, the coding detection module includes: a detection objective lens, a tube lens, a light modulator, a first relay lens group, a first image acquisition device and a processor. The multiple imaging results received by the coding detection module form multiple magnified images on the surface of the light modulator after passing through the detection objective lens and the tube lens. The light modulator generates multiple modulation patterns to modulate the intensity of the multiple magnified images. The modulated images are projected onto the first image acquisition device through the first relay lens group. The first image acquisition device acquires the modulated images to obtain a single image. The processor uses a compressed sensing algorithm to restore the single image.

[0048] Specifically, there is a certain angle between the detection objective lens and the refocusing objective lens, and this angle makes the focal plane of the detection objective lens conjugate with the excitation plane. The combination of the detection objective lens and the tube lens realizes the amplification of the imaging result. The first relay lens group is a 4F system composed of two convex lenses. The excitation plane passes through the microscopic imaging system composed of the refocusing objective lens and the tube lens, and forms an enlarged image on the surface of the light modulator. The light modulator modulates the intensity of this enlarged image, and the modulated image is projected onto the first image acquisition device such as a camera through the first relay lens group and recorded by it.

[0049] For the light modulator, a device that can realize intensity modulation of imaging and has sufficient speed can be used, that is, the speed at which the light modulator generates the modulation pattern needs to keep up with the speed at which the scanning galvanometer rotates, so as to facilitate the modulation of multiple imaging results. Among them, the pattern generated by the light modulator can be a pattern with the same number as the imaging results and orthogonal to each other, and the multiple modulation patterns are different from each other, and each modulation pattern modulates the corresponding imaging result respectively. In this embodiment, the light modulator uses a digital micromirror array or a rotating disk with intensity coding such as a rotating code disk.

[0050] The digital micromirror array is composed of many small aluminum reflective mirrors, each of which is called a pixel. Each mirror can deflect ±12° around the diagonal of each small mirror (or a pixel) in the positive direction, that is, the digital micromirror array has three states: +12°, 0°, and -12°. Therefore, when the digital micromirror array is in state 0°, a total internal reflection prism can be used between the tube lens and the light modulation device to reflect the magnified image output by the tube lens to the surface of the digital micromirror array, and reflect the modulated image of the digital micromirror array to the first relay lens group.

[0051] When the reflective mirror surface in the digital micromirror array is deflected, reflected light in two directions can be formed, and the reflected light in the two directions, that is, the modulated light information in the two directions is complementary. At this time, the first relay lens group and the first image acquisition device can be set in one direction, and the second relay lens group and the second image acquisition device can also be set in the other direction. The modulated image of the digital micromirror array is projected to the first image acquisition device by the first relay lens group, and the complementary image of the modulated image of the digital micromirror array is projected to the second image acquisition device by the second relay lens group. The processor uses a compressed sensing algorithm to restore the image collected by the first image acquisition device and the image collected by the second image acquisition device. Among them, the modulated images in the two directions are respectively collected by the first image acquisition device and the second image acquisition device. Because the two images are complementary, when the processor uses a compressed sensing algorithm to restore the images collected by the two image acquisition devices, the information of the two images can be integrated for restoration.

[0052] Optionally, the light modulation device may further include an intensity-encoded rotating disk, which is used to generate a plurality of modulation patterns by position variation to modulate the intensity of a plurality of magnified images.

[0053] In an optional embodiment, if Figure 4 As shown, the light sheet fluorescence microscopy system can work according to the following process: the thin layer beam generation module generates and outputs a thin layer beam to the excitation detection common module, the excitation detection common module modulates the thin layer beam by scanning the scanning galvanometer to form multiple thin layer beams in sequence, the multiple thin layer beams are irradiated on the sample to be tested to obtain multiple excitation planes, and the fluorescence excited by the multiple excitation planes passes through the excitation detection common module again to obtain multiple imaging results; the coding detection module uses multiple modulation patterns to modulate the intensity of the multiple imaging results, and collects the modulated imaging within an exposure time of the camera to obtain a single image, and the single image is restored by a compressed sensing algorithm to obtain multiple images. In order to reconstruct the volume image of the sample to be tested through multiple images, an affine transformation can be performed on the stacked image by scaling and shearing, and the transformation matrix is ​​the product of the scaling matrix and the shearing matrix.

