Super-resolution light-sheet fluorescence microscopy system and method

Abstract

A super-resolution light-sheet fluorescence microscopy method, comprising: using reversibly photoswitchable fluorescent proteins (RSFPs) to process a biological sample (1000) to be tested; using a light sheet as activation light to be incident onto said biological sample (1000), so as to generate an activated portion in the reversibly photoswitchable fluorescent proteins (RSFPs); using modulated striped structured light as excitation light to be incident onto the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs) towards a surface where the activated portion is located, such that the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs) on a plane where the light sheet is incident emits fluorescence, wherein the activation light and the excitation light have different wavelengths; and collecting a fluorescence image with structured light stripe information as a two-dimensional fluorescence image for super-resolution image processing. Further provided is a super-resolution light-sheet fluorescence microscopy system.

Description

Super-resolution light-sheet fluorescence microscopy system and method Technical Field

[0001] The present application generally relates to the technical field of super-resolution microscopy, and more particularly to two-dimensional or three-dimensional super-resolution light sheet fluorescence microscopy systems and methods. Background Art

[0002] In recent years, the demand for high-resolution three-dimensional imaging technology in life science research has been increasing. Achieving high-resolution, high-speed, low-damage, and long-duration three-dimensional imaging of microscopic life activities has important practical significance and broad application prospects for exploring the mechanisms of life activities and understanding physiological and pathological information.

[0003] Optical fluorescence microscopy, as an important observational technique, has provided strong support for the development of life science research. Light-sheet fluorescence microscopy (LSFM), a type of optical fluorescence microscopy, has gained popularity in the field of three-dimensional imaging in recent years. Unlike traditional three-dimensional imaging techniques (such as laser scanning confocal microscopy and multiphoton fluorescence microscopy), LSFM utilizes a thin light sheet for surface excitation and detection in a direction perpendicular to the light sheet. This surface excitation imaging is faster than traditional point scanning imaging and, when combined with high-precision devices such as high-speed scanning galvanometers and piezoelectric stages, enables rapid three-dimensional volumetric imaging.

[0004] However, the imaging resolution of light-sheet fluorescence microscopy itself is limited and it is difficult to be compatible with super-resolution microscopy technology. This is mainly because (1) in the light-sheet fluorescence microscopy system, the physical arrangement of the excitation objective and the detection objective, which are perpendicular to each other, requires the detection objective to have a long working distance to achieve imaging. This limits the selection range of the numerical aperture (NA) of the detection objective, resulting in the need to use a detection objective with a lower NA as the detection objective of the light-sheet fluorescence microscopy system, thereby affecting the two-dimensional resolution of the final fluorescence image, which is even lower than the two-dimensional resolution of the image obtained by traditional confocal microscopy. For example, the lateral resolution of light-sheet fluorescence microscopy is 350-500 nanometers, while the lateral resolution of confocal microscopy can reach 200 nanometers; (2) the imaging field of traditional light-sheet fluorescence microscopy and the axial resolution of three-dimensional imaging are mutually restricted. In order to achieve uniform light-sheet illumination over a large range, it is necessary to use an objective with a smaller NA to generate the light sheet, but this will result in a thicker light sheet, and the background signal in the final fluorescence image will be undesirably enhanced, thereby reducing the axial resolution of subsequent three-dimensional imaging. In addition, if structured illumination microscopy (SIM) is directly used in traditional light-sheet fluorescence microscopy, it can only improve the lateral resolution in a single direction in the final two-dimensional fluorescence image. To achieve isotropic lateral resolution improvement (i.e., two-dimensional resolution improvement), the direction of the structured light illumination stripes needs to be changed, which is difficult to achieve in traditional light-sheet fluorescence microscopy systems where the excitation and detection objectives are perpendicular to each other.

[0005] Summary of the Invention

[0006] The present application aims to propose a novel super-resolution light-sheet fluorescence microscopy system, which can overcome the shortcomings of the above-listed traditional light-sheet fluorescence microscopy systems and realize super-resolution microscopy, which can effectively improve the two-dimensional resolution of the acquired fluorescence images and the three-dimensional resolution of the acquired fluorescence images.

[0007] According to one aspect of the present application, a super-resolution light sheet fluorescence microscopy method is provided, comprising:

[0008] Using reversibly photoswitchable fluorescent proteins to process biological samples;

[0009] The light sheet is used as an activation light surface to illuminate the biological sample to be tested, so as to generate an activation portion in the reversibly photoswitchable fluorescent protein thereof;

[0010] allowing the modulated stripe structured light as excitation light to be incident on the activated portion of the reversibly photoswitchable fluorescent protein toward the plane where the activated portion is located, so that the activated portion of the reversibly photoswitchable fluorescent protein emits fluorescence on the plane where the light sheet is incident, wherein the activation light and the excitation light have different wavelengths;

[0011] A fluorescence image with structured light fringe information is collected as a two-dimensional fluorescence image for super-resolution image processing.

[0012] Optionally, when considering using a linear super-resolution reconstruction algorithm for super-resolution image reconstruction, for each part of the reversibly photoswitchable fluorescent protein to be activated by the activation light sheet, the activation light sheet is first incident and then the modulated stripe structured light is incident N·M times, where N and M are both integers greater than or equal to 3, and the stripe directions of the modulated stripe structured light incident each time differ from each other by 180 / M degrees when observed in the plane where the light sheet is located, and the stripe phases differ by 2π / N.

[0013] Optionally, after each time the modulated stripe structured light is incident on the activation part of the reversible light-switch fluorescent protein as excitation light, a stripe structured light with the same stripe direction as the modulated stripe structured light but a phase difference of π is incident on the activation part of the reversible light-switch fluorescent protein as excitation light.

[0014] Optionally, when a nonlinear super-resolution reconstruction algorithm is used for super-resolution image reconstruction, N and M are at least 5.

[0015] Optionally, the light sheet is formed by dithering a single Bessel beam or a multi-beam interference lattice.

[0016] Optionally, a light sheet is generated by using a single Bessel beam at a near-infrared wavelength as activation light and incident on a biological sample in a manner that induces a two-photon effect in the reversibly photoswitchable fluorescent protein.

[0017] Optionally, after the light sheet serving as activation light has been incident on the biological sample and before the modulated stripe structured light serving as excitation light is incident on the activation portion of the reversibly photoswitchable fluorescent protein, a light sheet for thinning formed by light having the wavelength of the excitation light is used to partially overlap on both sides of the activation portion to reduce the thickness of the activation portion.

[0018] Optionally, the relative position between the light sheet serving as the activation light and the biological sample changes at least in a direction perpendicular to the plane in which the light sheet is located, thereby allowing the modulated stripe structured light serving as the excitation light to be incident on the activation part of the reversibly photoswitchable fluorescent protein, stimulating fluorescence and collecting a fluorescence image with structured light stripe information as a three-dimensional fluorescence image stack for super-resolution image processing.

[0019] Optionally, the relative position between the light sheet and the biological sample is changed by moving the light sheet itself or by moving the biological sample.

