Super-resolution single objective lens optical sheet microscopic imaging optical system and imaging system thereof

Abstract

Super-resolution single objective light sheet microscopy imaging optical systems, methods and related imaging systems. The super-resolution single objective light sheet microscopy imaging optical system includes a light source module (100) configured to output a laser beam of a single or multiple wavelengths, a light sheet generating and phase adjusting module (200) configured to receive the laser beam of a single or multiple wavelengths and output two light sheets, a scanning module (300), a single objective lens (400) located downstream of the scanning module (300), the scanning module (300) configured to guide the two light sheets output from the light sheet generating and phase adjusting module (200) to the single objective lens (400), which causes the two light sheets to exit the single objective lens (400) and interfere with each other to generate structured light stripe regions (T1000, T2000, T3000), the single objective lens (400) configured to receive a fluorescent signal, and a fluorescence detection module (500) configured to record the fluorescent signal received by the single objective lens (400).

Description

[Technical field]

[0001] The present application relates generally to the field of super-resolution microscopy imaging technology, and more particularly to a super-resolution single-objective light sheet microscopy imaging optical system and imaging system thereof. [Background technology]

[0002] The emergence of optical microscopes has provided an intuitive means of observing the dynamic processes of biological activities, and promoted the breakthrough of life science research. The wave nature of light limits the imaging resolution of optical microscopes, which greatly hinders the observation of minute biological activities, so that breaking through the diffraction limit resolution has been a goal that has been pursued tirelessly in the field of optical microscopy research. In recent years, super-resolution microscopy imaging technologies that break through the diffraction limit have been developed, and current super-resolution microscopy technologies are mainly classified into three types: (1) stimulated emission depletion (STED) technology, (2) single molecule alignment reconstruction microscopy technology, and (3) structured illumination microscopy (SIM) technology. Super-resolution microscopy imaging systems based on STED technology require relatively high light intensity, which is prone to photobleaching and phototoxicity, causing significant damage to living biological samples, and super-resolution microscopy based on single molecule alignment reconstruction loses time resolution in exchange for spatial resolution, which is not favorable for high-speed dynamic imaging. Structured illumination microscopy (SIM) employs a wide-field illumination scheme, does not require high laser power, and is more suitable for high-speed biological imaging than the two super-resolution microscopy techniques mentioned above. However, for 3D structured illumination microscopy imaging, SIM illuminates the entire sample, which increases the probability of photobleaching and reconstruction artifacts due to the presence of defocused signals.

[0003] Unlike traditional wide-field imaging modes, light-sheet fluorescence microscopy generates a sheet-like light source to illuminate the object, and separates the excitation light path and the search light path from each other, thereby avoiding the generation of defocused signals and greatly reducing photobleaching. Traditional light-sheet microscope systems are also limited in their actual operation. The first is limited by the arrangement method of the excitation objective lens and the search objective lens, and standard slide glasses and perforated plates often cannot be used normally, and special designs are often required for the preparation and fixation of samples. The second is mechanically limited, and the objective lens needs a sufficient working distance to meet the imaging conditions, which limits the numerical aperture of the objective lens, i.e., the imaging resolution.

[0004] In recent years, the imaging technology of single-objective light sheets has been developed, and instead of the excitation objective and the search objective being perpendicular to each other and independent, the same objective is used as both the excitation objective and the search objective to collect the fluorescent signal, and the inclined image plane is searched through the remote focus imaging method. This overcomes the shortcomings of the sample limitation of the traditional light sheet microscope and can be used to image different types of samples. Although the light sheet fluorescence microscope is good at long-term and high-speed body imaging, its spatial resolution is not high. Therefore, if the single-objective light sheet microscope imaging technology can be combined with the structured light super-resolution microscope imaging technology, it will be very convenient for high-resolution and high-speed three-dimensional biological imaging, which is of great importance to the research of cells, tissues and embryos in the fields of life sciences such as developmental biology and neurobiology. Summary of the Invention [Problem to be solved by the invention]

[0005] The main objective of this application is to combine single-objective light sheet imaging technology with structured light illumination super-resolution microscope imaging technology to realize super-resolution single-objective light sheet microscope imaging, especially three-dimensional super-resolution imaging. [Means for solving the problem]

[0006] According to one aspect of the present application, there is provided a super-resolution single objective lens light sheet microscopy imaging optical system, a light source module configured to output a laser beam of single or multiple wavelengths; a light sheet generating and phase adjusting module configured to receive the single or multiple wavelength laser beams and output two light sheets; A scanning module; a single objective lens located downstream of the scanning module, the scanning module being configured to guide two optical sheets output from the optical sheet generating and phase adjusting module to the single objective lens, the single objective lens configured to make the two optical sheets exit the single objective lens and interfere with each other to generate a structured light stripe region, and to receive a fluorescent signal; and a fluorescence detection module configured to record the fluorescence signal received by the single objective lens.

[0007] Optionally, the scanning module is configured to scan the structured light stripe region along one direction, and the fluorescence detection module is configured to synchronously record the fluorescence signal received by the single objective lens during scanning.

[0008] Optionally, the fluorescent signal is a fluorescent signal generated by irradiating the structured light stripe region onto a sample to be detected.

[0009] Optionally, the light sheet generating and phase adjusting module comprises a spatial light modulator, a half wave plate, a polarizing beam splitter, a cylindrical lens and a mask.

[0010] Optionally, the spatial light modulator, the half-wave plate, and the polarizing beam splitter are arranged to form a phase grating, so that the single or multiple wavelength laser beams generate multiple stages of positive and negative light components after passing through the phase grating.

[0011] Optionally, the polarizing beam splitter is configured to reflect the single or multiple wavelength laser beams towards the half-wave plate and through the half-wave plate to the spatial light modulator; The spatial light modulator is configured to be switchable between at least two different states, and in the at least two different states, different patterns are defined in the spatial light modulator for generating multiple stages of positive and negative light components correspondingly.

