Super-resolution single objective lens optical sheet microimaging 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] This application generally relates to the field of super-resolution microscopic imaging technology, and particularly to a super-resolution single objective lens optical sheet microscopic imaging optical system and its imaging system.

Background Art

[0002] The emergence of optical microscopes has provided an intuitive observation means for the dynamic process of life activities and promoted the rapid development of life science research. Due to the wave nature of light, the imaging resolution of optical microscopes is limited, and the observation of fine life activities is greatly inhibited. Therefore, breaking through the diffraction-limited resolution has always been the goal pursued in the field of optical microscopy research. In recent years, super-resolution microscopic imaging technology that breaks through the diffraction limit has been developing. Current super-resolution microscopy techniques are mainly classified into three types: (1) Stimulated Emission Depletion (STED) microscopy technology, (2) single molecule alignment reconstruction microscopy technology, and (3) Structured Illumination Microscopy (SIM) technology. The super-resolution microscopic imaging system based on STED technology requires a relatively high light intensity, is prone to photo-bleaching and photo-toxicity, causes great damage to biological samples, and the super-resolution microscope 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 Microscope SIM adopts a wide-field illumination method, does not require a high laser power, and is more suitable for high-speed biological imaging than the above two types of super-resolution microscopy techniques. However, in the case of three-dimensional structured illumination microscopic imaging, SIM irradiates the entire sample, increasing the probability of photo-bleaching and reconstruction artifacts due to the presence of defocus signals.

[0003] Unlike conventional wide-field imaging modes, light-sheet fluorescence microscopy (FLFS) illuminates objects by generating a sheet-like light source and separating the excitation and search paths, thereby avoiding defocus signals and significantly reducing photobleaching. Conventional FLFS systems are similarly limited in their practical operation. Firstly, they are limited by the arrangement of the excitation and search objective lenses, often making it impossible to use standard slides or perforated plates properly, and requiring special designs for sample preparation and fixation. Secondly, they are mechanically limited; the objective lens requires a sufficient working distance to meet imaging conditions, which limits the numerical aperture of the objective lens, and thus the imaging resolution.

[0004] In recent years, imaging technology for single-objective optical sheet microscopes has advanced. Instead of using a system where the excitation and search objectives are perpendicular and independent to each other, the same objective lens is used as both the excitation and search objective to collect fluorescence signals, and the inclined image plane is searched through a remote focusing imaging method. This overcomes the sample limitations of conventional optical sheet microscopes and allows for imaging of different types of samples. Optical sheet fluorescence microscopes excel at imaging long-duration and high-speed objects, but their spatial resolution is not high. Therefore, if single-objective optical sheet microscope imaging technology can be combined with structural optical superresolution microscope imaging technology, it would be extremely useful for high-resolution, high-speed three-dimensional imaging of living organisms, and would be crucial for cell, tissue, and embryo research in life science fields such as developmental biology and neurobiology. [Overview of the project] [Problems that the invention aims to solve]

[0005] The main objective of this invention is to achieve super-resolution single-objective lens optical sheet imaging, particularly three-dimensional super-resolution imaging, by combining single-objective lens optical sheet imaging technology and structural light illumination super-resolution microscopy imaging technology. [Means for solving the problem]

[0006] According to one aspect of the present invention, a super-resolution single objective lens optical sheet microimaging optical system is provided. A light source module configured to output a laser beam of one or more wavelengths, An optical sheet generation and phase adjustment module configured to receive a laser beam of one or more wavelengths and output two optical sheets, Scanning module and A single objective lens located downstream of the scanning module, wherein the scanning module is configured to guide two optical sheets output from the optical sheet generation and phase adjustment module to the single objective lens, and the single objective lens is configured to receive fluorescence signals such that the two optical sheets are ejected from the single objective lens and interfere with each other to generate a structured optical stripe region, The system includes a fluorescence detection module configured to record the fluorescence signal received by the aforementioned single objective lens.

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

[0008] Selectively, the fluorescence signal is a fluorescence signal generated when the structural light stripe region is irradiated onto the sample to be detected.

[0009] Selectively, the optical sheet generation and phase adjustment module comprises a spatial light modulator, a half-wave plate, a polarizing beam splitter, a cylindrical lens, and a mask.

[0010] Selectively, 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 positive and negative optical components after passing through the phase grating.

[0011] Selectively, the polarizing beam splitter is configured to reflect the single or multiple wavelength laser beams toward the half-wave plate, pass through the half-wave plate, and enter the spatial light modulator. The spatial light modulator is configured to be switchable between at least two different states, and in each of these at least two different states, the spatial light modulator is provided with correspondingly different patterns for generating multiple stages of positive and negative light components.

[0012] The mask is configured to selectively remove the light components of other stages while retaining only the positive and negative light components of one stage, and the mask is located downstream of the cylindrical lens, generating the two optical sheets after the multiple stages of positive and negative light components have passed through the cylindrical lens and the mask.