[0020] According to another aspect of the present application, a super-resolution light sheet fluorescence microscopy system is provided, comprising:

[0021] Light source module;

[0022] Laser gating and splitting modules;

[0023] a first light sheet generating module;

[0024] Light sheet scanning module;

[0025] Activate the objective lens;

[0026] Structured light generation and polarization modulation module;

[0027] Detection objective lens;

[0028] a fluorescence detection module; and

[0029] Sample placement mobile module,

[0030] The first light sheet generation module, the light sheet scanning module, and the activation objective lens constitute a first light sheet activation optical path; the structured light generation and polarization modulation module, the fluorescence detection module, and the detection objective lens constitute a structured light excitation and detection optical path; and the laser gating and light splitting module is configured so that the laser beam emitted by the light source module selectively enters the first light sheet activation optical path or the structured light excitation and detection optical path;

[0031] The first light sheet activation light path is configured to generate a light sheet as activation light incident on the biological sample to be tested treated with the reversibly photoswitchable fluorescent protein to generate an activation portion in the reversibly photoswitchable fluorescent protein;

[0032] The structured light excitation and detection optical path is configured to generate modulated stripe structured light as excitation light incident on the activated portion of the reversibly photoswitchable fluorescent protein, so that the activated portion of the reversibly photoswitchable fluorescent protein emits fluorescence in a plane incident on the light sheet, wherein the activation light and the excitation light have different wavelengths;

[0033] The structured light excitation and detection optical path is further configured to collect a fluorescence image with structured light fringe information as a two-dimensional fluorescence image for super-resolution image processing.

[0034] Optionally, the first light sheet generating module comprises a first galvanometer mirror, and the light sheet is formed by shaking a single Bessel beam or a multi-beam interference lattice via the first galvanometer mirror.

[0035] Optionally, the system further includes:

[0036] a second light sheet generating module capable of emitting a laser beam independently of the light source module;

[0037] The second light sheet generation module, the first galvanometer of the first light sheet generation module, the light sheet scanning module and the activation objective lens constitute a second light sheet activation optical path.

[0038] The second light sheet generating module includes:

[0039] A laser capable of generating near-infrared wavelength laser light independent of the light source module;

[0040] a pair of lenses located downstream of the laser; and

[0041] A submodule or annular mask consisting of an axicon and a lens, the submodule or annular mask being located downstream of the pair of lenses. The submodule or annular mask is configured such that after the laser emits near-infrared wavelength laser light, the light is first collimated and expanded by the pair of lenses and then passes through the submodule or annular mask to generate an annular light beam, and the annular light beam forms a single Bessel light beam after passing through the activation objective lens. Simultaneously, the single Bessel light beam generates a light sheet as activation light through the vibration of the first galvanometer of the first light sheet generating module, which is incident on the biological sample in a manner that causes a two-photon effect in the reversibly photoswitchable fluorescent protein.

[0042] Optionally, after the light sheet serving as activation light has been incident on the biological sample and before the modulated stripe structured light serving as excitation light is incident on the activation portion of the reversibly photoswitchable fluorescent protein, a light sheet for thinning formed by light having the wavelength of the excitation light is used to partially overlap on both sides of the activation portion to reduce the thickness of the activation portion.

[0043] Optionally, the relative position between the light sheet serving as the activation light and the biological sample changes at least along the optical axis of the detection objective lens, so that the modulated stripe structured light serving as the excitation light is incident on the activation part of the reversibly photoswitchable fluorescent protein, fluorescence is excited, and a fluorescence image with structured light stripe information is collected as a three-dimensional fluorescence image stack for super-resolution image processing.

[0044] Optionally, the light sheet scanning module is configured to shift only the light sheet serving as activation light and synchronously move the detection objective lens along its optical axis or to move only the sample placement movement module, thereby changing the relative position between the light sheet and the biological sample at least along the optical axis of the detection objective lens.

[0045] By adopting the above-mentioned technical means of the present application, it is possible to obtain a two-dimensional fluorescence image or a three-dimensional fluorescence image stack that is helpful for realizing super-resolution microscopy. In the process of exciting and detecting the two-dimensional fluorescence image, a multi-directional and multi-phase excitation method of structured light stripes is adopted, which results in a significant improvement in the isotropic lateral resolution of the super-resolution image obtained after super-resolution reconstruction of the final two-dimensional fluorescence image. At the same time, by using the method of thinning the thickness of the activated part, the z-axis resolution of the three-dimensional fluorescence image stack can also be significantly improved. In addition, by adopting the technical means of the present application, the penetration depth of the biological tissue by the activation light sheet with a near-infrared wavelength is greatly improved and the damage to the biological tissue is reduced, which is helpful for the two-dimensional or three-dimensional observation of the biological tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following detailed description and the accompanying drawings will provide a more comprehensive understanding of the principles and various aspects of this application. It should be noted that the scales of the various drawings may vary for the purpose of clarity, but this does not affect the understanding of this application. In the accompanying drawings:

[0047] FIG1 schematically shows a framework diagram of a super-resolution light sheet microscopy imaging system according to an embodiment of the present application;

[0048] FIG2 schematically shows a light path diagram of a super-resolution light sheet microscopy imaging system according to an embodiment of the present application, corresponding to FIG1 ;

[0049] FIG3 schematically shows a light path diagram of an example of a light source module of a super-resolution light sheet microscopy imaging system according to the present application;

[0050] FIG4 schematically shows a light path diagram of a second light sheet generating module according to an embodiment of the present application;

[0051] FIG5 schematically illustrates a principle diagram of super-resolution light sheet fluorescence microscopy imaging of a biological sample according to one embodiment of the present application, wherein the obtained fluorescence image is super-resolved using a linear super-resolution reconstruction algorithm;

[0052] 6A , 6B, and 6C schematically illustrate comparisons of frequency domain expansion of stripe structured light as excitation light in conventional wide-field illumination and in a super-resolution light sheet fluorescence microscopy imaging method according to an embodiment of the present application;

[0053] FIG7 schematically shows a principle diagram of super-resolution light sheet fluorescence microscopy imaging of a biological sample according to another embodiment of the present application, wherein the acquired fluorescence image is super-resolved using a nonlinear super-resolution reconstruction algorithm;

[0054] 8A , 8B, and 8C schematically illustrate comparisons of frequency domain expansion of stripe structured light as excitation light in conventional wide-field illumination and in a super-resolution light sheet fluorescence microscopy imaging method according to an embodiment of the present application;

[0055] FIG9 schematically shows a principle diagram of a method for thinning an activated portion of a reversibly photoswitchable fluorescent protein using a light sheet for thinning according to an embodiment of the present application. DETAILED DESCRIPTION

[0056] In the various figures of this application, features with the same structure or similar functions are indicated by the same reference numerals.

[0057] FIG1 schematically shows a framework diagram of a super-resolution light sheet microscopy imaging system according to an embodiment of the present application. The super-resolution light sheet microscopy imaging system generally includes a light source module 100, a laser gating and light splitting module 200, a first light sheet generation module 300, a second light sheet generation module 110 capable of emitting a laser beam independently of the light source module (100), a light sheet scanning module 400, an activation objective lens 500, a structured light generation and polarization modulation module 600, a detection objective lens 700, a fluorescence detection module 800, and a sample placement and movement module 900.

[0058] FIG2 schematically shows an optical path diagram of a super-resolution light sheet microscopy imaging system according to an embodiment of the present application, corresponding to FIG1 . In the optical path, a sample placement and movement module 900 and a biological sample 1000 placed thereon are simplified.

[0059] The light source module 100 is configured to emit collimated laser beams of different wavelengths. For example, FIG3 schematically shows an optical path diagram of the light source module 100 according to an embodiment of the present application. The light source module 100 may include a plurality of lasers, for example, three lasers Laser1, Laser2, and Laser3 are shown. These can respectively emit laser beams of different wavelengths, such as laser beams of 405 nm (nanometers), 445 nm, and 488 nm wavelengths. In an alternative embodiment, the light source module 100 may include more lasers so that they can emit laser beams of more wavelengths, such as laser beams of wavelengths such as 405 nm, 445 nm, 488 nm, 561 nm, and 590 nm.

