A method and apparatus for lattice light sheet microscopy imaging based on galvanometer scanning

Abstract

This invention discloses a lattice light sheet microscopy imaging method based on galvanometer scanning, comprising: splitting a laser beam into three linearly polarized beams, which are then shaped by a cylindrical lens to form linearly polarized light; projecting the light onto a fluorescent sample through an excitation objective to generate a lattice fringe structured light illumination pattern through mutual interference; using an electro-optic modulator to change the phase of the interference lattice fringes, in structured light mode, achieving structured light illumination of the sample with step-phase-shifted lattice fringes, and in dithering mode, rapidly dithering the lattice interference fringes to achieve uniform illumination of the sample with a light sheet; and using a probe objective to collect the fluorescence signal emitted by the sample, obtaining fluorescence intensity information, and reconstructing a high-resolution light sheet microscopic image. This invention also discloses a lattice light sheet microscopy imaging device based on galvanometer scanning. This invention has high incident light energy utilization and high contrast of the interference lattice fringes, and can obtain resolution exceeding the diffraction limit under low incident light power conditions.

Description

Technical Field

[0001] This invention belongs to the field of optical super-resolution microscopy, and specifically relates to a lattice light sheet microscopy method and apparatus based on galvanometer scanning. Background Technology

[0002] Optical microscopy plays a vital role in life science research. Among them, light-sheet fluorescence microscopy is one of the most promising tools for three-dimensional long-term imaging of samples. Light-sheet microscopy employs a side-illumination strategy, illuminating the sample from the side and collecting the fluorescence signal using a probe objective perpendicular to the excitation optical axis. This avoids off-axis excitation of the sample, inherently possesses optical tomography capabilities, and can acquire images with good signal-to-noise ratios even under low-power illumination, avoiding premature photobleaching and phototoxicity. It can also perform long-term in vivo imaging of cellular samples. Furthermore, due to its wide-field detection, the imaging speed is fast, which is beneficial for capturing the dynamic processes of biological samples. However, light-sheet microscopy has relatively low imaging resolution, making it unsuitable for probing the fine structures of samples.

[0003] In recent years, researchers have proposed various resolution enhancement methods to improve the imaging resolution of light-sheet microscopes. For example, using a high numerical aperture (NA) excitation objective to generate a thin Gaussian sheet and then using a high NA probe objective to examine the sample can effectively improve resolution. However, this type of method causes the light sheet to converge and diverge rapidly, resulting in a smaller imaging field of view and impairing imaging speed. Subsequently, researchers have used Bessel beams to replace Gaussian beams, improving axial resolution while maintaining the imaging field of view. However, the side lobes of the Bessel sheet can cause off-axis excitation of the sample, reducing the optical tomography capability of the light-sheet microscope and introducing more phototoxicity to the sample.

[0004] To address the sidelobes of Bessel beams, patent application CN110220875A discloses a lattice sheet microscope. Utilizing the interference effect between Bessel beam arrays, it successfully suppresses sidelobes, resulting in thin lattice sheets with a large field of view, offering advantages such as high imaging resolution and fast imaging speed. However, to obtain these lattice sheets, the original lattice sheet microscopy system uses a binary spatial light modulator to modulate the incident beam and filters the diffracted beam using a mask, retaining only the positive and negative first-order diffracted light for lattice sheet generation. Due to the pixel gaps in the spatial light modulator, most of the incident light energy is concentrated in the unmodulated and unutilized zero-order diffracted light, resulting in very low light energy utilization of the system.

[0005] To address this issue, researchers proposed passing the generated annular beam through a custom-designed mask with only four slits, thereby obtaining the accurate light field distribution at the back focal plane of the excitation objective and forming a lattice light sheet to illuminate the sample. However, because the mask blocks most of the light source, the system's light energy utilization rate remains low, and because the mask is fixed, the switching of the beam's NA at the entrance pupil of the excitation objective is not flexible enough. Patent application CN110687670A provides a field-synthesized light sheet microscope that uses a scanning galvanometer to continuously or discretely scan the beam at the entrance pupil of the excitation objective, significantly improving the system's light energy utilization rate. However, the field-synthesized light sheet microscope is incompatible with structured illumination micro-imaging techniques, making it difficult to improve its imaging resolution. Furthermore, the need for scanning at the entrance pupil somewhat impairs the imaging speed. Summary of the Invention

[0006] This invention provides a lattice light sheet microscopy imaging method and apparatus based on galvanometer scanning, which utilizes galvanometer scanning to flexibly switch the light beam NA and uses the beam interference effect to generate structured light patterns on the sample surface.