[0013] Selectively, the structured optical stripe region comprises a structured optical stripe region in a first viewing angle and a structured optical stripe region in a second viewing angle. The state of the aforementioned spatial light modulator is, A first state in which, in the first state, the spatial light modulator has a first diffraction pattern defined such that two optical sheets for forming a structured light stripe region in the first field of view are output from the optical sheet generation and phase adjustment module, The second state includes a second state in which, in the second state, the spatial light modulator has a second diffraction pattern defined that is different from the first diffraction pattern, such that two optical sheets for forming a structured light stripe region in the second field of view are output from the optical sheet generation and phase adjustment module.

[0014] Selectively, the scanning module comprises a first galvanometer scanner and a second galvanometer scanner, wherein the first galvanometer scanner is arranged to receive two optical sheets output from the optical sheet generation and phase adjustment module and reflect them toward the second galvanometer scanner. The second galvanometer scanner is positioned to reflect the two optical sheets toward the single objective lens.

[0015] Selectively, the super-resolution single-objective lens optical sheet microimaging optical system further comprises a spectrometer between the single-objective lens and the second galvanoscanner, wherein the spectrometer is configured such that the two optical sheets are passable through the spectrometer but the fluorescence signal is reflected toward the fluorescence detection module, the fluorescence detection module comprising a camera for recording the fluorescence signal and a conical lens or microlens array located upstream of the camera.

[0016] Selectively, the super-resolution single-objective lens optical sheet microimaging optical system further comprises a spectrometer between the single-objective lens and the first galvanoscanner, wherein the spectrometer is configured such that the two optical sheets are passable through the spectrometer but the fluorescence signal is reflected toward the fluorescence detection module, the fluorescence detection module comprises a camera for recording the fluorescence signal and an eighth lens and a third lens arranged between the spectrometer and the camera to form a 4F system, wherein the third lens is closer to the spectrometer and a stepped plate is provided at the focal plane of the third lens.

[0017] The first and second lenses are selectively positioned between the single objective lens and the second galvanoscanner to constitute a 4F system, the fluorescence detection module further comprises a third lens located upstream of the conical lens or the microlens array, the first and third lenses being positioned between the single objective lens and the conical lens or the microlens array to constitute a 4F system, and the spectrometer being positioned between the first and second lenses.

[0018] Selectively, the super-resolution single-objective lens optical sheet microimaging optical system further comprises a fourth lens and a fifth lens arranged to constitute a 4F system between the second galvanoscanner and the first galvanoscanner, the second galvanoscanner conjugate with the first galvanoscanner G1 via the fourth and fifth lenses, and a spectrometer positioned between the fifth lens and the first galvanoscanner, the spectrometer configured such that the two optical sheets are passable through the spectrometer but the fluorescence signal is reflected toward the fluorescence detection module.

[0019] Selectively, the fluorescence detection module comprises a camera for recording the fluorescence signal, a second objective lens and a third objective lens located upstream of the camera and with their optical axes at a non-zero angle to each other, and a sixth lens and a seventh lens positioned between the spectrometer and the second objective lens to form a 4F system, wherein a galvanoscanner-reflector system is positioned between the spectrometer and the seventh lens, which is close to the second objective lens, such that the imaging direction of the fluorescence image at different field angles coincides.

[0020] Selectively, the galvanoscanner-reflector system comprises a third galvanoscanner and a fourth galvanoscanner or an additional reflector positioned between the spectrometer and the sixth lens, a second reflector, a third reflector and a fourth reflector positioned between the third galvanoscanner and the fourth galvanoscanner or the additional reflector, and a fifth reflector positioned between the sixth lens and the seventh lens, wherein the third galvanoscanner is operable to allow fluorescence to propagate to the second objective lens in one of the first and second fields of view via the third galvanoscanner, the second reflector, the fourth reflector, the fourth galvanoscanner or additional reflector, the sixth lens, the fifth reflector and the seventh lens in sequence, and to the second objective lens in the other of the first and second fields of view.

[0021] Optionally, the operation of the first galvanometer scanner and / or the second galvanometer scanner is used to scan and move the structured light stripe region.

[0022] Optionally, in the case of two-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 scan and move 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 scan and move in opposite directions along the same straight line.

[0024] Optionally, the optical sheet generation and phase adjustment module further includes a beam expansion filter sub-module for expanding and / or filtering the laser beam of the single or multiple wavelengths, and the beam expansion filter sub-module is configured such that after the laser beam of the single or multiple wavelengths enters the optical sheet generation and phase adjustment module, it first enters the beam expansion filter sub-module.

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

[0026] [[ID=2」 Optionally, 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, two lenses are arranged to form a 4F system.

[0027] According to another aspect of the present application, a super-resolution single objective lens optical sheet microscopic imaging system is further provided, a stage having a plane for placing a detection target sample, the super-resolution single objective lens optical sheet microscopic imaging optical system in which the optical axis of the single objective lens forms a 90-degree angle with the plane.