[0060] In the example shown, each laser Laser1, Laser2, Laser3 is equipped with a group of lenses L18, L19 and L20, L21 and L22, L23, so that the laser beams emitted from lasers Laser1, Laser2, Laser3 are expanded and collimated into parallel lights of equal diameter through the corresponding lens groups. The reflector M5 and the dichroic mirrors DM5, DM6 combine the three parallel lights of equal diameter from different lasers Laser1, Laser2, Laser3 into one laser beam as the laser beam output of the light source module 100.

[0061] As shown in the figure, the laser gating and splitting module 200 according to the present application mainly includes an acousto-optic tunable filter AOTF (Acousto-Optic Tunable Filter), a half-wave plate HWP (Half Wave Plate), and a polarizing beam splitter PBS (Polarizing Beam Splitter), which are arranged in sequence on the optical path of the laser beam output by the light source module 100, so that the laser beam output by the light source module 100 can be incident on the acousto-optic tunable filter AOTF at a suitable angle and direction. The acousto-optic tunable filter AOTF is configured to selectively allow light of a specific wavelength to pass through and can control the output optical power. Therefore, in an embodiment of the present application, since the laser beam contains lasers of different wavelengths, the laser beam can ensure that the laser of a specific wavelength can be output as needed after passing through the acousto-optic tunable filter AOTF. In this way, the laser light of a specific wavelength is adjusted by the half-wave plate HWP and then split by the polarization beam splitter prism PBS, and respectively enters the first light sheet activation optical path composed of the first light sheet generation module 300, the light sheet scanning module 400 and the activation objective lens 500, and the structured light excitation and detection optical path composed of the structured light generation and polarization modulation module 600, the detection objective lens 700, and the fluorescence detection module 800.

[0062] It should be noted that the control of optical components such as the acousto-optic tunable filter (AOTF) of the super-resolution light sheet microscopy system described in this application specification can be achieved via a computer in any suitable manner familiar to those skilled in the art, and will not be described in detail here.

[0063] As shown in the figure, the light sheet generation module 300 mainly includes a lens L119, cylindrical lenses CL1 and CL2, a polarization beam splitter prism PBS, a spatial light modulator SLM, a half-wave plate HWP, and an annular mask mask 1. In the optical path diagram introducing the present application, relevant lenses such as L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L17, L18, L19, L20, L21, L22, L23, L119, etc. and reflectors such as M1, M2, and M3 are also essential components of the optical system of the present application. However, those skilled in the art will understand the conventional configuration and function of these lenses and reflectors in the optical system when observing the optical path diagram, so their description will be omitted herein. For example, when the lenses involved below in this article are understood to have a relay function with reference to the accompanying drawings, those skilled in the art should be aware that the number of lenses can be increased or decreased as needed, and the explanation of these lenses having a relay function will be omitted.

[0064] Lens L119 and the two cylindrical lenses CL1 and CL2 are configured to stretch the parallel light from the laser gating and light splitting module 200 in two mutually perpendicular directions, causing the cylindrical lens CL2 to emit rectangular parallel light. Therefore, it can be considered that lens L119 and the two cylindrical lenses CL1 and CL2 constitute a subsystem that converts a circular light beam into rectangular parallel light. Of course, those skilled in the art will appreciate that other suitable beam expansion optical systems capable of stretching parallel light in two directions can also be used as the beam expansion subsystem in the technical solution of this application.

[0065] In the first light sheet activation optical path, the spatial light modulator (SLM) in the light sheet generation module 300 can be a binary spatial light modulator. For example, the spatial light modulator (SLM) can include a liquid crystal panel composed of multiple liquid crystal pixels, each of which can be in either an on or off state. Under computer control, each pixel on the spatial light modulator (SLM) can be programmed to change state, generating different diffraction patterns (i.e., patterns formed by the computer-controlled liquid crystal pixels on the spatial light modulator (SLM) changing their on or off states) through a designed algorithm. As a result, light entering the spatial light modulator (SLM) and passing through lens L3 and an annular mask (mask1) generates an annular beam. In an alternative embodiment, the pattern can also be generated using other suitable optical devices, such as a digital micromirror device (DMD).

[0066] The annular mask plate mask1 is engraved with concentric annular holes of different inner and outer diameters. The annular mask plate mask1 is arranged to be conjugate with the back pupil of the activated objective lens 500 after passing through three groups of relay lenses L4, L5, L6, L7, L8, and L9, and the annular holes of different inner and outer diameters of the annular mask plate mask1 correspondingly generate annular beams of different numerical apertures at the back pupil of the activated objective lens 500. As a result, different lattice beams or single Bessel beams are generated at the focal plane of the activated objective lens 500. The annular mask plate mask1 is placed at the focal position of the light beam, and the center of the annular hole of the annular mask plate mask1 is located on the optical axis.

[0067] As shown in the figure, light sheet generation module 300 also includes a first galvanometer Galvo1, and lenses L4 and L5. The first galvanometer Galvo1 is arranged in a conjugate relationship with the annular mask mask1, and lenses L4 and L5 are arranged between the first galvanometer Galvo1 and the annular mask mask1 to form a 4F system. Persons skilled in the art can refer to any technical references known in the art, including technical dictionaries, manuals, and textbooks, for any references to optical terms such as "galvanometer (or laser galvanometer)," "conjugate," and "4F," or their operating principles, in this specification.

[0068] As shown in the figure, the light sheet scanning module 400 includes a second galvanometer mirror Galvo2, and lenses L6, L7, L8, and L9. The first galvanometer mirror G1 and the second galvanometer mirror G2 are also arranged in a conjugate relationship. Lenses L6 and L7 are also arranged between the first galvanometer mirror G1 and the second galvanometer mirror G2 to form a 4F system. Furthermore, the second galvanometer mirror G2 is also arranged in a conjugate relationship with the back pupil of the active objective lens 500. Lenses L8 and L9 are also arranged between the second galvanometer mirror G2 and the back pupil of the active objective lens 500 to form a 4F system.

[0069] The technical solution of the present application generates a light sheet by dithering a single Bessel beam or a multi-beam interference lattice. For example, the first galvanometer Galvo1 of the light sheet generation module 300 is configured to rotate around an axis as needed and / or the second galvanometer Galvo2 of the light sheet scanning module 400 is also configured to rotate around an axis as needed. For example, the first galvanometer Galvo1 rotates around the axis by a corresponding angle under the drive of a computer-controlled motor (not shown). For example, when only the first galvanometer Galvo1 rotates forward and backward around an axis, by dithering the first galvanometer Galvo1 (i.e., rotating in both forward and reverse directions around an axis), the single Bessel beam or the multi-beam interference lattice moves along the x-axis to generate a light sheet parallel to the xy plane, or a "virtual light sheet." Compared with the traditional method of generating a light sheet using only a cylindrical lens, the light sheet generated by dithering a single Bessel beam or a multi-beam interference lattice in the present application is thinner, resulting in a higher z-axis resolution of the three-dimensional fluorescence image stack.

[0070] In an alternative embodiment, the light sheet can also be generated by utilizing the optical property of a cylindrical lens that acts on light in one dimension.