[0007] To achieve the aforementioned objectives, the specific technical solution adopted by this invention is as follows:

[0008] A lattice light sheet microscopy imaging method based on galvanometer scanning includes the following steps:

[0009] 1) The laser beam is split into three linearly polarized beams with the same vibration direction;

[0010] 2) The three-line polarized light is shaped into a linear beam, which is then projected onto the fluorescent sample through the excitation objective lens. The mutual interference produces a lattice fringe structure illumination pattern.

[0011] 3) The phase of the interference lattice fringes is changed multiple times, and the detection objective lens collects multiple fluorescence intensity images at the corresponding phases;

[0012] 4) Data processing is performed using multiple fluorescence intensity images to reconstruct a super-resolution image.

[0013] In step 3), the phase of the interference lattice fringes needs to be changed at least five times to illuminate the sample with structured light fringes or a uniform light sheet.

[0014] In this invention, the fluorescence intensity information obtained by projecting a single structured light pattern onto a sample contains five frequency components. To separate these five frequency components, five equations are required. Traditional lattice light sheet microscopy systems utilize an x-scanning galvanometer conjugate to the back focal plane of the excitation objective to step the generated lattice beam along the x-direction, achieving phase shifting of the lattice fringe structured light illumination pattern. This invention, however, rapidly alters the optical path of the beam using an electro-optic modulator, thereby changing the phase of the interference lattice fringes projected onto the sample, resulting in five phase-shifted fluorescence images, thus allowing for the extraction and shifting of the frequency components.

[0015] Preferably, the electro-optic modulator can control the phase shift of the interference lattice fringes to 0°, 72°, 144°, 216°, and 288°. These are merely optimal examples; theoretically, the phase shift angle can be any value, as long as the sample receives uniform illumination. Alternatively, the electro-optic modulator can rapidly change the phase of the beam to form a uniform illumination sheet. This operation matches the dithering mode of a lattice sheet microscope, offering faster imaging speeds but relatively lower imaging resolution compared to structured light illumination.

[0016] This invention is fully compatible with the image reconstruction algorithms used in traditional lattice light sheet microscopy, and image data processing and reconstruction can be implemented based on existing algorithms.

[0017] The present invention provides a lattice light sheet microscopy imaging device based on galvanometer scanning, including an excitation optical path module and an imaging optical path module;

[0018] The excitation optical path module has the following components arranged in sequence:

[0019] A laser emits a laser beam;

[0020] A central beam shaping system is used to shape the beam into a linear vibrating beam and illuminate the sample from the center of the back focal plane of the excitation objective.

[0021] A galvanometer beam splitter system is used to split a laser beam into two linearly polarized beams that have symmetrical propagation directions and the same vibration direction and are incident on the sample from the edge of the back focal plane of the excitation objective.

[0022] z-scanning galvanometer is used to scan the beam and scan the sample axially;

[0023] The excitation objective lens is used to focus three linear beams onto the sample surface to generate interference lattice fringe patterns and excite the sample fluorescence intensity signal.

[0024] The imaging optical path module includes:

[0025] The probe objective is used to collect the fluorescence signal of the sample;

[0026] A camera is used to record the fluorescence intensity signal.

[0027] A computer is used to control the central beam shaping system and the galvanometer beam splitting system to change the phase, numerical aperture, and rotation direction of the interference lattice pattern; to control the z-scan galvanometer to perform axial scanning of the sample; to control the fluorescence intensity signal acquired by the camera; and to process the data to obtain a super-resolution image.

[0028] Preferably, the central beam shaping system includes:

[0029] The first polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams.

[0030] The polarizing beam splitter comprises a first electro-optic modulator, a first cylindrical lens, and a first annular mask, sequentially arranged in the reflected optical path. The first electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes appearing on the sample plane. The first cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the x-direction and remains parallel along the z-direction. The first annular mask, located at the focal plane of the first cylindrical lens, is used to filter the beam.

[0031] The transmission optical path sequentially arranged in the first polarizing beam splitter includes a first quarter-wave plate and a galvanometer beam splitting system. The first quarter-wave plate is used to convert the transmitted p-polarized light into circularly polarized light.

[0032] Preferably, the galvanometer beam splitting system includes:

[0033] A polarizing beam splitter is used to split incident circularly polarized light into two linearly polarized light beams.

[0034] A second quarter-wave plate, a reflecting mirror, a second cylindrical lens, a first scanning galvanometer, and a first scanning lens are sequentially arranged in the transmission optical path of the polarizing beam splitter. The second quarter-wave plate is used to convert linearly polarized light into circularly polarized light. The reflecting mirror is used to reflect the circularly polarized light so that it passes through the second quarter-wave plate again and becomes s-polarized light, which can be reflected again by the second polarizing beam splitter. The second cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and remains parallel in the x-direction. The first scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The first scanning lens is used to convert the linear beam into linearly polarized light that is compressed along the x-direction and remains parallel in the z-direction.