[0028] By employing the technical means of this invention, two optical sheets are generated by a specially designed optical sheet generation and phase adjustment module, thereby forming structural optical stripes through optical sheet interference and ensuring that the optical path of the entire optical system is easily stabilized. Furthermore, this invention achieves two-dimensional or three-dimensional super-resolution microimaging using a single objective lens, with the excitation objective lens and the search objective lens being the same objective lens, allowing the use of standard slides and perforated plates, and making it easier to position the sample to be detected compared to conventional optical sheet systems where the excitation objective lens and the search objective lens are separated, thus improving the applicability of the optical system. Moreover, the optical system according to this invention combines the advantages of optical sheet imaging and structural optical imaging, reducing the probability of photobleaching and the effects of reconstruction artifacts due to the presence of defocus signals, and providing a foundation for realizing high-resolution, high-speed three-dimensional biomicroimaging. [Brief explanation of the drawing]

[0029] The principles and embodiments of this application will be better understood from the following detailed description and drawings. Note that the proportions in the drawings may differ for illustrative purposes, but this will not affect the understanding of this application. [Figure 1] This is a schematic block diagram illustrating the super-resolution single objective lens optical sheet microimaging optical system according to the present invention. [Figure 2] A schematic diagram of an example of the optical path of a light source module relating to this application is shown. [Figure 3] A schematic diagram of an example of an optical sheet generation and phase adjustment module relating to the present invention is shown. [Figure 4] A schematic diagram of an example of a scanning module and a single objective lens relating to this application is shown. [Figure 5A] A schematic diagram of a partial optical path of an example of a fluorescence detection module relating to this application is shown. [Figure 5B] A schematic diagram of a partial optical path of another example of the fluorescence detection module relating to this application is shown. [Figure 5C]A schematic diagram of a partial optical path of another example of the fluorescence detection module relating to this application is shown. [Figures 6A-6F] The present invention schematically illustrates the distribution of the pupil plane after the objective lens when two optical sheets pass through the single objective lens at different field angles in the super-resolution single objective lens optical sheet microimaging optical system. [Figure 7A] This diagram schematically illustrates the scanning process at one field of view in a two-dimensional super-resolution imaging mode. [Figure 7B] Figure 7A schematically shows the image data information of the acquired fluorescence signal. [Figure 7C] The scanning process at other field of view angles in the 2D super-resolution imaging mode is schematically shown. [Figure 7D] Figure 7C schematically shows the image data information of the acquired fluorescence signal. [Figure 8A] This diagram schematically illustrates the scanning process at one field of view in a 3D super-resolution imaging mode. [Figure 8B] Figure 8A schematically shows the image data information of the acquired fluorescence signal. [Figure 8C] The scanning process at other field of view angles in the 3D super-resolution imaging mode is schematically shown. [Figure 8D] Figure 8C schematically shows the image data information of the acquired fluorescence signal. [Figure 9] A schematic diagram of the optical path of a super-resolution single objective lens optical sheet microimaging optical system according to one embodiment of the present invention is shown. [Figure 10] A schematic diagram of the optical path of a super-resolution single-objective lens optical sheet microimaging optical system according to another embodiment of the present invention is shown. [Figure 11] A schematic diagram of the optical path of a fluorescence detection module according to one embodiment of the present invention is shown. [Modes for carrying out the invention]

[0030] In the drawings of this application, features that have the same structure or similar function are indicated by the same reference numerals.

[0031] Figure 1 is a schematic block diagram showing a super-resolution single-objective lens optical sheet microimaging system according to one embodiment of the present application, and Figure 9 is a schematic optical path diagram of the super-resolution single-objective lens optical sheet microimaging system according to the embodiment of the present application. In the present application, the super-resolution single-objective lens optical sheet microimaging system mainly comprises a light source module 100, an optical sheet generation and phase adjustment module 200, a scanning module 300, a single-objective lens 400, and a fluorescence detection module 500. When the super-resolution single-objective lens optical sheet microimaging system of the present invention is in operation, the light source module 100 outputs a laser beam of one or more wavelengths and incidents it onto the optical sheet generation and phase adjustment module 200. The optical sheet generation and phase adjustment module 200 generates two optical sheets having a predetermined spatial relationship. These two optical sheets are modulated as needed via the scanning module 300, and can then be irradiated onto a sample (not shown) placed on the stage of the super-resolution single-objective lens optical sheet microimaging system via the single-objective lens 400. In this process, the two optical sheets interfere in the plane on which the sample is located to form structural light, which is then irradiated onto the sample. Simultaneously, fluorescence generated from the irradiated sample is received by the same objective lens 400 and collected by the fluorescence detection module 500. Since the excited fluorescence signal contains super-resolution information due to the structural light irradiation, a predetermined scanning process can be performed in the scanning module 300 to ultimately achieve two-dimensional or three-dimensional super-resolution single-objective lens optical sheet microimaging.

[0032] Figure 2 schematically shows an optical path diagram of an example of a light source module 100 according to the present invention. In the present invention, 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, usable lasers may include lasers with wavelengths of 405 nm (nanometers), 445 nm, 488 nm, 561 nm, and 640 nm. In the example shown, the light source module 100 is shown to include three lasers, Laser1, Laser2, and Laser3. These three lasers, Laser1, Laser2, and Laser3, may emit laser light of different wavelengths from each other. 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 beam-widened into parallel light of equal diameter through their respective lens groups. The reflector M1 and dichroic mirrors (also called spectrometers) DM1 and DM2 combine the parallel light of equal diameter from each path of the different lasers and integrate them into a single laser beam.