[0071] The light sheet can be incident on the biological sample placed on the sample placement and movement module 900 via the objective lens (in the technical solution of the present application, via the activation objective lens 500). If necessary, the second galvanometer Galvo2 can be rotated to enable the light sheet to move along the optical axis of the detection objective lens 700. At the same time, the detection objective lens 700 is synchronously moved along its optical axis, i.e., the z-axis, to ensure real-time focusing. In an alternative embodiment, the light sheet can be kept stationary and only the sample placement and movement module 900 can be translated. Although the light sheet does not move, the relative position between the sample and the light sheet at least changes along the optical axis of the detection objective lens 700.

[0072] As shown in the figure, the second light sheet generating module 110 mainly includes a laser L which is independent of the light source module 100 and can generate near-infrared wavelength laser light. nr , placed in the laser L nr A pair of lenses L16 and L17 downstream, an axicon located downstream of the pair of lenses L16 and L17, and a lens L118 and a dichroic mirror DM1 located downstream of the axicon lens Axicon in sequence, wherein the dichroic mirror DM1 is arranged in the optical path between the mask plate mask1 and the first galvanometer Galvo1 of the light sheet generation module 300, so that the light beam from the lens L3 can directly pass through the dichroic mirror DM1 after passing through the mask plate mask1, but the light beam from the lens L118 continues to propagate after being reflected toward the lens L4 via the dichroic mirror DM1.

[0073] Laser L nr Independent of the light source module 100. nr The generated laser beam, collimated and expanded by lenses L16 and L17, passes through the axicon lens and then lens L118, generating a parallel annular beam. After passing through lenses L4 and L5 of the light-sheet generation module 300, the first galvanometer Galvo 1, and the light-sheet scanning module 400, this parallel annular beam emerges from the activated objective lens 500 as a Bessel beam due to the conjugate design of the back focal plane of the axicon lens and the focal plane of the activated objective lens 500.

[0074] Figure 4 schematically illustrates an optical path diagram of a second light-sheet generation module 110 according to another embodiment of the present application. This second light-sheet generation module 110 differs from the second light-sheet generation module shown in Figure 2 in that the axicon lens and lens L118 are replaced by an annular mask Am. This allows a collimated near-infrared laser beam to directly generate a parallel annular beam after passing through the annular mask Am. After passing through lenses L4 and L5, the first galvanometer Galvo 1, and the light-sheet scanning module 400 of the light-sheet generation module 300, this parallel annular beam emerges as a Bessel beam from the activated objective lens 500.

[0075] Therefore, it can be considered that the second light sheet generation module 110, the first galvanometer Gavol1 of the light sheet generation module 300, the light sheet scanning module 400, and the activation objective lens 500 constitute the second light sheet activation light path. Although the second light sheet activation light path partially overlaps with the first light sheet activation light path in terms of the composition of optical devices, the two light paths can only work in one of the two modes, that is, to generate an activation light sheet.

[0076] As shown in the figure, the structured light generation and polarization modulation module 600 mainly includes a spatial light modulator SLM, a half-wave plate HWP, a polarization beam splitter prism PBS, a mask mask2, and a half-wave plate HWP2 located downstream of the mask mask2. It should be clear to those skilled in the art that the spatial light modulator SLM can also be replaced by other suitable optical devices such as a digital micromirror device (DMD).

[0077] According to the technical solution of the present application, the spatial light modulator SLM of the light sheet generation module 300 and / or the spatial light modulator SLM of the structured light generation and polarization modulation module 600 can be configured to control the on and off of the first light sheet activation optical path and / or the structured light excitation and detection optical path as needed, that is, by controlling the corresponding spatial light modulator SLM to load the corresponding pattern to control whether the laser beam can be emitted from the spatial light modulator SLM and pass through the mask templates mask1 and mask2 to the downstream of the optical path.

[0078] Furthermore, in the illustrated example, the spatial light modulator (SLM) in the structured light generation and polarization modulation module 600 can be a binary spatial light modulator, and the half-wave plate (HWP) in the structured light generation and polarization modulation module 600 can be an achromatic half-wave plate. The spatial light modulator (SLM), the half-wave plate (HWP), and the polarization beam splitter (PBS) can form a phase grating. After being reflected by the polarization beam splitter (PBS), the parallel light is linearly polarized, rotated by π / 8 by the half-wave plate (HWP), and then incident on the spatial light modulator (SLM). For example, a spatial light modulator (SLM) may include a liquid crystal panel composed of multiple liquid crystal pixels, each of which can be in either an on or off state. Thus, under computer control, each pixel on the SLM can be programmed to change state, thereby rotating the polarization direction of the incident light leftward or rightward by π / 4. This polarization direction is then rotated again by π / 8 after passing through a half-wave plate (HWP). The polarization states of the two linearly polarized lights reflected from the SLM form angles of ±45° with the output polarization direction of the spatial light modulator (PBS). Finally, after reflection from the spatial light modulator (PBS), the corresponding on and off pixels on the SLM are modulated to have a phase difference of π. For the reasons described above, the spatial light modulator (SLM), the half-wave plate (HWP), and the polarization beam splitter (PBS) form a phase grating. By designing algorithms to generate different diffraction patterns (i.e., patterns formed by the computer-controlled on and off states of the liquid crystal pixels on the SLM), desired sine or cosine structured light fringes of varying directions and phases can be generated on a biological sample.

[0079] In addition, a mask plate mask2 is arranged at the focal position of the light beam, and the mask plate mask2 is configured to retain only the positive and negative first-order light components and filter out the light components of other orders. Unlike the mask plate mask1, the mask plate mask2 can be configured to have a plurality of through holes distributed around a center concentric with the optical axis, such as 6 through holes or 10 through holes. These through holes are evenly spaced in the circumferential direction and aligned in pairs along the radial direction. When the laser beam passes through the above-mentioned phase grating in the structured light excitation and detection light path, two laser beams are generated that can pass through one group of through holes in these through hole groups. After passing through the half-wave plate HWP2, the two laser beams are emitted from the detection objective lens 700 to form two light spots, which are incident toward the surface where the part activated by the activated light sheet is located and interfere with each other to generate stripe structured light.

[0080] The half-wave plate HWP2 located downstream of the mask mask2 can be divided into multiple regions (e.g., 6 or 10 regions), each symmetrically distributed. The fast axis directions of the liquid crystal molecules in each region are aligned, but the principal optical axes of each region are different. This zone-by-zone design of the half-wave plate HWP2 allows the polarization direction of linearly polarized light incident on different regions to change differently, thereby achieving the same λ / 2 retardation across the entire half-wave plate HWP2. These regions correspond to the through-holes of each group of mask mask2, respectively. This ensures that the polarization directions of the two beams of light passing through any group of through-holes, after passing through the half-wave plate HWP2, dichroic mirror DM2, lenses L14 and L15, and detection objective lens 700, are perpendicular to the direction connecting the two light spots illuminated by the detection objective lens 700, achieving the most efficient fringe modulation on the surface of the biological sample 1000. Those skilled in the art will appreciate that other optical modules capable of generating modulated fringe structured light can also be used as the structured light generation and polarization modulation module 600 in the technical solution of this application.

[0081] As shown in the figure, the fluorescence detection module 800 primarily comprises a dichroic mirror DM2, a camera, and a reflector M3. According to the technical solution of this application, the modulated structured light fringes output by the structured light generation and polarization modulation module 600 are illuminated by the detection objective lens 700 onto the surface of the biological sample. The fluorescence is then collected by the same detection objective lens 700, passing through lens L15, reflector M3, lens L14, dichroic mirror DM2, and lens L13 before entering the camera. This fluorescence image, containing the structured light fringes, is then reconstructed using a structured light super-resolution reconstruction algorithm to produce a super-resolution image.