[0012] Optionally, the mask is configured to leave only one stage of positive and negative light components and remove the other stages of light components, the mask is positioned downstream of the cylindrical lens, and the two optical sheets are generated after the multiple stages of positive and negative light components pass through the cylindrical lens and the mask.

[0013] Optionally, the structured light stripe region comprises a structured light stripe region at a first viewing angle and a structured light stripe region at a second viewing angle; The state of the spatial light modulator is a first state, in which a first diffraction pattern is defined in the spatial light modulator, such that in the first state, two optical sheets for forming a structured light stripe region at the first viewing angle are output from the optical sheet generating and phase adjusting module; a second state in which a second diffraction pattern different from the first diffraction pattern is defined in the spatial light modulator, such that in the second state, two optical sheets for forming a structured light stripe region at the second viewing angle are output from the optical sheet generation and phase adjustment module.

[0014] Optionally, the scanning module includes a first galvanometer scanner and a second galvanometer scanner, the first galvanometer scanner being arranged to receive the two optical sheets output from the optical sheet generating and phase adjusting module and reflect them toward the second galvanometer scanner; The second galvanometer scanner is positioned to reflect the two optical sheets towards the single objective lens.

[0015] Optionally, the super-resolution single objective lens light sheet microscopy imaging optical system further comprises a spectrometer between the single objective lens and the second galvanometer scanner, the spectrometer being configured such that the two light sheets are capable of transmitting through the spectrometer but the fluorescent signal is reflected towards the fluorescent detection module, the fluorescent detection module comprising a camera for recording the fluorescent signal and a conical lens or a microlens array positioned upstream of the camera.

[0016] Optionally, the super-resolution single objective lens light sheet microscopy imaging optical system further comprises a spectrometer between the single objective lens and the first galvanometer scanner, the spectrometer being configured such that the two optical sheets are capable of transmitting through the spectrometer but the fluorescence signal is reflected towards the fluorescence detection module, the fluorescence detection module comprising a camera for recording the fluorescence signal, and an eighth lens and a third lens arranged to form a 4F system between the spectrometer and the camera, the third lens being closer to the spectrometer and having a step plate at the focal plane of the third lens.

[0017] Optionally, a first lens and a second lens are arranged to form a 4F system between the single objective lens and the second galvanometer scanner, and the fluorescence detection module further includes a third lens located upstream of the conical lens or the microlens array, the first lens and the third lens being located between the single objective lens and the conical lens or the microlens array to form a 4F system, and the spectrometer is located between the first lens and the second lens.

[0018] Optionally, the super-resolution single objective lens optical sheet microscopy imaging optical system further comprises a fourth lens and a fifth lens arranged to form a 4F system between the second galvanometer scanner and the first galvanometer scanner, the second galvanometer scanner being conjugated with the first galvanometer scanner G1 via the fourth lens and the fifth lens, and a spectrometer being arranged between the fifth lens and the first galvanometer scanner, the spectrometer being configured such that the two optical sheets are capable of transmitting through the spectrometer but the fluorescence signal is reflected towards the fluorescence detection module.

[0019] Optionally, the fluorescence detection module includes a camera for recording the fluorescence signal, a second objective lens and a third objective lens located upstream of the camera and having optical axes arranged at a non-zero angle to each other, and a sixth lens and a seventh lens arranged to form a 4F system between the spectrometer and the second objective lens, and a galvanometer scanner-reflector system is arranged between the spectrometer and the seventh lens close to the second objective lens so that the imaging directions of fluorescence images at different viewing angles coincide.

[0020] Optionally, the galvanometer scanner-reflector system includes a third galvanometer scanner and a fourth galvanometer scanner or an additional reflector disposed between a spectrometer and the sixth lens, a second reflector, a third reflector, and a fourth reflector disposed between the third galvanometer scanner and the fourth galvanometer scanner or the additional reflector, and a fifth reflector disposed between the sixth lens and the seventh lens, wherein the third galvanometer scanner is operable such that, at one of the first field of view and the second field of view, the fluorescence propagates through the third galvanometer scanner, the second reflector, the fourth reflector, the fourth galvanometer scanner or the additional reflector, the sixth lens, the fifth reflector, and the seventh lens in that order to the second objective lens, and, at the other of the first field of view and the second field of view, the fluorescence propagates through the third galvanometer scanner, the third reflector, the fourth galvanometer scanner or the additional reflector, the sixth lens, the fifth reflector, and the seventh lens in that order to the second objective lens.

[0021] Optionally, the structured light stripe region is scanned by operation of the first galvanometer scanner and / or the second galvanometer scanner.

[0022] Optionally, in the case of two-dimensional super-resolution imaging, the structured light stripe area at the first viewing angle and the structured light stripe area at the second viewing angle are scanned along directions perpendicular to each other.

[0023] Optionally, in the case of three-dimensional super-resolution imaging, the structured light stripe region at the first viewing angle and the structured light stripe region at the second viewing angle are scanned and moved in opposite directions along the same straight line.

[0024] Optionally, the optical sheet generation and phase adjustment module further comprises a beam expansion filter submodule for expanding and / or filtering the single or multiple wavelength laser beam, the beam expansion filter submodule being configured such that the single or multiple wavelength laser beam is initially incident on the beam expansion filter submodule after entering the optical sheet generation and phase adjustment module.

[0025] Optionally, the mask and the first galvanometer scanner are disposed in a conjugate relationship.

[0026] Optionally, two lenses are arranged between the mask and the first galvanometer scanner, and / or between the first galvanometer scanner and the second galvanometer scanner, and / or between the second galvanometer scanner and the single objective lens to form a 4F system.

[0027] According to another aspect of the present application, there is further provided a super-resolution single objective lens light sheet microscopic imaging system, a stage having a flat surface on which a sample to be detected is placed; and a super-resolution single objective lens light sheet microscopy imaging optical system, the optical axis of which forms a 90 degree angle with the plane.