[0054] The present invention also provides a light sheet fluorescence microscopy system. Figure 5 As shown, the system includes a thin layer beam generating module 1100 , an excitation and detection common module 1200 , and a coding detection module 1300 .

[0055] The thin-layer light beam generating module 1100 includes a laser 101, a beam expansion system composed of a first convex lens 102 and a second convex lens, a reflector 104, and a cylindrical lens 105. The laser 101 emits a laser beam, which becomes a parallel light beam after passing through the beam expansion system, and reaches the cylindrical lens 105 after being reflected by the reflector 104. The beam is compressed into a thin sheet in one direction and remains parallel in the other direction, thereby generating a Gaussian light sheet.

[0056] The excitation detection common module 1200 includes a dichroic mirror 106, a first relay lens 107, a second relay lens 108, a scanning galvanometer 109, a third relay lens 110, a fourth relay lens 111, a main objective lens 112, and a refocusing objective lens 114. The Gaussian light sheet generated by the thin-layer light beam generating module 1100 enters the excitation light path after being reflected by the dichroic mirror 106, and the light sheet deviates from the center of the dichroic mirror 106 to ensure that the final light sheet can be tilted. After that, the Gaussian light sheet passes through the first relay lens 107 and the second relay lens 108 in turn to reach the scanning galvanometer 109, and after being reflected by the scanning galvanometer 109, it passes through the third relay lens 110 and the fourth relay lens 111 again, reaches the edge of the rear focal plane of the main objective lens 112, and is tilted after passing through the main objective lens 112 to form an inclined excitation plane in the sample 113 to be tested. When the scanning galvanometer 109 rotates, the excitation plane will move on the sample to be tested 113 to achieve three-dimensional scanning of the sample.

[0057] The generated fluorescence passes through the main objective lens 112, the fourth relay lens 111, and the third relay lens 110 in sequence, reaches the scanning galvanometer mirror 109, and after being reflected by the scanning galvanometer mirror 109, passes through the second relay lens 108, the first relay lens 107, the dichroic mirror 106, and the refocusing objective lens 114 in sequence, forming an image of the sample near the focal plane of the refocusing objective lens. The focal lengths of the main objective lens 112, the fourth relay lens 111, the third relay lens 110, the second relay lens 108, the first relay lens 107, and the refocusing objective lens 114 should achieve perfect imaging. When the focal lengths of the four relay lenses are equal, the ratio of the focal lengths of the main objective lens 112 and the refocusing objective lens 114 should be equal to the ratio of the refractive index after the refocusing objective lens 114 to the refractive index after the main objective lens 112.

[0058] The code detection module 1300 includes: a detection objective lens 115, a detection tube lens 116, a total internal reflection prism 117, a digital micromirror array 118, a fifth relay lens 119, a sixth relay lens 120 and a first camera 121. The fluorescence after exciting the detection common module 1200 is incident on the digital micromirror array 118 through the detection objective lens 115, the detection tube lens 116, and the total internal reflection prism 117, and is modulated by the intensity pattern on the digital micromirror array 118. After passing through the fifth relay lens 119 and the sixth relay lens 120, an image is formed on the first camera 121. During one exposure time of the first camera 121, the position of the scanning galvanometer 109 and the pattern on the digital micromirror array 118 change multiple times to achieve code acquisition.

[0059] The present invention also provides a light sheet fluorescence microscopy system. Figure 6 As shown, the system includes a thin layer beam generating module 1100 , an excitation and detection common module 1200 , and a coding detection module 1300 .

[0060] The thin-layer light beam generating module 1100 includes a laser 101, a beam expansion system composed of a first convex lens 102 and a second convex lens, a reflector 104, and a cylindrical lens 105. The laser 101 emits a laser beam, which becomes a parallel light beam after passing through the beam expansion system, and reaches the cylindrical lens 105 after being reflected by the reflector 104. The beam is compressed into a thin sheet in one direction and remains parallel in the other direction, thereby generating a Gaussian light sheet.