[0082] On the basis of obtaining a two-dimensional fluorescence image containing structured light stripe information, a two-dimensional super-resolution image can be reconstructed through a structured light two-dimensional super-resolution reconstruction algorithm; and after obtaining a three-dimensional fluorescence image stack, a super-resolution image stack can be reconstructed through a reconstruction algorithm. It should be clear to those skilled in the art that the super-resolution reconstruction algorithm can be implemented with any reconstruction method well known in the field of microscopic image processing technology. For example, commercial software Matlab can be used to implement image reconstruction, and commercial interactive microscopic image analysis software such as Imaris and Amira can be used to implement image display and analysis on a computer. Therefore, it is not within the scope of discussion in this article. One of the main purposes of the super-resolution light sheet fluorescence microscopy imaging system of the present application is to obtain fluorescence images (two-dimensional fluorescence images or three-dimensional fluorescence image stacks) that can be used to implement super-resolution reconstruction.

[0083] The present application proposes to introduce reversibly photoswitchable fluorescent proteins RSFPs (Reversibly Photoswitchable Fluorescent Proteins) into the biological sample to be observed, so as to realize super-resolution light sheet fluorescence microscopy. The introduction of reversibly photoswitchable fluorescent proteins into biological samples can be achieved by any suitable method known in the art, such as transfection. Reversibly photoswitchable fluorescent proteins RSFPs are fluorescent proteins that can only be stimulated to produce fluorescence after activation. For example, reversibly photoswitchable fluorescent proteins RSFPs can be activated under irradiation with activating light of a first wavelength (or activation wavelength) and are in an activated state. Then, after irradiation with excitation light of a second wavelength (or excitation wavelength) different from the first wavelength, fluorescence is stimulated to be produced and the cells are in a bright state. After emitting fluorescence, the reversibly photoswitchable fluorescent proteins RSFPs will immediately change to an inactive state, i.e., a dark state. In the dark state, even if the reversibly photoswitchable fluorescent proteins RSFPs are exposed to light of a non-activating wavelength, fluorescence will not be stimulated. When irradiated with activation light of an activation wavelength again, this reversible light-switch fluorescent protein RSFPs can be in an activated state again, waiting to be irradiated with excitation light of an excitation wavelength to stimulate fluorescence and become in a bright state again.

[0084] Depending on the physical or chemical properties of reversibly photoswitchable fluorescent proteins (RSFPs), different types of reversibly photoswitchable fluorescent proteins (RSFPs) may have different activation or excitation wavelengths. In the embodiments described herein, the activation wavelength of the reversibly photoswitchable fluorescent proteins (RSFPs) used is illustratively selected to be 405 nm, and the excitation wavelength of the excitation light is illustratively selected to be 488 nm.

[0085] FIG5 schematically shows a principle diagram of super-resolution light sheet fluorescence microscopy imaging of a biological sample according to an embodiment of the present application. This method can be implemented using the super-resolution light sheet fluorescence microscopy imaging system described above.

[0086] As shown, a biological sample 1000, treated with reversibly photoswitchable fluorescent proteins (RSFPs), is placed on the stage of a sample placement and movement module 900. According to the technical solution of the present application, the sample placement and movement module 900 can be configured to translate in three dimensions as needed. For clarity, FIG4 illustrates a three-dimensional Cartesian coordinate system, where the x-axis is perpendicular to the paper or screen, the y-axis is parallel to the optical axis of the activation objective lens 500, and the z-axis is parallel to the optical axis of the detection objective lens 700.

[0087] First, after the activation objective lens 500 emits a single Bessel beam or a multi-beam interference lattice at the activation wavelength, it is vibrated by the first galvanometer Galvo 1 to form an activation light sheet. This activation light sheet is primarily located in a plane parallel to the xy plane and is incident on the biological sample 1000, thereby activating the portion of reversibly photoswitchable fluorescent proteins (RSFPs) in the biological sample 1000 illuminated by the activation light sheet. Therefore, the incidence of the activation light sheet on the biological sample 1000 can be considered to be a form of surface incidence.

[0088] For example, emitting a single Bessel beam or a multi-beam interference lattice at the activation wavelength from the activation objective lens 500 can be achieved by controlling the acousto-optic tunable filter (AOTF) of the laser gating and spectroscopic module 200 of the super-resolution light sheet microscopy imaging system shown in FIG2 to allow only a 405 nm collimated laser beam to pass through, and controlling the spatial light modulator (SLM) to allow only the light sheet activation optical path to be conductive. Thus, as previously described, the collimated laser beam at the activation wavelength, after passing through the light sheet activation optical path, is emitted from the activation objective lens 500 as a single Bessel beam or a multi-beam interference lattice. As the light sheet generation module 300 operates (vibrates the first galvanometer Galvo 1), an activation light sheet is generated.

[0089] After generating an activation light sheet to illuminate the biological sample 1000 to activate the reversibly photoswitchable fluorescent proteins RSFPs, the detection objective lens 700 emits a modulated stripe structured light of an excitation wavelength to illuminate the portion of the biological sample 1000 that has been activated, thereby exciting the portion to produce fluorescence. The fluorescence is then collected and imaged by the detection objective lens 700 via the fluorescence detection module 800. This process can be called fluorescence detection of the reversibly photoswitchable fluorescent proteins RSFPs.

[0090] Taking the mask mask2 with 6 through holes as an example, according to optical principles, these 6 through holes can be formed into three groups of through hole pairs, each group having an angle of 180° / 3=60°, and each pair of through holes is aligned with each other along the radial direction.

[0091] Each time the reversibly photoswitchable fluorescent proteins (RSFPs) are activated by the activation light sheet and illuminated with excitation light, the two laser beams emitted by the spatial light modulator (SLM) to form the stripe structured light are controlled to pass through different pairs of through-holes in the three pairs of through-holes of mask 2. This allows three fluorescence images with different stripe directions to be obtained for the same plane (e.g., parallel to the xy plane) where the reversibly photoswitchable fluorescent proteins (RSFPs) are activated (e.g., corresponding to the three fluorescence images in the stripe directions shown in the lower portion of Figure 5). For conventional stripe structured light excitation, if the stripe information does not contain high-frequency information, the fluorescence image is processed using only a linear super-resolution reconstruction algorithm. In this case, for each stripe direction, the spatial light modulator (SLM) is used to control the generation of three stripe structured lights with different stripe phases as the excitation light, and three fluorescence images are obtained (each fluorescence image carries a different phase of the stripe information). Thus, a total of nine fluorescence images containing stripe information are obtained. The phase difference between the stripes is the same, 2π / 3; the difference between the stripe directions is also the same, 180° / 3 = 60°. The stripe information contained in these fluorescence images can significantly improve the isotropic lateral resolution of the final image through subsequent two-dimensional super-resolution image processing.

[0092] Figures 6A, 6B, and 6C schematically illustrate spectrum diagrams comparing the excitation structured light fringes of conventional wide-field illumination and the super-resolution light sheet fluorescence microscopy imaging method of an embodiment of the present application. In Figure 6A, a spectrum diagram of fluorescence image information of conventional wide-field illumination is schematically shown, where wide-field means that the mask mask2 shown in Figure 2 is replaced by a mask plate with a central circular hole, and the central circular hole of the mask plate is located on the optical axis. Figure 6B schematically illustrates a spectrum diagram of a fluorescence image containing three phase information in one direction, and Figure 6C schematically illustrates a spectrum diagram of a fluorescence image containing three phase information in three directions, where these three directions correspond to three groups of through-hole pairs of the mask plate mask2. From the comparison between Figure 6C and Figure 6A, it can be seen that due to the increase in the superposition part of the frequency domain information, it is obvious that the super-resolution light sheet fluorescence microscopy imaging method of the present application has more advantages in improving the resolution of the final super-resolution image of the biological sample 1000.