[0028] By adopting the technical means of the present application, two optical sheets are generated by a specially designed optical sheet generation and phase adjustment module, and a structured light stripe is formed by optical sheet interference, ensuring that the optical path of the entire optical system is easily stable. In addition, the present application realizes two-dimensional or three-dimensional super-resolution microscopic imaging using a single objective lens, and the excitation objective lens and the search objective lens are the same objective lens, and standard slide glass, perforated plate, etc. can be applied. Compared with the conventional optical sheet system in which the excitation objective lens and the search objective lens are separated, the sample to be detected is easily arranged, and the applicability of the optical system is improved. In addition, the optical system of the present application combines the advantages of optical sheet imaging and structured light imaging, and can reduce the probability of photobleaching and the effect of reconstruction artifacts caused by the presence of defocused signals, providing a basis for the realization of high-resolution and high-speed three-dimensional biological microscopic imaging. [Brief description of the drawings]

[0029] The principles and aspects of the present application will become more fully understood from the following detailed description and the following drawings, in which the proportions of the drawings may vary for clarity of illustration, but this does not affect the understanding of the present application. [Figure 1] FIG. 1 is a block diagram illustrating a super-resolution single-objective lens light sheet microscopy imaging optical system according to the present application. [Diagram 2] 1 is a schematic diagram showing an optical path of an example of a light source module according to the present application; [Diagram 3] 1 is a schematic diagram of a light path of an example of a light sheet generating and phase adjusting module according to the present application; [Figure 4] 1 is a schematic diagram of an example of a scanning module and a single objective lens according to the present application; [Figure 5A] 1 is a schematic diagram showing a light path of a portion of an example of a fluorescence detection module according to the present application. [Figure 5B] 13 is a schematic diagram showing a light path of a portion of another example of a fluorescence detection module according to the present application. [Figure 5C]13 is a schematic diagram showing a light path of a portion of another example of a fluorescence detection module according to the present application. [Figures 6A-6F] 13A and 13B are schematic diagrams showing the distribution of the pupil plane after the objective lens and when two optical sheets pass through a single objective lens at different field angles in the super-resolution single objective lens optical sheet microscopy imaging optical system of the present application. [Figure 7A] Schematic diagram of the scanning process at one viewing angle in two-dimensional super-resolution imaging mode. [Figure 7B] 7B shows a schematic diagram of image data information of acquired fluorescent signals corresponding to FIG. 7A. [Figure 7C] 3A-3C are schematic diagrams showing the scanning process at other viewing angles in the two-dimensional super-resolution imaging mode. [Figure 7D] 7D and 7E are schematic diagrams showing image data information of acquired fluorescent signals corresponding to FIG. 7C. [Figure 8A] Schematic diagram of the scanning process at one viewing angle in 3D super-resolution imaging mode. [Figure 8B] 8B shows a schematic diagram of image data information of acquired fluorescent signals corresponding to FIG. 8A. [Figure 8C] 3A-3C are schematic diagrams illustrating the scanning process at other viewing angles in the 3D super-resolution imaging mode. [Figure 8D] FIG. 8D shows a schematic diagram of image data information of the acquired fluorescent signal corresponding to FIG. 8C. [Figure 9] Schematic diagram of an optical path of a super-resolution single objective lens light sheet microscopic imaging optical system according to one embodiment of the present application. [Figure 10] Schematic diagram of an optical path diagram of a super-resolution single objective lens light sheet microscopic imaging optical system according to another embodiment of the present application. [Figure 11] 1 is a schematic diagram showing an optical path of a fluorescence detection module according to an embodiment of the present application; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In the various drawings of this application, features that are structurally identical or functionally similar are designated with the same reference numerals.

[0031] 1 is a block diagram showing a super-resolution single objective lens optical sheet microscopic imaging optical system according to an embodiment of the present application, and FIG. 9 shows a schematic optical path diagram of the super-resolution single objective lens optical sheet microscopic imaging optical system according to the embodiment of the present application. In the present application, the super-resolution single objective lens optical sheet microscopic imaging optical system mainly includes a light source module 100, an optical sheet generating and phase adjusting module 200, a scanning module 300, a single objective lens 400, and a fluorescence detection module 500. During operation of the super-resolution single objective light sheet microscopic imaging optical system of the present application, the light source module 100 outputs a laser beam of a single or multiple wavelengths to be incident on the light sheet generating and phase adjusting module 200, which generates two light sheets having a predetermined spatial positional relationship therewith, and the two light sheets are modulated as necessary via the scanning module 300, and can be irradiated via the single objective 400 to a sample (not shown) placed on the stage of the super-resolution single objective light sheet microscopic imaging system. In this process, the two light sheets interfere with each other on the plane where the sample is located to form structured light and irradiate the sample. At the same time, the fluorescence generated from the irradiated sample is received by the same objective 400 and collected by the fluorescence detection module 500, and the excited fluorescent signal contains super-resolution information due to the structured light irradiation, so that by realizing a predetermined scanning process in the scanning module 300, two-dimensional or three-dimensional super-resolution single objective light sheet microscopic imaging can be finally realized.

[0032] FIG. 2 is a schematic diagram of an example of a light path of a light source module 100 according to the present application. In the present application, the light source module 100 may include one or more lasers. For example, each laser may emit a laser beam of a different wavelength. For example, available lasers may include lasers with wavelengths of 405 nm (nanometers), 445 nm, 488 nm, 561 nm, 640 nm, etc. In the illustrated example, the light source module 100 is shown to include three lasers, Laser 1, Laser 2, and Laser 3. These three lasers Laser 1, Laser 2, and Laser 3 may emit laser light of different wavelengths. These three lasers, Laser1, Laser2, and Laser3, are equipped with lens groups L1 and L2, L3 and L4, and L5 and L6, respectively, so that the laser light emitted from each laser can be expanded into a parallel beam of equal diameter through each lens group. A reflector M1 and dichroic mirrors (also called spectrometers) DM1 and DM2 combine the parallel beams of equal diameter from each path from the different lasers and combine them into a single laser beam.