[0061] The excitation detection common module 1200 includes a dichroic mirror 106, a first relay lens 107, a second relay lens 108, a scanning galvanometer 109, a third relay lens 110, a fourth relay lens 111, a main objective lens 112, and a refocusing objective lens 114. The Gaussian light sheet generated by the thin-layer light beam generating module 1100 enters the excitation light path after being reflected by the dichroic mirror 106, and the light sheet deviates from the center of the dichroic mirror 106 to ensure that the final light sheet can be tilted. After that, the Gaussian light sheet passes through the first relay lens 107 and the second relay lens 108 in turn to reach the scanning galvanometer 109, and after being reflected by the scanning galvanometer 109, it passes through the third relay lens 110 and the fourth relay lens 111 again, reaches the edge of the rear focal plane of the main objective lens 112, and is tilted after passing through the main objective lens 112 to form an inclined excitation plane in the sample 113 to be tested. When the scanning galvanometer 109 rotates, the excitation plane will move on the sample to be tested 113 to achieve three-dimensional scanning of the sample.

[0062] The generated fluorescence passes through the main objective lens 112, the fourth relay lens 111, and the third relay lens 110 in sequence, reaches the scanning galvanometer mirror 109, and after being reflected by the scanning galvanometer mirror 109, passes through the second relay lens 108, the first relay lens 107, the dichroic mirror 106, and the refocusing objective lens 114 in sequence, forming an image of the sample near the focal plane of the refocusing objective lens. The focal lengths of the main objective lens 112, the fourth relay lens 111, the third relay lens 110, the second relay lens 108, the first relay lens 107, and the refocusing objective lens 114 should achieve perfect imaging. When the focal lengths of the four relay lenses are equal, the ratio of the focal lengths of the main objective lens 112 and the refocusing objective lens 114 should be equal to the ratio of the refractive index after the refocusing objective lens 114 to the refractive index after the main objective lens 112.

[0063] The coding detection module 1300 includes: a detection objective lens 115, a detection tube lens 116, a digital micromirror array 118, a seventh relay lens 122, an eighth relay lens 123, a ninth relay lens 124, a tenth relay lens 125, a second camera 126, and a third camera 127. The fluorescence after exciting the detection common module 1200 passes through the detection objective lens 115, the detection tube lens 116, and the total internal reflection prism 117, and is incident on the digital micromirror array 118. After being modulated by the intensity pattern on the digital micromirror array 118, the corresponding path of the intensity pattern passes through the seventh relay lens 122 and the eighth relay lens 123, and is imaged on the second camera 126. The complementary pattern of the intensity pattern passes through the ninth relay lens 124 and the tenth relay lens 125, and is imaged on the third camera 127. The second camera 126 and the third camera 127 are exposed simultaneously. During one exposure time of the second camera 126 and the third camera 127 , the position of the scanning galvanometer 209 and the pattern on the digital micromirror array 118 change multiple times to achieve code acquisition.

[0064] The present invention also provides a light sheet fluorescence microscopy system. Figure 7 As shown, the system includes a thin layer beam generating module 1100 , an excitation and detection common module 1200 , and a coding detection module 1300 .

[0065] The thin-layer light beam generating module 1100 includes a laser 101, a beam expansion system composed of a first convex lens 102 and a second convex lens, a reflector 104, and a cylindrical lens 105. The laser 101 emits a laser beam, which becomes a parallel light beam after passing through the beam expansion system, and reaches the cylindrical lens 105 after being reflected by the reflector 104. The beam is compressed into a thin sheet in one direction and remains parallel in the other direction, thereby generating a Gaussian light sheet.