[0093] It should be noted that since fluorescence images corresponding to three stripe directions and three stripe phases need to be obtained for the same plane where the reversibly photoswitchable fluorescent proteins (RSFPs) are activated, that is, the RSFPs need to be activated and excited nine times, after the activated plane of the RSFPs has been excited by the excitation structured light stripes to produce fluorescence, the portion of the RSFPs in that plane that produces fluorescence will be in a dark state, while the portion not illuminated by the excitation structured light stripes will remain in an activated state. Therefore, to ensure that clear fluorescence images can continue to be obtained for the same plane after a fluorescence image corresponding to a certain direction and a certain phase has been obtained, after illuminating the plane with the excitation structured light stripes, it is necessary to illuminate the plane again with an excitation structured light stripe with the same direction as the excitation structured light stripe but with a phase difference of π as the excitation structured light stripe to ensure that the entire plane is in a dark state, thereby preparing the plane for the next activation and excitation, and preventing residual activated RSFP stripes from affecting the quality of fluorescence images in other directions.

[0094] Although the linear super-resolution reconstruction algorithm is sufficient to reconstruct the fluorescence images with three stripe directions and three stripes to obtain a satisfactory super-resolution reconstructed image, the super-resolution reconstructed image result can also be obtained by using the linear super-resolution reconstruction algorithm if the fluorescence images with more directions and phases are used.

[0095] Therefore, for post-fluorescence image reconstruction using a linear super-resolution reconstruction algorithm, for each portion of the reversibly photoswitchable fluorescent protein (RSFP) to be activated and excited, a modulated stripe structured light can be incident N·M times as the excitation light, where N and M are integers greater than or equal to 3. The stripe directions of the modulated stripe structured light in each incident pattern differ by 180 / M degrees when observed in the plane of the activation light sheet, and the stripe phases differ by 2π / N. In this case, the mask 2 is also configured to have 2M through-holes, and these through-holes form M pairs of through-hole pairs. Each pair of through-holes is radially symmetrical, and the phases of the through-holes in each pair differ by 360° / 2M degrees.

[0096] Limited by the principle of linear structured light imaging, the super-resolution light-sheet fluorescence microscopy shown in Figures 5 and 6C is linear two-dimensional super-resolution fluorescence microscopy. However, as is well known to those skilled in the art, if the striped structured light used as excitation light can induce a saturation effect in the fluorescent protein, resulting in a nonlinear positive correlation between the fluorescence emission intensity and the excitation light intensity, the acquired information-carrying stripes will be narrower, and thus higher-frequency stripe information will be included in the acquired fluorescence image. Then, for this fluorescence image containing high-frequency information, nonlinear super-resolution reconstruction algorithms can be used to obtain super-resolution images with higher resolution.

[0097] Figure 7 schematically illustrates a schematic diagram of a method for super-resolution light-sheet fluorescence microscopy of a biological sample according to another embodiment of the present application. This method can be implemented, for example, using the super-resolution light-sheet fluorescence microscopy system described above. The following describes only the differences between this method and Figure 5 ; for other similarities, refer to the description of the aforementioned method embodiment.

[0098] In the embodiment shown in Figure 7, the stripe structured light used as excitation light induces a saturation effect in the fluorescent protein, resulting in narrower information-carrying stripes and, consequently, the inclusion of higher-frequency stripe information in the acquired fluorescence image. Consequently, the acquired fluorescence image can contain higher-frequency stripe information. The energy requirements for excitation light to stimulate high-frequency fluorescence information vary depending on the physical and chemical properties of the reversibly photoswitchable fluorescent proteins (RSFPs). Therefore, examples are not provided herein. Based on the properties of reversibly photoswitchable fluorescent proteins (RSFPs), those skilled in the art should be able to clearly understand the excitation light intensity required to stimulate high-frequency fluorescence information.

[0099] In addition, in the embodiment shown in FIG7 , at least twenty-five fluorescence images need to be obtained for the same plane (parallel to xy) in which the reversible photoswitch fluorescent proteins RSFPs are activated (for example, fluorescence images of five stripe directions of a certain phase are shown below FIG7 ), and each fluorescence image carries stripe information for one stripe direction and one stripe phase. Therefore, 10 through holes are formed in the mask mask2, which form five groups of through hole pairs, and each pair of through holes is aligned with each other along the radial direction. FIG8A , 8B, and 8C schematically illustrate spectrum diagrams comparing the excitation structured light stripes of conventional wide-field illumination and the super-resolution light sheet fluorescence microscopy imaging method of the embodiment of the present application. FIG8A schematically illustrates the spectrum diagram of conventional wide-field illumination. FIG8B schematically illustrates the spectrum diagram of a fluorescence image containing five phase information in one direction. FIG8C schematically illustrates the spectrum diagram of a fluorescence image containing five phase information in five directions, where these five directions correspond to the five groups of through hole pairs of the mask mask2. From the comparison between Figure 8C and Figure 8A, it can be seen that due to the expansion of the spectrum, the super-resolution light sheet fluorescence microscopy method of the present application is more advantageous in improving the resolution of the final super-resolution image of the biological sample 1000. And from the comparison between Figure 8C and Figure 6C, in the frequency domain, because more high-frequency information is collected, more fluorescence image details are reflected. Therefore, after the fluorescence image represented by the spectrum diagram shown in Figure 8C is processed by the nonlinear super-resolution reconstruction algorithm, the resolution of the super-resolution image generated must be better than that of linear structured light imaging.

[0100] Therefore, for the post-fluorescence image reconstruction using the nonlinear super-resolution reconstruction algorithm, each part of the reversibly photoswitchable fluorescent protein RSFPs to be activated and excited can be incident N·M times by the modulated stripe structured light as the excitation light, where N and M are integers greater than or equal to 5, and the stripe directions of the modulated stripe structured light incident each time differ from each other by 180 / M degrees when observed in the plane where the activation light sheet is located, and the stripe phases differ by 2π / N.

[0101] The laser L of the second light sheet generating module 110 nr The second light sheet generation module 110 is configured to emit a laser beam capable of inducing a two-photon effect in reversibly photoswitchable fluorescent proteins (RSFPs). In the embodiment of the present application, since the wavelength of the activation light for the reversibly photoswitchable fluorescent proteins (RSFPs) is selected to be 405 nm, the wavelength of the activation light capable of inducing a two-photon effect in the reversibly photoswitchable fluorescent proteins (RSFPs) is selected to be 780 nm. Those skilled in the art will appreciate that the wavelength of the activation light required to induce a two-photon effect in fluorescent proteins is generally within the near-infrared band, and therefore the second light sheet generation module 110 may also be referred to as the near-infrared light sheet generation module 110.