[0033] In a preferred or alternative embodiment, an acousto-optical tunable filter AOTF is disposed in the optical path of the combined laser beam downstream of the dichroic mirror M2, so that the combined laser beam can be incident on the acousto-optical tunable filter AOTF. The role of the acousto-optical tunable filter AOTF is to selectively ensure the passage of light of a specific wavelength and to be able to control the power of the light output therethrough.

[0034] FIG. 3 is a schematic diagram of an example of the optical path of the optical sheet generating and phase adjusting module 200 according to the present application. In the present application, the optical sheet generating and phase adjusting module 200 mainly includes a spatial light modulator SLM, a half-wave plate HWP, a polarizing beam splitter PBS, a cylindrical lens CL3, a mask Mask, and a beam expanding submodule 210. The beam expanding submodule 210 includes a first cylindrical lens CL1 and a second cylindrical lens CL2, which one-dimensionally expand the incident parallel light from the light source module 100 so that the incident parallel light becomes a rectangular parallel light when emitted. The first cylindrical lens CL1 and the second cylindrical lens CL2 constitute a beam expanding system as will be apparent to those skilled in the art.

[0035] In the illustrated example, the spatial light modulator SLM may be a binary spatial light modulator, and the half-wave plate HWP may be an achromatic half-wave plate. The spatial light modulator SLM, the half-wave plate HWP, and the polarizing beam splitter PBS are arranged to form a phase grating, so that the laser beam input from the beam expansion submodule 210 generates multiple stages of positive and negative light components after passing through the phase grating. Specifically, the spatial light modulator SLM, the half-wave plate HWP, and the polarizing beam splitter PBS are arranged to first reflect the laser beam input from the beam expansion submodule 210 as linearly polarized light from the polarizing beam splitter PBS toward the half-wave plate HWP and the spatial light modulator SLM. The reflected linearly polarized light then passes through the half-wave plate HWP and is rotated by a phase of π / 8 before entering the spatial light modulator SLM. A plurality of pixel points are scattered on the spatial light modulator SLM, and each pixel point can be switched between an ON or OFF state as necessary. In this way, by intentionally designing in advance, pixel points in different states on the spatial light modulator SLM can reflect the incident light by rotating it left or right by π / 4 in the polarization direction. The reflected light passes through the half-wave plate HWP again, rotates the phase accordingly, and finally passes through the polarizing beam splitter PBS and is emitted. In this way, the light emitted from the polarizing beam splitter PBS is modulated to have a π phase difference from each other due to the presence of pixel points in different ON or OFF states of the spatial light modulator SLM. In other words, the diffraction pattern displayed on the spatial light modulator SLM can be treated as a phase grating, and different diffraction patterns can be generated by designing an algorithm (for example, by controlling the ON or OFF state of the pixel points), and a cosine stripe light output can be realized.

[0036] The multiple stages of positive and negative light components generated through the phase grating are incident on a cylindrical lens CL3 to generate an optical sheet. A mask is disposed downstream of the cylindrical lens CL3. The mask is particularly disposed at the focal position of the cylindrical lens CL3, and removes the light components of the other stages while leaving only the positive and negative light components of one stage, thereby generating two optical sheets. The two optical sheets are spaced apart from each other with respect to the optical axis.

[0037] FIG. 4 is a schematic diagram of an example of a light path of a scanning module 300 according to the present application. In the scanning module 300, a light path or a part of a light path is defined, for example, shown as three segments Lp1, Lp2, and Lp3. A first galvanometer scanner (or called a laser galvanometer scanner) G1 is provided at the boundary between the light path segments Lp1 and Lp2, and a second galvanometer scanner (or called a laser galvanometer scanner) G2 is provided at the boundary between the light path segments Lp2 and Lp3. The light path segment Lp1 is configured to receive the light path of the light sheet from the light sheet generating and phase adjusting module 200. Those skilled in the art may refer to technical documents known to those skilled in the art, such as technical dictionaries, manuals, textbooks, etc., for related optical terms such as "galvanometer scanner (or laser galvanometer scanner)", "conjugate", and "4F" in this specification and their operating principles. In addition, the first galvanometer scanner G1 and the second galvanometer scanner G2 are also arranged in a conjugate relationship.

[0038] In a preferred embodiment, in the optical path segment Lp1, the lenses L7 and L8 are arranged between the mask Mask and the first galvanometer scanner G1 to form a 4F system. L8 and L9, and L9 and L10 are also arranged in a 4F system, and a single objective lens 400 is arranged downstream of the scanning module 300, for example, the single objective lens 400 is arranged coaxially with the optical path segment Lp3. The single objective lens 400 (rear pupil) is arranged to be in a conjugate relationship with the second galvanometer scanner G2. In a preferred embodiment, in the optical path segment Lp3, the lenses L11 and L12 are arranged between the second galvanometer scanner G2 and the single objective lens 400 to form a 4F system. With the above optical path arrangement, the rear pupil plane of the objective lens 400 is conjugate with the first galvanometer scanner G1, the second galvanometer scanner G2, and the mask Mask.

[0039] In the present application, the first galvanometer scanner G1 and the second galvanometer scanner G2 are arranged to rotate about one axis as required for scanning.

[0040] Between the lenses L11 and L12, a spectroscope (or dichroic mirror) DM4 is disposed in the optical path segment Lp3. The spectroscope (or dichroic mirror) DM4 is configured to directly transmit the light beam traveling through the optical path segments Lp1 and Lp2 in order. After being emitted through the single objective lens 400, the light beam is irradiated onto a sample to generate fluorescence. The generated fluorescence enters the single objective lens 400, travels along the optical path segment Lp3 to the spectroscope DM4, and is reflected toward the camera 510.