[0066] The excitation detection common module 1200 includes a dichroic mirror 106, a first relay lens 107, a second relay lens 108, a scanning galvanometer 109, a third relay lens 110, a fourth relay lens 111, a main objective lens 112, and a refocusing objective lens 114. The Gaussian light sheet generated by the thin-layer light beam generating module 1100 enters the excitation light path after being reflected by the dichroic mirror 106, and the light sheet deviates from the center of the dichroic mirror 106 to ensure that the final light sheet can be tilted. After that, the Gaussian light sheet passes through the first relay lens 107 and the second relay lens 108 in turn to reach the scanning galvanometer 109, and after being reflected by the scanning galvanometer 109, it passes through the third relay lens 110 and the fourth relay lens 111 again, reaches the edge of the rear focal plane of the main objective lens 112, and is tilted after passing through the main objective lens 112 to form an inclined excitation plane in the sample 113 to be tested. When the scanning galvanometer 109 rotates, the excitation plane will move on the sample to be tested 113 to achieve three-dimensional scanning of the sample.

[0067] The generated fluorescence passes through the main objective lens 112, the fourth relay lens 111, and the third relay lens 110 in sequence, reaches the scanning galvanometer mirror 109, and after being reflected by the scanning galvanometer mirror 109, passes through the second relay lens 108, the first relay lens 107, the dichroic mirror 106, and the refocusing objective lens 114 in sequence, forming an image of the sample near the focal plane of the refocusing objective lens. The focal lengths of the main objective lens 112, the fourth relay lens 111, the third relay lens 110, the second relay lens 108, the first relay lens 107, and the refocusing objective lens 114 should achieve perfect imaging. When the focal lengths of the four relay lenses are equal, the ratio of the focal lengths of the main objective lens 112 and the refocusing objective lens 114 should be equal to the ratio of the refractive index after the refocusing objective lens 114 to the refractive index after the main objective lens 112.

[0068] The coding detection module 1300 includes: a detection objective lens 115, a detection tube lens 116, a rotating disk 128 with intensity coding, a fifth relay lens 119, a sixth relay lens 120 and a first camera 121. The fluorescence after exciting the detection common module 1200 is incident on the rotating disk 128 with intensity coding through the detection objective lens 115 and the detection tube lens 116, and after being modulated by the intensity pattern on the rotating disk 128, it passes through the fifth relay lens 119 and the sixth relay lens 120 to form an image on the first camera 121. During one exposure time of the first camera 121, the position of the scanning galvanometer 109 and the pattern on the rotating disk 128 change multiple times to achieve coding acquisition.

[0069] Although the exemplary embodiments and their advantages have been described in detail, those skilled in the art may make various changes, substitutions and modifications to these embodiments without departing from the spirit of the present invention and the scope of protection defined by the appended claims, and such modifications and variations all fall within the scope defined by the appended claims. For other examples, those of ordinary skill in the art should readily understand that the order of the process steps may be changed while maintaining the scope of protection of the present invention.

[0070] In addition, the scope of application of the present invention is not limited to the processes, mechanisms, manufactures, material compositions, means, methods and steps of the specific embodiments described in the specification. From the disclosure of the present invention, it will be easily understood by those skilled in the art that the processes, mechanisms, manufactures, material compositions, means, methods or steps that currently exist or will be developed in the future, which perform substantially the same functions as the corresponding embodiments described in the present invention or obtain substantially the same results, can be applied according to the present invention. Therefore, the claims attached to the present invention are intended to include these processes, mechanisms, manufactures, material compositions, means, methods or steps within their scope of protection.

Claims

1. A light sheet fluorescence microscopy system, characterized in that: include: The excitation and detection common module is used to receive the thin layer light beam input from the outside and modulate it to form multiple thin layer light beams in sequence. The multiple thin layer light beams are irradiated on the sample to be tested to obtain multiple excitation planes. The fluorescence excited by the multiple excitation planes passes through the excitation and detection common module again to obtain multiple imaging results. The coding detection module is used to receive multiple imaging results, perform intensity modulation on the multiple imaging results using multiple modulation patterns respectively, collect the modulated imaging to obtain a single image, and restore the single image using a compressed sensing algorithm.

2. The light sheet fluorescence microscopy system according to claim 1, characterized in that: The coding detection module is also used to perform geometric transformation on the image stack restored by the compressed sensing algorithm to obtain an imaging result with the same viewing angle as the sample to be tested.