[0033] In a preferred or selectable embodiment, an acousto-optically tuned filter (AOTF) is positioned downstream of the dichroic mirror M2 in the optical path of the integrated laser beam, so that the integrated laser beam can be incident on the acousto-optically tuned filter (AOTF). The role of the acousto-optically tuned filter (AOTF) is to selectively ensure the passage of light of a specific wavelength and to control the power of the light output through it.

[0034] Figure 3 schematically shows an optical path diagram of an example of an optical sheet generation and phase adjustment module 200 according to the present invention. In the present invention, the optical sheet generation and phase adjustment module 200 mainly comprises a spatial light modulator SLM, a half-wave plate HWP, a polarizing beam splitter PBS, a cylindrical lens CL3, a mask, and a beam expansion submodule 210. The beam expansion submodule 210 comprises a first cylindrical lens CL1 and a second cylindrical lens CL2, which one-dimensionally stretch the incident parallel light from the light source module 100 so that it becomes rectangular parallel light upon emission. The first cylindrical lens CL1 and the second cylindrical lens CL2 constitute a beam expansion system as will be apparent to those skilled in the art.

[0035] In the example shown, the spatial light modulator SLM may be a binarizing 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 optical 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 so that the laser beam input from the beam expansion submodule 210 is first reflected as linearly polarized light from the polarizing beam splitter PBS towards the half-wave plate HWP and the spatial light modulator SLM. The reflected linearly polarized light then rotates by a phase of π / 8 via the half-wave plate HWP before being incident on the spatial light modulator SLM. The spatial light modulator SLM has multiple pixel points, and each pixel point can be switched ON or OFF as needed. Thus, by intentionally designing it in advance, pixel points in different states on the spatial light modulator (SLM) can reflect incident light by rotating it left or right by π / 4 in the polarization direction. The reflected light then passes through the half-wave plate (HWP) again, its phase is rotated 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 phase difference of π from each other, depending on the presence of pixel points in different ON or OFF states on the spatial light modulator (SLM). In other words, the diffraction pattern displayed on the spatial light modulator (SLM) can be treated as a phase-type grating, and by designing an algorithm (for example, by controlling the ON or OFF state of the pixel points), different diffraction patterns can be generated, and cosine stripe light output can be realized.

[0036] Multiple stages of positive and negative light components generated through the phase grating are incident on the cylindrical lens CL3, generating an optical sheet. A mask is placed downstream of the cylindrical lens CL3. The mask is positioned specifically at the focal point of the cylindrical lens CL3, removing all but one stage of positive and negative light components, thereby generating two optical sheets. These two optical sheets are spaced apart from each other with respect to the optical axis.

[0037] Figure 4 schematically shows an optical path diagram of an example of a scanning module 300 according to the present application. The scanning module 300 has an optical path or a part of an optical path defined, for example, shown as three segments Lp1, Lp2, and Lp3. A first galvanoscanner (or laser galvanoscanner) G1 is provided at the boundary between optical path segments Lp1 and Lp2, and a second galvanoscanner (or laser galvanoscanner) G2 is provided at the boundary between optical path segments Lp2 and Lp3. Optical path segment Lp1 is configured to receive the optical path of the optical sheet from the optical sheet generation and phase adjustment module 200. Those skilled in the art can refer to technical documents known to those skilled in the art, such as technical dictionaries, manuals, and textbooks, for information on related optical terms such as "galvanoscanner (or laser galvanoscanner)," "conjugate," and "4F," and their operating principles. The first galvanoscanner G1 and the second galvanoscanner G2 are also arranged in a conjugate relationship.

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

[0039] In this application, the first galvanoscanner G1 and the second galvanoscanner G2 are arranged to rotate around a single axis as needed for scanning.

[0040] A spectrometer (or dichroic mirror) DM4 is positioned between lenses L11 and L12 in the optical path segment Lp3. The spectrometer (or dichroic mirror) DM4 is configured to directly transmit the light beam traveling sequentially through optical path segments Lp1 and Lp2. This light beam is emitted after passing through the single objective lens 400, irradiates the sample to generate fluorescence, and the generated fluorescence, after passing through the single objective lens 400, travels along the optical path segment Lp3 to the spectrometer DM4 and is reflected toward the camera 510.