[0041] 5A is a schematic diagram of an example of a light path of a portion of a fluorescence detection module 500 according to the present application. In the fluorescence detection module 500, one light path segment Lp4 is defined. The light path segment Lp4 is reflected by the spectrometer DM4 and is perpendicular to the light path segment Lp3. The fluorescence detection module 500 is provided with a camera 510 for capturing the fluorescence. In a preferred embodiment, in the light path segment Lp4, lenses L13 and L14 are disposed between the camera 510 and the spectrometer DM4, and the lenses L13 and L12 form a 4F system. A step plate 520 for increasing the depth of field is arranged on the focal plane of the lens L13 (a plane conjugate with the rear pupil plane of the objective lens). For the technical content of the step plate 520, please refer to the disclosed technical literature such as Sara Abrahamsson et al., "A new approach to extended focus for high-speed, high-resolution biological microscopy", Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, Volume 6090, 60900N (https: / / doi.org / 10.1117 / 12.647022). A lens L14 is arranged downstream of the step plate 520, and together with L13, a 4F system is formed, and a camera 510 is arranged on the rear focal plane of the lens L14. The fluorescence detection module 500 further includes an optical path segment through which the fluorescence received from the single objective lens 400 proceeds to the spectrometer DM4.

[0042] In the optical system according to the embodiment of the present application, the single objective lens 400 is adopted as both the excitation objective lens and the receiving objective lens, so that the received fluorescent image is tilted. In order to receive such a tilted image, the conventional receiving optical path requires a special objective lens and a complex steering optical path design to accommodate image formation. In the fluorescence detection module 500 of the present application, the adoption of the step plate 520 can cancel such a complex steering optical path design. In an alternative detection mode embodiment, as shown in FIG. 5B, the idea of ​​Fourier domain light field imaging is used to place a microlens array 520' on the back focal plane of L13, and a camera 510 is placed behind the microlens array 520' to perform searching, and the collected light field image is reconstructed to obtain three-dimensional information of the object to perform searching. In another alternative detection embodiment, the idea of ​​the conical lens 520'' generating a Bessel beam is utilized to extend the imaging depth of field, and the conical lens 520'' is placed behind L13 according to the actual situation, and the camera 510 searches directly, as shown in FIG. 5C.

[0043] As mentioned above, the two optical sheets generated by the optical sheet generating and phase adjusting module 200 enter the single objective lens 400 after passing through the scanning module 300, and at the same time, they intersect each other due to the focusing action of the lens in the single objective lens 400, and interfere at the intersection to generate a structured light stripe (as shown in the T area of ​​Figures 6A, 6C, and 6E). In order to clearly explain the technology of the present application, a three-dimensional Cartesian coordinate system XYZ or a two-dimensional coordinate system projected onto a corresponding two-dimensional plane is shown in the corresponding drawings. Figures 6B, 6D, and 6F respectively show the distribution situation of the optical sheet in the back pupil of the objective lens when the optical sheet shown in Figures 6A, 6C, and 6E is generated. When different patterns are provided on the spatial light modulator SLM, they are respectively emitted from the single objective lens 400, and structured light stripe areas with different viewing angles are formed. As a result, the super-resolution single objective lens microscopic imaging system using the optical system of the present application can realize two-dimensional or three-dimensional super-resolution.

[0044] Furthermore, in the case of two-dimensional super-resolution imaging, as shown in FIG. 7A, a sample 2000 is placed on a glass slide 1000. First, the light source module 100 is started, and the parameters of the optical sheet generating and phase adjusting module 200 and the scanning module 300 are adjusted so that two optical sheets (for example, see FIG. 6A) are emitted from a single objective lens 400 to form a structured light stripe region T1000. For example, the distribution of the two optical sheets and the rear pupil of the objective lens at the viewing angle is as shown in FIG. 6B, and the structured light stripe region T1000 generated by interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the glass slide (see FIG. 7A). For example, such a structured light stripe region T1000 can be scanned along the Y axis. The scanning process is realized by a galvanometer scanner in the scanning module, for example, the galvanometer scanner G1 controls the scanning along the X axis, and the galvanometer scanner G2 controls the scanning along the Y axis. To realize the scanning along the Y axis, the galvano scanner G1 remains fixed at the initial position, and the galvano scanner G2 rotates at a set interval angle, thereby realizing the movement of the structured light stripe region T1000 along the Y direction, and the fluorescence detection module 500 records the fluorescence image data at each position where the angle is increased, which is the fluorescence image data at the viewing angle (for example, the first viewing angle) corresponding to the structured light stripe region T1000 (shown in FIG. 7B). Next, the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300 are adjusted so that the two optical sheets (for example, see FIG. 6C) are emitted from the single objective lens 400 to form the structured light stripe region T2000. For example, the distribution of the two optical sheets and the rear pupil of the objective lens at the viewing angle is as shown in FIG. 6D, and the structured light stripe region T2000 generated by the interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the slide glass, as shown in FIG. 7C. For example, such a structured light stripe region T1000 can be scanned along the X-axis.To achieve such scanning, the galvano scanner G2 remains fixed at an initial position, and the galvano scanner G1 rotates at a set interval angle, thereby realizing the movement of the structured light stripe region T2000 along the X direction, and the fluorescence detection module 500 records the fluorescence image data at each increased angle position, which is the fluorescence image data at the viewing angle (e.g., the second viewing angle) corresponding to the structured light stripe region T2000 (as shown in FIG. 7D). It is clear to those skilled in the art that the specific embodiment of which direction the structured light stripe region moves along can be adjusted and manipulated by the galvano scanners G1 and / or G2 as needed.

[0045] Finally, the image data at the two viewing angles are fused and the data is processed using a super-resolution reconstruction algorithm to obtain an imaging result with super-resolution effect in the XY plane.