3. The light sheet fluorescence microscopy system according to claim 1, characterized in that: The coding detection module includes: a detection objective lens, a tube lens, a light modulation device, a first relay lens group, a first image acquisition device and a processor. The multiple imaging results received by the coding detection module form multiple magnified images on the surface of the light modulator after passing through the detection objective lens and the tube lens. The light modulator generates multiple modulation patterns to modulate the intensity of the multiple magnified images. The modulated images are projected onto the first image acquisition device through the first relay lens group. The first image acquisition device acquires the modulated images to obtain a single image. The processor restores the single image using a compressed sensing algorithm.

4. The light sheet fluorescence microscopy system according to claim 3, characterized in that: The optical modulation device includes a digital micromirror array, and the coding detection module also includes a total internal reflection prism arranged between the tube lens and the optical modulation device, and the total internal reflection prism is used to reflect the magnified image output by the tube lens to the surface of the digital micromirror array, and reflect the image modulated by the digital micromirror array to the first relay lens group.

5. The light sheet fluorescence microscopy system according to claim 3, characterized in that: The optical modulation device includes a digital micromirror array, and the coding detection module also includes a second relay lens group and a second image acquisition device. The image modulated by the digital micromirror array is projected by the first relay lens group to the first image acquisition device, and the complementary image of the image modulated by the digital micromirror array is projected by the second relay lens group to the second image acquisition device. The processor uses a compressed sensing algorithm to restore the images captured by the first image acquisition device and the images captured by the second image acquisition device.

6. The light sheet fluorescence microscopy system according to claim 3, characterized in that: The optical modulation device comprises a rotating disk with intensity coding, and the rotating disk is used to generate multiple modulation patterns by position variation to modulate the intensity of multiple magnified images.

7. The light sheet fluorescence microscopy system according to claim 1, characterized in that: The excitation detection common module includes: a main objective lens, a third relay lens group, a scanning galvanometer, a fourth relay lens group, a dichroic mirror, and a refocusing objective lens, which are arranged in sequence. The main objective lens is close to the sample to be tested, and the refocusing objective lens is close to the coding detection module. The dichroic mirror receives the thin layer light beam and reflects it. The reflected thin layer light beam passes through the fourth relay lens group, the scanning galvanometer mirror, the third relay lens group and the main objective lens in sequence, is emitted from the edge of the main objective lens, and is irradiated on the sample to be tested, thereby generating an excitation plane with an angle with the focal plane of the objective lens in the sample to be tested. The thin layer fluorescence generated by the excitation plane of the sample to be tested passes through the main objective lens, the third relay lens group, the scanning galvanometer mirror, the fourth relay lens group and the refocusing objective lens in sequence, and is copied to the focal plane of the refocusing objective lens to form an image identical to the excitation plane. The scanning galvanometer rotates around the central axis during the imaging process to change the propagation direction of the thin-layer light beam. The thin-layer light beams with different propagation directions are irradiated on the sample to be tested to obtain multiple excitation planes. The thin-layer fluorescence generated by the multiple excitation planes of the sample to be tested passes through the main objective lens, the third relay lens group, the scanning galvanometer, the fourth relay lens group and the refocusing objective lens in sequence, and is copied to the focal plane of the refocusing objective lens to form multiple imaging results that are the same as the excitation planes.

8. The light sheet fluorescence microscopy system according to claim 7, characterized in that: The back focal plane of the main objective lens, the center of the scanning galvanometer, and the back focal plane of the refocusing objective lens are conjugate.

9. The light sheet fluorescence microscopy system according to claim 7, characterized in that: The front focal plane of the main objective lens, the middle plane of the third relay lens group, the middle plane of the fourth relay lens group, and the front focal plane of the refocusing objective lens are conjugate.

10. The light sheet fluorescence microscopy system according to claim 1, characterized in that: The system further comprises: a thin layer light beam generating module, wherein the thin layer light beam generating module comprises: a light source, a beam expander and a cylindrical lens, The light beam generated by the light source is expanded by the beam expander to generate a parallel light beam, and the cylindrical lens focuses the parallel light beam in one direction to form a thin layer light beam.

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