[0041] Figure 5A schematically shows an optical path diagram of a partial example of a fluorescence detection module 500 according to the present invention. The fluorescence detection module 500 has one optical path segment Lp4. The optical path segment Lp4 is reflected by a spectrometer DM4 and is perpendicular to optical path segment Lp3. The fluorescence detection module 500 is equipped with a camera 510 for capturing fluorescence. In a preferred embodiment, lenses L13 and L14 are positioned between the camera 510 and the spectrometer DM4 in the optical path segment Lp4, so that lenses L13 and L12 constitute a 4F system. A stepped plate 520 is positioned at the focal plane of lens L13 (the plane conjugate to the rear pupil plane of the objective lens) for the purpose of increasing the depth of field. For technical details of the stepped plate 520, please refer to disclosed technical literature such as "A new approach to extended focus for high-speed, high-resolution biological microscopy" by Sara Abrahamsson et al., Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIII, Volume 6090, 60900N (https: / / doi.org / 10.1117 / 12.647022). Downstream of the stepped plate 520 is lens L14, which together with L13 forms the 4F system, and camera 510 is positioned at the rear focal plane of lens L14. The fluorescence detection module 500 further includes an optical path segment through which 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 invention, since a single objective lens 400 is used as both the excitation objective lens and the light-receiving objective lens, the received fluorescence image is tilted. In order to receive such a tilted image, conventional light-receiving optical paths require special objective lenses and complex steering optical path designs to accommodate image formation. In the fluorescence detection module 500 of the present invention, such a complex steering optical path design can be canceled by employing a stepped plate 520. In an embodiment of an alternative detection method, as shown in Figure 5B, the concept of Fourier domain optical field imaging is used to place a microlens array 520' at the back focal plane of L13, and a camera 510 is placed behind the microlens array 520' to perform a search. The collected optical field image is then reconstructed to obtain three-dimensional information of the object and perform the search. In other alternative detection method embodiments, the idea of ​​a cone lens 520'' generating a Bessel beam is used to expand the depth of field of the image, and as shown in Figure 5C, the cone lens 520'' is positioned behind L13 depending on the actual situation, and the camera 510 performs the search directly.

[0043] As described above, the two optical sheets generated by the optical sheet generation and phase adjustment module 200 pass through the scanning module 300 and enter the single objective lens 400. Simultaneously, they intersect with each other due to the focusing action of the lenses within the single objective lens 400, and interfere at the intersection (as shown in the T region of Figures 6A, 6C, and 6E) to generate structural optical stripes. To clearly illustrate the technology of this 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 show the distribution of the optical sheets in the posterior pupil of the objective lens when the optical sheets shown in Figures 6A, 6C, and 6E are generated, respectively. When different patterns are given to the spatial light modulator SLM, they are emitted from the single objective lens 400, forming structural optical stripe regions with different field of view angles. As a result, the super-resolution single objective lens microimaging system using the optical system of this application can achieve two-dimensional or three-dimensional super-resolution.

[0044] Furthermore, in the case of two-dimensional super-resolution imaging, a sample 2000 is placed on a glass slide 1000, as shown in Figure 7A. First, the light source module 100 is activated, and the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300 are adjusted so that two optical sheets (see, for example, Figure 6A) are emitted from the single objective lens 400 to form a structured optical stripe region T1000. For example, the distribution of the two optical sheets and the posterior pupil of the objective lens at the field of view is as shown in Figure 6B, and the structured optical stripe region T1000 generated by interference after passing through the objective lens 400 can illuminate the sample 2000 on the glass slide (see Figure 7A). For example, such a structured optical stripe region T1000 can be scanned along the Y axis. The scanning process is realized by galvanometer scanners in the scanning module, for example, galvanometer scanner G1 controls scanning along the X axis, and galvanometer scanner G2 controls scanning along the Y axis. To achieve scanning along the Y-axis as described above, the galvanoscanner G1 remains fixed in its initial position, while the galvanoscanner G2 rotates at set intervals, thereby enabling movement of the structural light stripe region T1000 along the Y-direction. At each position where the angle increases, the fluorescence detection module 500 records fluorescence image data, resulting in fluorescence image data at the field of view corresponding to the structural light stripe region T1000 (e.g., the first field of view) (shown in Figure 7B). Next, the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300 are adjusted so that two optical sheets (e.g., see Figure 6C) are emitted from the single objective lens 400 to form the structural light stripe region T2000. For example, the distribution of the two optical sheets and the posterior pupil of the objective lens at the field of view is as shown in Figure 6D, and as shown in Figure 7C, the structural light stripe region T2000 generated by interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the slide glass. For example, such a structured optical stripe region T1000 can be scanned along the X-axis.To achieve this scanning, the galvanoscanner G2 remains fixed in its initial position, while the galvanoscanner G1 rotates at set intervals, thereby enabling movement of the structural light stripe region T2000 along the X direction. At each position where the angle increases, the fluorescence detection module 500 records fluorescence image data, resulting in fluorescence image data at a field of view corresponding to the structural light stripe region T2000 (e.g., a second field of view) (shown in Figure 7D). It will be apparent to those skilled in the art that a specific embodiment of the direction along which the structural light stripe region moves can be controlled by adjusting the galvanoscanners G1 and / or G2 as needed.

[0045] Finally, by fusing image data from two field angles and processing the data using a super-resolution reconstruction algorithm, an image with a super-resolution effect in the XY plane can be obtained.