[0102] Taking Figure 2 as an example, first, the acousto-optic tunable filter AOTF and the spatial light modulator SLM of the light sheet generation module 300 are set so that no activation light is generated in the first light sheet activation light path. Then, in the second light sheet activation light path, when the collimated laser beam with a wavelength of 780nm generated by the laser Lnr passes through the axicon lens Axicon and then passes through the lens L118, a parallel annular light beam can be generated. After the parallel annular light beam passes through the first galvanometer Galvo1 of the light sheet generation module 300 and the light sheet scanning module 400, a Bessel light beam can be emitted from the activation objective lens 500 due to the conjugate design of the back focal plane of the axicon lens Axicon and the focal plane of the activation objective lens 500. The Bessel light beam forms an activation light sheet after being shaken by the first galvanometer Galvo1. Through the laser L nr The power setting of the activation light sheet is sufficient to induce a two-photon effect in reversibly photoswitchable fluorescent proteins (RSFPs). Compared to lasers operating in the visible light range, the near-infrared wavelength used to generate the activation light sheet increases the depth of the activated portion of the RSFPs and reduces damage to biological samples. In addition to utilizing the two-photon effect to generate the activation light sheet, the structured light excitation and detection optical paths can be referenced to the previously described embodiments.

[0103] The above embodiments primarily relate to two-dimensional super-resolution light-sheet fluorescence microscopy (e.g., in a plane parallel to the xy plane). As the activated light sheet moves in intervals along the z-axis, the linear or nonlinear two-dimensional super-resolution light-sheet fluorescence microscopy method described above is repeatedly applied to the portion of reversibly photoswitchable fluorescent proteins (RSFPs) activated by the activated light sheet at each z-axis position. A stack of fluorescence images reflecting the three-dimensional characteristics of a biological sample can be obtained. Within the stack of fluorescence images, the two-dimensional image at each z-axis position can be super-resolved using a super-resolution reconstruction algorithm. It should be pointed out that when imaging is performed using the super-resolution light sheet fluorescence microscopy imaging system of the present application, the relative position between the biological sample and the activation light sheet can be changed along the optical axis of the detection objective lens 700 or along the z-axis in the following two ways: (1) rotating the second galvanometer Galvo2 so that the activation light sheet moves along the optical axis of the detection objective lens 700, while the detection objective lens 700 moves synchronously along the optical axis to ensure focusing; and (2) keeping the activation light sheet stationary, but using the sample placement and movement module 900 to move the biological sample 1000.

[0104] In order to improve the z-axis resolution (resolution along the optical axis of the detection objective lens 700 ) of the three-dimensional fluorescence image stack, the activated portion of the reversibly photoswitchable fluorescent proteins RSFPs illuminated by the activation light sheet each time needs to be as thin as possible.

[0105] FIG9 schematically illustrates a method for thinning an activated portion of reversibly photoswitchable fluorescent proteins RSFPs illuminated by an activation light sheet according to an embodiment of the present application.

[0106] As shown in the figure, the reversibly photoswitchable fluorescent proteins (RSFPs) of a biological sample 1000 are first illuminated with an activation light sheet (shown in the solid frame 2000) to produce an activated portion. Then, a laser beam at an excitation wavelength, forming a thinning light sheet (shown in the dashed frame 2100), is used to illuminate both sides of the activated portion of the biological sample 1000 along its thickness. Each irradiation region overlaps the activated portion. Consequently, upon exposure to the excitation wavelength, the overlapping portion transitions from a bright state to a dark state. The activated portion of the RSFPs that does not overlap with the thinning light sheet remains activated and has a significantly reduced thickness, as shown in the figure. Consequently, utilizing the information provided regarding structured light excitation and detection optical pathways, the thinned activated portion of the RSFPs can be re-excited with stripe structured light as the excitation light, thereby acquiring a fluorescence image containing stripe information.

[0107] In the embodiment of Figure 9, the generation of the activation light sheet can refer to the contents described in Figures 2, 5, and 7, including using a single Bessel beam or multi-beam interference lattice dithering of the activation wavelength or using a single Bessel beam dithering with a wavelength that can cause a two-photon effect in reversibly photoswitchable fluorescent proteins RSFPs to generate an activation light sheet incident on a biological sample.

[0108] Taking the super-resolution light sheet fluorescence microscopy system shown in Figure 2 as an example, when generating a light sheet for thinning, the acousto-optic tunable filter AOTF of the laser gating and splitting module 200 is controlled to allow only a collimated laser beam of 488nm (excitation wavelength) to pass through, and the spatial light modulator SLM in the structured light excitation and detection optical path (or in an alternative embodiment, a digital micromirror device for replacing the spatial light modulator SLM) is set to make the structured light excitation and detection optical paths non-conductive. In this way, the first light sheet generating optical path generates thinning light sheets, as shown in the dashed frame 2100 of FIG. 9 , to partially overlap and illuminate both sides of the portion of the reversibly photoswitchable fluorescent protein (RSFP) illuminated by the activated light sheet. This causes the overlapping portion to be prematurely excited to produce fluorescence and then change to an inactive, or dark, state, thereby thinning the thickness of the portion of the reversibly photoswitchable fluorescent protein (RSFP) activated by the activated light sheet (along the z-axis, i.e., along the optical axis of the detection objective lens 700). Subsequently, the thinned activated portion of the reversibly photoswitchable fluorescent protein (RSFP) (as shown in the shaded area in FIG. 9 ) is illuminated by the excitation structured light stripes generated by the structured light excitation and detection optical path, thereby obtaining an excited fluorescence image. The obtained fluorescence image stack is then reconstructed using an algorithm to obtain a super-resolution image reflecting the three-dimensional features of the biological sample. Super-resolution can be achieved in the z-axis direction due to the further thinning of the activated portion of the reversibly photoswitchable fluorescent protein (RSFP). It should be clear to those skilled in the art that the generation of excitation structured light stripes in the structured light excitation and detection optical path and the acquisition of the fluorescence image can be implemented with reference to the description of the aforementioned embodiments.

[0109] In an alternative embodiment, the thinned light sheet can also be generated by controlling a specific pattern generated by a spatial light modulator (SLM) in any manner known to those skilled in the art. It should be clear to those skilled in the art that the spatial light modulator (SLM) can also be replaced by other suitable optical devices such as a digital micromirror device (DMD).

[0110] Those skilled in the art should be aware that the position change of the light sheet for thinning along the optical axis of the detection objective lens 700 or along the z-axis can be achieved in the following two ways: (1) rotating the second galvanometer Galvo2 so that the light sheet moves along the optical axis of the detection objective lens 700, and at the same time the detection objective lens 700 also moves synchronously along its optical axis to ensure focusing; and (2) keeping the light sheet for thinning stationary, but using the sample placement and movement module 900 to move the biological sample 1000.

[0111] Although specific embodiments of the present application are described in detail herein, they are provided for illustrative purposes only and should not be construed as limiting the scope of the present application. Furthermore, it will be apparent to those skilled in the art that the various embodiments described herein may be used in combination with one another. Various substitutions, variations, and modifications may be contemplated without departing from the spirit and scope of the present application.

Claims

1. A super-resolution light sheet fluorescence microscopy method, comprising: Reversibly photoswitchable fluorescent proteins (RSFPs) are used to treat the biological sample (1000) to be tested; The light sheet is used as an activation light surface to irradiate the biological sample to be tested (1000) to generate activation parts in the reversible light switch fluorescent protein (RSFPs) thereof; Allow the modulated stripe structured light as excitation light to be incident on the activated part of the reversible photoswitch fluorescent protein (RSFPs) toward the surface where the activated part is located, so that the activated part of the reversible photoswitch fluorescent protein (RSFPs) emits fluorescence on the plane where the light sheet is incident, wherein the activation light and the excitation light have different wavelengths; A fluorescence image with structured light fringe information is collected as a two-dimensional fluorescence image for super-resolution image processing.