[0046] Furthermore, in the case of three-dimensional super-resolution imaging, the distribution of the structured light stripe region T1000 at the first viewing angle is as shown in FIG. 8A, for example, the structured light stripe region at the first viewing angle can be provided as shown in FIG. 7A. And the distribution of the structured light stripe region T3000 at the third viewing angle is as shown in FIG. 8C. For example, by adjusting the parameters of the optical sheet generating and phase adjusting module 200 and the scanning module 300, two optical sheets (for example, see FIG. 6E) are emitted from the single objective lens 400 to form the structured light stripe region T3000. For example, the distribution of the two optical sheets and the rear pupil of the objective lens at the viewing angle is as shown in FIG. 6F, and the structured light stripe region T3000 generated by interference after passing through the objective lens 400 can be irradiated on the sample 2000 on the slide glass, as shown in FIG. 8C. For example, first, the light source module 100 is started, and the parameters of the optical sheet generating and phase adjusting module 200 and the scanning module 300 are adjusted so that two optical sheets (for example, see FIG. 6A) are emitted from the single objective lens 400 to form a structured light stripe region T1000. For example, the distribution of the two optical sheets and the rear pupil of the objective lens at the viewing angle is as shown in FIG. 6B, and the structured light stripe region T1000 generated by the interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the slide glass, as shown in FIG. 8A. For example, such a structured light stripe region T1000 can be scanned along the Y axis. The scanning process is realized by a galvanometer scanner in the scanning module 300, for example, the galvanometer scanner G1 controls the scanning along the X axis, and the galvanometer scanner G2 controls the scanning along the Y axis. To achieve scanning along the Y axis, the galvanometer scanner G1 remains fixed at its initial position, and the galvanometer scanner G2 rotates at a set interval angle, thereby achieving movement of the structured light stripe region T1000 along the Y direction, and the fluorescence image data is recorded by the fluorescence detection module 500 at each increased angle position, which becomes the fluorescence image data at a viewing angle (e.g., a first viewing angle) corresponding to the structured light stripe region T1000 (shown in Figure 8B).Then, adjust the parameters of the optical sheet generating and phase adjusting module 200 and the scanning module 300 so that the two optical sheets (see, for example, FIG. 6E) are emitted by the single objective lens 400 to form a structured light stripe region T3000. For example, the distribution of the rear pupil of the objective lens at the two optical sheets and the viewing angle is as shown in FIG. 6F, and the structured light stripe region T3000 generated by interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the slide glass, as shown in FIG. 8C. Unlike the two-dimensional super-resolution imaging, the structured light stripe region T3000 can be scanned along the same Y-axis, but the direction is opposite to the direction in which the structured light stripe region T1000 moves. For example, the galvano scanner G2 is controlled to rotate in the opposite direction in the same manner, and the galvano scanner G1 is fixed at the initial position, and the galvano scanner G2 records the fluorescence image data at each different angle position by the fluorescence detection module 500, which is the fluorescence image data at the viewing angle (for example, the third viewing angle) corresponding to the structured light stripe region T3000 (shown in FIG. 8D). The collected images at each viewing angle are used to realize two-dimensional super-resolution imaging by a one-dimensional direction structured light illumination optical sheet microscopy device using a deep learning method, and the two-dimensional super-resolution images at the two viewing angles are fused to obtain a three-dimensional super-resolution image with a super-resolution effect in the XYZ directions. For the specific content of the deep learning method, please refer to patent document CN113917677A.

[0047] Those skilled in the art should appreciate that the analysis and processing of 3D super-resolution microscopic images can be achieved by any 3D image reconstruction method well known in the field of microscopic image processing technology, and can be implemented on a computer using commercial interactive microscopic image analysis software such as Imaris and Amira.

[0048] The slide glass 1000 shown in Figures 7A, 7C and 8A, 8C can be placed on a stage. Alternatively, the stage defines a plane for placing the sample 2000 to be detected. Thus, the super-resolution microscopic imaging system can generally include a stage and the super-resolution single objective light sheet microscopic imaging optical system described above, where the optical axis of the single objective 400 forms a 90 degree angle with the plane.

[0049] For three-dimensional super-resolution imaging, FIG. 10 is a schematic diagram of a light path of a super-resolution single-objective lens optical sheet microscopic imaging optical system according to another embodiment of the present application. Comparing the embodiment shown in FIG. 10 with FIG. 9 and FIG. 1, it can be seen that the main difference between the optical system shown in FIG. 10 and the optical system shown in FIG. 9 is the difference in the fluorescence detection module. Therefore, for simplicity, the same reference numerals as those described above in the embodiment shown in FIG. 10 can refer to the description of the previous embodiment. The optical system shown in FIG. 10 includes a fluorescence detection module 600 instead of the fluorescence detection module 500, and FIG. 11 shows a light path diagram of a part of the fluorescence detection module 600.

[0050] 10, the spectrometer DM4 and the associated fluorescence detection module 500 are deleted compared to the embodiment shown in FIG. 9, but the spectrometer DM3 is disposed between the lens L9 and the first galvano scanner G1, so that the optical sheet light can propagate in the direction from the first galvano scanner G1 to the lens L9, but the fluorescence received by the single objective lens 400 passes through the lenses L12, L11, the second galvano scanner G2, and the lenses L10, L9, and is then reflected by the spectrometer DM3 toward the fluorescence detection module 600 (FIG. 11). If the propagation directions of the excitation light and the fluorescence are not taken into consideration, the optical paths of the scanning module 300 and the single objective lens 400 partially overlap with the optical path of the fluorescence detection module 600. FIG. 11 shows only a portion of the optical path of the fluorescence detection module 600 that does not overlap.

[0051] As shown in FIG. 11, the fluorescence detection module 600 includes a third galvanometer scanner G3 and a fourth galvanometer scanner G4, and three reflectors, namely, a second reflector M2, a third reflector M3, and a fourth reflector M4, are arranged between the third galvanometer scanner G3 and the fourth galvanometer scanner G4. In addition, downstream of the fourth galvanometer scanner G4, a lens L15, a fifth reflector M5, a lens L16, objective lenses OBJ2 and OBJ3, a lens L17, and a camera 610 are arranged in this order. The optical axis of the objective lens OBJ2 and the optical axis of the objective lens OBJ3 form a certain angle. As described above, since the fluorescent image is tilted, the angle between the optical axis of the objective lens OBJ2 and the optical axis of the objective lens OBJ3 is configured to be just right to cancel this tilt.