[0046] Furthermore, in the case of three-dimensional super-resolution imaging, the distribution of the structural optical stripe region T1000 in the first field of view is as shown in Figure 8A. For example, the structural optical stripe region in the first field of view can be provided as shown in Figure 7A. Also, the distribution of the structural optical stripe region T3000 in the third field of view is as shown in Figure 8C. For example, by adjusting the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300, two optical sheets (see, for example, Figure 6E) are ejected from the single objective lens 400 to form the structural optical stripe region T3000. For example, the distribution of the two optical sheets and the posterior pupil of the objective lens in that field of view is as shown in Figure 6F, and as shown in Figure 8C, the structural optical stripe region T3000 generated by interference after passing through the objective lens 400 can be irradiated onto the sample 2000 on the slide glass. For example, first, the light source module 100 is activated, and the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300 are adjusted so that two optical sheets (see, for example, Figure 6A) are ejected from the single objective lens 400 to form a structured optical stripe region T1000. For example, the distribution of the two optical sheets and the posterior pupil of the objective lens at the field of view is as shown in Figure 6B, and as shown in Figure 8A, the structured optical stripe region T1000 generated by interference after passing through the objective lens 400 can be illuminated onto the sample 2000 on the slide glass. For example, such a structured optical 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, galvanometer scanner G1 controls scanning along the X axis, and galvanometer scanner G2 controls scanning along the Y axis. To achieve scanning along the Y-axis as described above, the galvanoscanner G1 remains fixed in its initial position, while the galvanoscanner G2 rotates at set intervals, thereby enabling movement along the Y-direction of the structural light stripe region T1000. At each position where the angle increases, the fluorescence detection module 500 records fluorescence image data, resulting in fluorescence image data at a field of view (e.g., a first field of view) corresponding to the structural light stripe region T1000 (shown in Figure 8B).Next, the parameters of the optical sheet generation and phase adjustment module 200 and the scanning module 300 are adjusted so that two optical sheets (see, for example, Figure 6E) are emitted from the single objective lens 400 to form a structured optical stripe region T3000. For example, the distribution of the two optical sheets and the posterior pupil of the objective lens at the field of view is as shown in Figure 6F, and as shown in Figure 8C, the structured optical stripe region T3000 generated by interference after passing through the objective lens 400 can be illuminated onto the sample 2000 on the slide glass. Unlike two-dimensional super-resolution imaging, the structured optical stripe region T3000 can be scanned along the same Y-axis, but in the opposite direction to the direction in which the structured optical stripe region T1000 moves. For example, by controlling the galvanoscanner G2 to rotate in the opposite direction, while keeping the galvanoscanner G1 fixed in its initial position, the galvanoscanner G2 records fluorescence image data at each different angle using the fluorescence detection module 500, resulting in fluorescence image data at a field of view (e.g., the third field of view) corresponding to the structural light stripe region T3000 (shown in Figure 8D). The collected images at each field of view are used to achieve two-dimensional super-resolution imaging with a one-dimensional directional structural light illumination optical sheet microscopy device using a deep learning method, and by fusing the two-dimensional super-resolution images at the two field of view, a three-dimensional super-resolution image with a super-resolution effect in the XYZ directions can be obtained. For details on the specific deep learning method, please refer to Patent Document CN113917677A.

[0047] Those skilled in the art should demonstrate that the analysis and processing of three-dimensional super-resolution microscopic images can be achieved using any three-dimensional image reconstruction method well known in the field of microscopic image processing technology, and can be performed on a computer using, for example, commercial interactive microscopic image analysis software such as Imaris or Amira.

[0048] The glass slides 1000 shown in Figures 7A, 7C and 8A, 8C can be placed on a stage. Alternatively, the stage may have a plane for placing the sample 2000 to be detected. Thus, the super-resolution microimaging system may generally include a stage and the super-resolution single-objective lens optical sheet microimaging optical system described above, where the optical axis of the single-objective lens 400 is at a 90-degree angle to the plane.

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

[0050] Furthermore, in the embodiment shown in Figure 10, compared to the embodiment shown in Figure 9, the spectrometer DM4 and associated fluorescence detection module 500 are removed. However, by placing the spectrometer DM3 between lens L9 and the first galvanoscanner G1, the optical sheet light can propagate from the first galvanoscanner G1 to lens L9. The fluorescence received by the single objective lens 400 passes through lenses L12, L11, the second galvanoscanner G2, and lenses L10, L9 before being reflected by the spectrometer DM3 toward the fluorescence detection module 600 (Figure 11). If the propagation direction of excitation light and fluorescence is not considered, 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. Figure 11 shows only the portion of the optical path of the fluorescence detection module 600 that does not overlap.

[0051] As shown in Figure 11, the fluorescence detection module 600 includes a third galvanoscanner G3 and a fourth galvanoscanner G4, with three reflectors, namely the second reflector M2, the third reflector M3, and the fourth reflector M4, positioned between the third and fourth galvanoscanners G3 and G4. Downstream of the fourth galvanoscanner 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 that order. The optical axes of objective lens OBJ2 and objective lens OBJ3 form a constant angle. As mentioned above, the fluorescence image is tilted, so the angle between the optical axes of objective lens OBJ2 and objective lens OBJ3 is configured to justify this tilt.

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

[0053] The third galvanoscanner G3, fourth galvanoscanner G4, second reflector M2, and fourth reflector M4 are configured such that when reflected fluorescence in the first field of view is received by the fluorescence detection module 600, the third galvanoscanner G3 is switched to one angle, causing the fluorescence reflected by the spectrometer DM3 to propagate through the path of the third galvanoscanner G3, second reflector M2, fourth reflector M4, and fourth galvanoscanner G4, pass through lens L15, and be received by camera 610. When reflected fluorescence in the second field of view is received by the fluorescence detection module 600, the third galvanoscanner G3 is switched to another angle, causing the fluorescence reflected by the spectrometer DM3 to propagate through the path of the third galvanoscanner G3, third reflector M3, and fourth galvanoscanner G4, pass through lens L15, and be received by camera 610. Because the fluorescence signals received at two different field of view passed through a different number of mirrors upon reaching camera 610, the imaging direction of the final fluorescence images at the different field of view coincides, eliminating the need for subsequent changes to the associated search optical path when switching between different field of view.