2. The method according to claim 1, characterized in that When considering super-resolution image reconstruction using a linear super-resolution reconstruction algorithm, for each part to be activated of the reversibly photoswitchable fluorescent protein (RSFPs) that is incident with an activation light sheet, the activation light sheet is first incident and then the modulated stripe structured light is incident N·M times, where N and M are both integers greater than or equal to 3, and the stripe directions of the modulated stripe structured light incident each time differ from each other by 180 / M degrees when observed in the plane where the light sheet is located, and the stripe phases differ by 2π / N.

3. The method according to claim 2, characterized in that After each time the modulated stripe structured light is incident on the activated part of the reversibly photoswitchable fluorescent protein (RSFPs) as excitation light, a stripe structured light with the same stripe direction as the modulated stripe structured light but a phase difference of π is incident on the activated part of the reversibly photoswitchable fluorescent protein (RSFPs) as excitation light.

4. The method according to claim 2 or 3, characterized in that: When a nonlinear super-resolution reconstruction algorithm is considered for super-resolution image reconstruction, N and M are at least 5.

5. The method according to claim 1 or 2 or 3 or 4, characterized in that: The light sheet is formed by dithering a single Bessel beam or a multi-beam interference lattice.

6. The method according to claim 5, characterized in that A light sheet is generated by using a single Bessel beam at a near-infrared wavelength as activation light to irradiate a biological sample in a manner that induces a two-photon effect in the reversibly photoswitchable fluorescent proteins (RSFPs).

7. The method according to claim 5 or 6, characterized in that: After a light sheet as activation light has been incident on the biological sample (1000) and before a modulated stripe structure light as excitation light is incident on the activation part of the reversible photoswitch fluorescent protein (RSFPs), a light sheet for thinning formed by light having the wavelength of the excitation light is irradiated on both sides of the activation part with partial overlap to thin the thickness of the activation part.

8. The method according to any one of claims 1 to 7, characterized in that: The relative position between the light sheet serving as the activation light and the biological sample changes at least in a direction perpendicular to the plane in which the light sheet is located, so that the modulated stripe structured light serving as the excitation light is incident on the activated part of the reversible photoswitchable fluorescent proteins (RSFPs), fluorescence is excited, and a fluorescence image with structured light stripe information is collected as a three-dimensional fluorescence image stack for super-resolution image processing.

9. The method according to claim 8, characterized in that The relative position between the light sheet and the biological sample is changed by moving the light sheet itself or by moving the biological sample (1000).

10. A super-resolution light sheet fluorescence microscopy system, comprising: A light source module (100); Laser gating and light splitting module (200); A first light sheet generating module (300); Light sheet scanning module (400); Activate the objective lens (500); Structured light generation and polarization modulation module (600); Detection objective lens (700); Fluorescence detection module (800); as well as Sample placement and movement module (900), The first light sheet generation module (300), the light sheet scanning module (400) and the activation objective lens (500) form a first light sheet activation optical path, the structured light generation and polarization modulation module (600), the fluorescence detection module (800) and the detection objective lens (700) form a structured light excitation and detection optical path, and the laser gating and light splitting module (200) is configured so that the laser beam emitted by the light source module (100) selectively enters the first light sheet activation optical path or the structured light excitation and detection optical path; The first light sheet activation light path is configured to generate a light sheet as activation light incident on a biological sample to be tested (1000) treated with reversibly photoswitchable fluorescent proteins (RSFPs) to generate activation parts in the reversibly photoswitchable fluorescent proteins (RSFPs); The structured light excitation and detection optical path is configured to generate modulated stripe structured light as excitation light incident on the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs), so that the activated portion of the reversibly photoswitchable fluorescent proteins (RSFPs) emits fluorescence on the plane incident on the light sheet, wherein the activation light and the excitation light have different wavelengths; The structured light excitation and detection optical path is also configured to collect a fluorescence image with structured light fringe information as a two-dimensional fluorescence image for super-resolution image processing.

11. The system according to claim 10, characterized in that When considering super-resolution image reconstruction using a linear super-resolution reconstruction algorithm, for each part to be activated of the reversibly photoswitchable fluorescent protein (RSFPs) that is incident with an activation light sheet, the activation light sheet is first incident and then the modulated stripe structured light is incident N·M times, where N and M are both integers greater than or equal to 3, and the stripe directions of the modulated stripe structured light incident each time differ from each other by 180 / M degrees when observed in the plane where the light sheet is located, and the stripe phases differ by 2π / N.

12. The system according to claim 11, characterized in that After each time the modulated stripe structured light is incident on the activated part of the reversibly photoswitchable fluorescent protein (RSFPs) as excitation light, a stripe structured light with the same stripe direction as the modulated stripe structured light but a phase difference of π is incident on the activated part of the reversibly photoswitchable fluorescent protein (RSFPs) as excitation light.

13. The system according to claim 11 or 12, characterized in that When a nonlinear super-resolution reconstruction algorithm is considered for super-resolution image reconstruction, N and M are at least 5.

14. The system according to claim 10 or 11 or 12 or 13, characterized in that: The first light sheet generating module (300) comprises a first galvanometer (Galvo1), and the light sheet is formed by shaking a single Bessel beam or a multi-beam interference lattice via the first galvanometer (Galvo1).

15. The system according to claim 14, characterized in that Also includes: a second light sheet generating module (110) capable of emitting a laser beam independently of the light source module (100); The second light sheet generating module (110), the first galvanometer (Galvo1) of the first light sheet generating module (300), the light sheet scanning module (400) and the activation objective lens (500) form a second light sheet activation optical path. The second light sheet generating module (110) comprises: A laser (L) capable of generating near-infrared wavelength laser light independent of the light source module (100) nr ); Located at the laser (L nr ) downstream of the pair of lenses (L16, L17); and a submodule or annular mask (Am) composed of an axicon lens (Axicon) and a lens (L118), wherein the submodule or annular mask (Am) is located downstream of the pair of lenses (L16, L17), and the submodule or annular mask (Am) is configured to nr ) emits a near-infrared wavelength laser, which is first collimated and expanded by the pair of lenses (L16, L17) and then passes through the submodule or the annular mask (Am) to generate an annular light beam, and the annular light beam passes through the activation objective lens to form a single Bessel light beam. At the same time, the single Bessel light beam is generated by the vibration of the first galvanometer (Galvo1) of the first light sheet generating module (300) to generate a light sheet as activation light to cause a two-photon effect in the reversible light switch fluorescent protein (RSFPs) and enter the biological sample.

16. The system according to claim 15, characterized in that After a light sheet as activation light has been incident on the biological sample (1000) and before a modulated stripe structure light as excitation light is incident on the activation part of the reversible photoswitch fluorescent protein (RSFPs), a light sheet for thinning formed by light having the wavelength of the excitation light is irradiated on both sides of the activation part with partial overlap to thin the thickness of the activation part.

17. The system according to any one of claims 10 to 16, characterized in that: The relative position between the light sheet as activation light and the biological sample changes at least along the optical axis of the detection objective lens (700), so that the modulated stripe structured light as excitation light is incident on the activation part of the reversible photoswitchable fluorescent proteins (RSFPs), fluorescence is excited, and a fluorescence image with structured light stripe information is collected as a three-dimensional fluorescence image stack for super-resolution image processing.

18. The system according to claim 17, characterized in that The light sheet scanning module (400) is configured to shift only the light sheet serving as activation light and synchronously move the detection objective lens along its optical axis or to move only the sample placement movement module (900), thereby changing the relative position between the light sheet and the biological sample at least along the optical axis of the detection objective lens (700).

Images