[0052] In this embodiment, the rear pupil plane of the objective lens 400 is conjugated to the second galvanometer scanner G2 via lenses L12 and L11, the second galvanometer scanner G2 is conjugated to the first galvanometer scanner G1 via lenses L10 and L9, the focal plane of the lens L9 is conjugated to the rear pupil plane of the objective lens OBJ2 via lenses L15 and L16, and the image at the objective lens OBJ2 is formed on the camera 610 via the objective lens OBJ3 and lens L17.

[0053] The third galvanometer scanner G3, the fourth galvanometer scanner G4, the second reflector M2, and the fourth reflector M4 are configured such that, when reflected fluorescence at a first field of view angle is received by the fluorescence detection module 600, the third galvanometer scanner G3 is switched to one angle, so that the fluorescence reflected by the spectrometer DM3 propagates through the path of the third galvanometer scanner G3, the second reflector M2, the fourth reflector M4, and the fourth galvanometer scanner G4, and is received by the camera 610 via the lens L15, and when reflected fluorescence at a second field of view angle is received by the fluorescence detection module 600, the third galvanometer scanner G3 is switched to another angle, so that the fluorescence reflected by the spectrometer DM3 propagates through the path of the third galvanometer scanner G3, the third reflector M3, and the fourth galvanometer scanner G4, and is received by the camera 610 via the lens L15. Because the fluorescence signals received at the two different viewing angles have passed through different numbers of mirrors when reaching the camera 610, the final imaging directions of the fluorescence images at the different viewing angles are consistent, eliminating the need for subsequent modification of the associated search light path when switching between different viewing angles.

[0054] Although specific embodiments of the present application have been described in detail in this specification, these are provided only for the purpose of interpretation and are not considered to limit the scope of the present application. In addition, it is clear to those skilled in the art that each embodiment described in this specification can be used in combination with each other. Various substitutions, modifications, and alterations can be envisioned without departing from the spirit and scope of the present application.

Claims

1. A super-resolution single objective lens light sheet microscopy imaging optical system, comprising: a light source module (100) configured to output a laser beam of single or multiple wavelengths; a light sheet generation and phase adjustment module (200) configured to receive the single or multiple wavelength laser beams and output two light sheets; A scanning module (300); a single objective lens (400) located downstream of the scanning module (300), the scanning module (300) configured to guide two optical sheets output from the optical sheet generating and phase adjusting module (200) to the single objective lens (400), the single objective lens (400) configured to make the two optical sheets exit the single objective lens (400) and interfere with each other to generate a structured light stripe region (T), and receive a fluorescent signal; a fluorescence detection module (500, 600) configured to record the fluorescence signal received by the single objective lens (400), A super-resolution single objective lens light sheet microscopic imaging optical system.

2. the scanning module (300) is configured to scan the structured light stripe region (T) along one direction (X or Y), and the fluorescence detection module (500, 600) is configured to synchronously record the fluorescence signal received by the single objective lens (400) during scanning; The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 1 .

3. The fluorescent signal is a fluorescent signal generated by irradiating the structured light stripe region (T) on a detection target sample.

3. The super-resolution single objective lens optical sheet microscopic imaging optical system according to claim 1 or 2.

4. The optical sheet generating and phase adjusting module (200) includes a spatial light modulator (SLM), a half-wave plate (HWP), a polarizing beam splitter (PBS), a cylindrical lens (CL3) and a mask (Mask); 3. The super-resolution single objective lens optical sheet microscopic imaging optical system according to claim 1 or 2.

5. The spatial light modulator (SLM), the half-wave plate (HWP), and the polarizing beam splitter (PBS) are arranged to form a phase grating, so that the single or multiple wavelength laser beams generate multiple stages of positive and negative light components after passing through the phase grating. The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 4 .

6. the polarizing beam splitter (PBS) is configured to reflect the single or multiple wavelength laser beams towards the half wave plate (HWP) and through the half wave plate (HWP) to be incident on the spatial light modulator (SLM); The spatial light modulator (SLM) is configured to be switchable between at least two different states, and in the at least two different states, different patterns are defined in the spatial light modulator (SLM) for generating multiple stages of positive and negative light components correspondingly. The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 5 .

7. The mask (Mask) is configured to be capable of removing light components of other stages while leaving only the light components of one stage of positive and negative, the mask (Mask) is located downstream of the cylindrical lens (CL3), and the two optical sheets are generated after the positive and negative light components of the multiple stages pass through the cylindrical lens (CL3) and the mask (Mask). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 6 .

8. The structured light stripe region (T) comprises a structured light stripe region (T1000) at a first viewing angle and a structured light stripe region (T2000 or T3000) at a second viewing angle; The state of the spatial light modulator (SLM) is a first state, in which a first diffraction pattern is defined in the spatial light modulator (SLM) in the first state, such that two optical sheets for forming a structured light stripe region (T1000) at the first viewing angle are output from the optical sheet generating and phase adjusting module (200); a second state in which a second diffraction pattern different from the first diffraction pattern is defined in the spatial light modulator (SLM) in the second state, such that two optical sheets for forming a structured light stripe region (T2000 or T3000) at the second viewing angle are output from the optical sheet generating and phase adjusting module (200). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 7.

9. The scanning module (300) includes a first galvanometer scanner (G1) and a second galvanometer scanner (G2), and the first galvanometer scanner (G1) is arranged to receive two optical sheets output from the optical sheet generating and phase adjusting module (200) and reflect the two optical sheets toward the second galvanometer scanner (G2); The second galvanometer scanner (G2) is arranged to reflect the two optical sheets toward the single objective lens (400). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 8.