[0054] While specific embodiments of the present application have been described in detail herein, these are provided solely for interpretive purposes and are not intended to limit the scope of the application. Furthermore, it will be apparent to those skilled in the art that the embodiments described herein can be used in combination with one another. Various substitutions, modifications, and alterations can be conceived without departing from the spirit and scope of the application.

Claims

1. A super-resolution single objective lens optical sheet microimaging optical system, A light source module (100) configured to output a laser beam of one or more wavelengths, An optical sheet generation and phase adjustment module (200) configured to receive a laser beam of one or more wavelengths and output two optical sheets, Scanning module (300), A single objective lens (400) located downstream of the scanning module (300), wherein the scanning module (300) is configured to guide two optical sheets output from the optical sheet generation and phase adjustment module (200) to the single objective lens (400), and the single objective lens (400) is configured to receive fluorescence signals such that the two optical sheets are emitted from the single objective lens (400) and interfere with each other to generate a structured optical stripe region (T), The system comprises a first fluorescence detection module (500) configured to record the fluorescence signal received by the single objective lens (400), The optical sheet generation and phase adjustment module (200) comprises a spatial light modulator (SLM), a half-wave plate (HWP), a polarizing beam splitter (PBS), a cylindrical lens (CL3), and a mask (Mask). A super-resolution single objective lens optical sheet microimaging optical system characterized by its features.

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

3. The aforementioned fluorescence signal is a fluorescence signal generated when the structural light stripe region (T) is irradiated onto the sample to be detected. The super-resolution single objective lens optical sheet microimaging optical system according to feature 1.

4. 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 a laser beam of one or more wavelengths generates multiple stages of positive and negative optical components after passing through the phase grating. The super-resolution single objective lens optical sheet microimaging optical system according to feature 1.

5. The polarizing beam splitter (PBS) is configured to reflect the single or multiple laser beams toward the half-wave plate (HWP), pass through the half-wave plate (HWP), and 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 each of these at least two different states, the spatial light modulator (SLM) has correspondingly defined different patterns for generating multiple stages of positive and negative light components. The super-resolution single objective lens optical sheet microimaging optical system according to feature 4.

6. The mask is configured to remove the light components of other stages while retaining only the positive and negative light components of one stage, and the mask is located downstream of the cylindrical lens (CL3), and the two optical sheets are generated after the multiple stages of positive and negative light components have passed through the cylindrical lens (CL3) and the mask. The super-resolution single objective lens optical sheet microimaging optical system according to feature 5.

7. The structured optical stripe region (T) comprises a structured optical stripe region (T1000) in a first viewing angle and a structured optical stripe region (T2000 or T3000) in a second viewing angle. The state of the spatial light modulator (SLM) is as follows: A first state in which, in the first state, the spatial light modulator (SLM) has a first diffraction pattern defined such that two optical sheets for forming a structured optical stripe region (T1000) in the first field of view are output from the optical sheet generation and phase adjustment module (200), The second state includes a second state in which, in the second state, the spatial light modulator (SLM) has a second diffraction pattern defined that is different from the first diffraction pattern, such that two optical sheets for forming a structured light stripe region (T2000 or T3000) in the second field of view are output from the optical sheet generation and phase adjustment module (200), The super-resolution single objective lens optical sheet microimaging optical system according to feature 6.

8. The scanning module (300) comprises a first galvanometer scanner (G1) and a second galvanometer scanner (G2), wherein the first galvanometer scanner (G1) is arranged to receive light from two optical sheets output from the optical sheet generation and phase adjustment module (200) and reflect it toward the second galvanometer scanner (G2). The second galvanoscanner (G2) is positioned to reflect the two optical sheets toward the single objective lens (400). The super-resolution single objective lens optical sheet microimaging optical system according to feature 7.

9. The system further comprises a spectrometer (DM4) between the single objective lens (400) and the second galvanoscanner (G2), wherein the spectrometer (DM4) is configured such that the two optical sheets are passable through the spectrometer but the fluorescence signal is reflected toward the first fluorescence detection module (500), and the first fluorescence detection module (500) comprises a camera (510) for recording the fluorescence signal and a conical lens (520'') or microlens array (520'') located upstream of the camera (510). The super-resolution single objective lens optical sheet microimaging optical system according to feature 8.

10. The system further comprises a spectrometer (DM4) between the single objective lens (400) and the first galvanoscanner (G1), wherein the spectrometer (DM4) is configured such that the two optical sheets are passable through the spectrometer but the fluorescence signal is reflected toward the first fluorescence detection module (500), and the first fluorescence detection module (500) comprises a camera (510) for recording the fluorescence signal and an eighth lens (L14) and a third lens (L13) arranged between the spectrometer (DM4) and the camera (510) to form a 4F system, wherein the third lens (L13) is closer to the spectrometer (DM4) and a stepped plate (520) is provided on the focal plane of the third lens (L13). The super-resolution single objective lens optical sheet microimaging optical system according to feature 8.

11. A first lens (L12) and a second lens (L11) are arranged between the single objective lens (400) and the second galvanoscanner (G2) to form a 4F system, and the first fluorescence detection module (500) further comprises a third lens (L13) located upstream of the conical lens (520) or the microlens array, 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 to form 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 optical sheet microimaging optical system according to feature 9.

12. A second fluorescence detection module (600) is configured to record the fluorescence signal received by the single objective lens (400), The system further comprises a fourth lens (L10) and a fifth lens (L9) arranged between the second galvanoscanner (G2) and the first galvanoscanner (G1) to constitute a 4F system, wherein the second galvanoscanner (G2) is conjugate to the first galvanoscanner G1 via the fourth lens (L10) and the fifth lens (L9), and a spectrometer (DM3) is positioned between the fifth lens (L9) and the first galvanoscanner (G1), wherein the spectrometer (DM3) is configured such that the two optical sheets are passable through the spectrometer, but the fluorescence signal is reflected toward the second fluorescence detection module (600). The super-resolution single objective lens optical sheet microimaging optical system according to feature 8.

13. The second fluorescence detection module (600) comprises 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 with their optical axes at a non-zero angle to each other, and a sixth lens (L15) and a seventh lens (L16) positioned between the spectrometer (DM3) and the second objective lens (OBJ2) to form a 4F system, wherein a galvanoscanner-reflector system is positioned between the spectrometer (DM3) and the seventh lens (L16), which is close to the second objective lens (OBJ2), such that the imaging direction of the fluorescence image at different field angles coincides. The super-resolution single objective lens optical sheet microimaging optical system according to feature 12.

14. The galvanoscanner-reflector system comprises a third galvanoscanner (G3) and a fourth galvanoscanner (G4) or an additional reflector positioned between the spectrometer (DM3) and the sixth lens (L15); a second reflector (M2), a third reflector (M3), and a fourth reflector (M4) positioned between the third galvanoscanner (G3) and the fourth galvanoscanner (G4) or the additional reflector; and a fifth reflector (M5) positioned between the sixth lens (L15) and the seventh lens (L16), wherein the third galvanoscanner (G3) is configured such that fluorescence is detected in one of the first and second fields of view. The fluorescence is operable to propagate to the second objective lens (OBJ2) via the second reflector (M2), the fourth reflector (M4), the fourth galvanoscanner (G4) or an additional reflector, the sixth lens (L15), the fifth reflector (M5), and the seventh lens (L16) in sequence, in the other of the first and second fields of view, via the third galvanoscanner (G3), the third reflector (M3), the fourth galvanoscanner (G4) or an additional reflector, the sixth lens (L15), the fifth reflector (M5), and the seventh lens (L16) in sequence to the second objective lens (OBJ2). The super-resolution single objective lens optical sheet microimaging optical system according to feature 13.

15. The operation of the first galvanoscanner (G1) and / or the second galvanoscanner (G2) causes the structural optical stripe region (T1000, T2000, or T3000) to be scanned and moved. The super-resolution single objective lens optical sheet microimaging optical system according to feature 8.

16. In the case of two-dimensional super-resolution imaging, the structural light stripe region (T1000) in the first field of view and the structural light stripe region (T2000) in the second field of view are scanned and moved along directions perpendicular to each other. The super-resolution single objective lens optical sheet microimaging optical system according to feature 7.

17. In the case of three-dimensional super-resolution imaging, the structural light stripe region (T1000) in the first field of view and the structural light stripe region (T3000) in the second field of view are scanned and moved in opposite directions along the same straight line. The super-resolution single objective lens optical sheet microimaging optical system according to feature 16.

18. The optical sheet generation and phase adjustment module (200) further comprises a beam expansion filter submodule (210) for expanding and / or filtering the single or multiple wavelength laser beam, the beam expansion filter submodule (210) being configured to first incident on the single or multiple wavelength laser beam after it has entered the optical sheet generation and phase adjustment module (200). The super-resolution single objective lens optical sheet microimaging optical system according to feature 17.

19. The mask and the first galvanoscanner (G1) are arranged in a conjugate relationship. The super-resolution single objective lens optical sheet microimaging optical system according to feature 18.

20. Between the mask and the first galvanoscanner (G1), and / or between the first galvanoscanner (G1) and the second galvanoscanner (G2), and / or between the second galvanoscanner (G2) and the single objective lens (400), two lenses are arranged to constitute a 4F system. The super-resolution single objective lens optical sheet microimaging optical system according to feature 19.

21. A super-resolution single objective lens optical sheet microimaging system, A stage having a flat surface for placing the sample to be detected, The system comprises a super-resolution single-objective lens optical sheet microimaging optical system according to any one of claims 1 to 20, wherein the optical axis of the single objective lens (400) forms a 90-degree angle with the plane, A super-resolution single-objective lens optical sheet microimaging system characterized by its features.

Images