10. The method further comprises a spectrometer (DM4) between the single objective lens (400) and the second galvano scanner (G2), the spectrometer (DM4) being configured such that the two optical sheets are capable of transmitting through the spectrometer but the fluorescent signal is reflected towards the fluorescent detection module (500), the fluorescent detection module (500) comprising a camera (510) for recording the fluorescent signal and a conical lens (520'') or a microlens array (520') located upstream of the camera (510). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 9.

11. The optical system further includes a spectrometer (DM4) between the single objective lens (400) and the first galvano scanner (G1), the spectrometer (DM4) being configured such that the two optical sheets are capable of transmitting through the spectrometer but the fluorescent signal is reflected toward the fluorescent detection module (500), the fluorescent detection module (500) includes a camera (510) for recording the fluorescent signal, and an eighth lens (L14) and a third lens (L13) arranged to form a 4F system between the spectrometer (DM4) and the camera (510), the third lens (L13) being closer to the spectrometer (DM4), and a step plate (520) being provided on the focal plane of the third lens (L13). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 9.

12. A first lens (L12) and a second lens (L11) are arranged to configure a 4F system between the single objective lens (400) and the second galvano scanner (G2), and the fluorescence detection module (500) further includes a third lens (L13) located upstream of the conical lens (520) or the microlens array, and the first lens (L12) and the third lens (L13) are located between the single objective lens (400) and the conical lens (520'') or the microlens array (520') to configure a 4F system, and the spectrometer (DM4) is located between the first lens (L12) and the second lens (L11). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 10.

13. The optical system further includes a fourth lens (L10) and a fifth lens (L9) arranged to form a 4F system between the second galvanometer scanner (G2) and the first galvanometer scanner (G1), the second galvanometer scanner (G2) is conjugated with the first galvanometer scanner G1 via the fourth lens (L0) and the fifth lens (L9), and a spectrometer (DM3) is arranged between the fifth lens (L9) and the first galvanometer scanner (G1), and the spectrometer (DM3) is configured such that the two optical sheets are capable of transmitting through the spectrometer but the fluorescent signal is reflected toward the fluorescent detection module (600). The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 9.

14. The fluorescence detection module (600) includes a camera (610) for recording the fluorescence signal, a second objective lens (OBJ2) and a third objective lens (OBJ3) located upstream of the camera (610) and having optical axes arranged at a non-zero angle to each other, and a sixth lens (L15) and a seventh lens (L16) arranged to configure a 4F system between the spectrometer (DM3) and the second objective lens (OBJ2), and a galvanometer scanner-reflector system is arranged between the spectrometer (DM3) and the seventh lens (L16) close to the second objective lens (OBJ2) so that the imaging directions of fluorescent images at different viewing angles coincide. The super-resolution single objective lens light sheet microscopic imaging optical system of claim 13.

15. The galvanometer scanner-reflector system includes a third galvanometer scanner (G3) and a fourth galvanometer scanner (G4) or an additional reflector arranged between a spectrometer (DM3) and the sixth lens (L15), a second reflector (M2), a third reflector (M3), and a fourth reflector (M4) arranged between the third galvanometer scanner (G3) and the fourth galvanometer scanner (G4) or the additional reflector, and a fifth reflector (M5) arranged between the sixth lens (L15) and the seventh lens (L16). The third galvanometer scanner (G3) detects whether the fluorescence is reflected by the third galvanometer scanner (G3) at one of the first field of view angle and the second field of view angle. and in the other of the first field of view and the second field of view, the fluorescence is operable to propagate to the second objective lens (OBJ2) via the third galvanometer scanner (G3), the third reflector (M3), the fourth galvanometer scanner (G4) or an additional reflector, the sixth lens (L15), the fifth reflector (M5), and the seventh lens (L16) in that order. The super-resolution single objective lens light sheet microscopic imaging optical system of claim 14.

16. The structured light stripe region (T1000, T2000 or T3000) is scanned and moved by the operation of the first galvano scanner (G1) and / or the second galvano scanner (G2); The super-resolution single objective lens light sheet microscopic imaging optical system according to any one of claims 9 to 15.

17. In the case of two-dimensional super-resolution imaging, the structured light stripe area (T1000) at the first viewing angle and the structured light stripe area (T2000) at the second viewing angle are scanned and moved along directions perpendicular to each other. The super-resolution single objective lens light sheet microscopic imaging optical system according to claim 3 .

18. In the case of three-dimensional super-resolution imaging, the structured light stripe area (T1000) at the first viewing angle and the structured light stripe area (T3000) at the second viewing angle are scanned and moved in opposite directions along the same straight line. The super-resolution single objective lens light sheet microscopic imaging optical system of claim 17.

19. The light sheet generating and phase adjusting module (200) further comprises a beam expansion filter sub-module (210) for expanding and / or filtering the single or multiple wavelength laser beam, the beam expansion filter sub-module (210) being configured such that the single or multiple wavelength laser beam is initially incident on the beam expansion filter sub-module (210) after entering the light sheet generating and phase adjusting module (200). The super-resolution single objective lens light sheet microscopy imaging optical system of claim 18.

20. The mask and the first galvanometer scanner are arranged in a conjugate relationship.

20. The super-resolution single objective lens light sheet microscopic imaging optical system of claim 19.

21. Two lenses are arranged between the mask and the first galvanometer scanner (G1), and / or between the first galvanometer scanner (G1) and the second galvanometer scanner (G2), and / or between the second galvanometer scanner (G2) and the single objective lens (400) to configure a 4F system. The super-resolution single objective lens light sheet microscopic imaging optical system of claim 20.

22. 1. A super-resolution single objective lens light sheet microscopy imaging system, comprising: a stage having a flat surface on which a sample to be detected is placed; and a super-resolution single objective light sheet microscopy imaging optical system according to any one of claims 1 to 21, wherein the optical axis of the single objective (400) forms an angle of 90 degrees with the plane. A super-resolution single objective lens light sheet microscopic imaging system.

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