[0035] A second electro-optic modulator, a second cylindrical lens, a second scanning galvanometer, and a second scanning lens are sequentially arranged on the reflected optical path of the second polarizing beam splitter. The second electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes on the sample plane. The third cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and kept parallel in the x-direction. The second scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The second scanning lens is used to convert the linear beam into linearly polarized light that is compressed along the x-direction and kept parallel in the z-direction.

[0036] In this invention, the galvanometer beam splitting system includes a triangular reflector for combining two linearly polarized beams emitted from the first scanning lens and the second scanning lens.

[0037] Preferably, the galvanometer beam splitting system includes a second annular mask plate for filtering the two line-polarized beams of the combined beam.

[0038] Between the laser and the central beam shaping system are, in sequence, a collimating lens for collimating the laser beam; a polarizer for converting the laser beam into linearly polarized light; and a half-wave plate for changing the rotation direction of the linearly polarized light and adjusting the intensity ratio of the two linearly polarized beams transmitted and reflected from the polarizing beam splitter.

[0039] A non-polarizing beam splitter, a z-scanning galvanometer, a third scanning lens, and a first tube mirror are sequentially placed between the galvanometer beam splitter system and the excitation objective. The non-polarizing mirror is used to combine the three linearly polarized beams into the subsequent system; the z-scanning galvanometer is used to control the beam to scan the sample under test along the z-direction; the third scanning lens and the tube mirror constitute a 4f system, used to conjugate the beam from the z-scanning galvanometer to the back focal plane of the excitation objective.

[0040] The excitation objective is used to transmit three linearly polarized beams with the same vibration direction to the surface of the sample to be tested for interference, generating interference lattice fringes.

[0041] The imaging optical path module is arranged with a detector objective, a filter, a second tube mirror, and a camera in sequence. The detector objective is perpendicular to the excitation objective and is used to collect the fluorescence signal of the sample; the filter is used to filter out stray light in the fluorescence emitted by the sample; and the tube mirror is used to focus the fluorescence signal onto the camera.

[0042] In another technical solution, the present invention also provides a lattice light sheet microscopic imaging device based on galvanometer scanning, including an excitation optical path module and an imaging optical path module;

[0043] The excitation optical path module has the following components arranged in sequence:

[0044] A laser emits a laser beam;

[0045] A central beam shaping system is used to shape the beam into a linear vibrating beam and illuminate the sample from the center of the back focal plane of the excitation objective.

[0046] A galvanometer beam splitter system is used to split a laser beam into two linearly polarized beams that have symmetrical propagation directions and the same vibration direction and are incident on the sample from the edge of the back focal plane of the excitation objective.

[0047] z-scanning galvanometer is used to scan the beam and scan the sample axially;

[0048] The excitation objective lens is used to focus three linear beams onto the sample surface to generate interference lattice fringe patterns and excite the sample fluorescence intensity signal.

[0049] The imaging optical path module includes:

[0050] The probe objective is used to collect the fluorescence signal of the sample;

[0051] A camera is used to record the fluorescence intensity signal.

[0052] A computer is used to control the central beam shaping system and the galvanometer beam splitting system to change the phase, numerical aperture, and rotation direction of the interference lattice pattern; to control the z-scan galvanometer to perform axial scanning of the sample; to control the fluorescence intensity signal acquired by the camera; and to process the data to obtain a super-resolution image.

[0053] Preferably, the central beam shaping system includes:

[0054] The first polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams.

[0055] The polarizing beam splitter comprises a first electro-optic modulator, a first cylindrical lens, and a first annular mask, sequentially arranged in the reflected optical path. The first electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes appearing on the sample plane. The first cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the x-direction and remains parallel along the z-direction. The first annular mask, located at the focal plane of the first cylindrical lens, is used to filter the beam.

[0056] The transmission optical path sequentially arranged in the first polarizing beam splitter includes a first quarter-wave plate and a galvanometer beam splitting system. The first quarter-wave plate is used to convert the transmitted p-polarized light into circularly polarized light.

[0057] Preferably, the galvanometer beam splitting system includes:

[0058] The first polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams.

[0059] A second cylindrical lens, a first scanning galvanometer, a first scanning lens, and a second half-wave plate are sequentially arranged on the transmission optical path of the first polarizing beam splitter. The second cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and kept parallel in the x-direction. The first scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The first scanning lens is used to convert the linear beam into linearly polarized light that is compressed along the x-direction and kept parallel in the z-direction. The second half-wave plate is used to change the polarization state of the beam to an s-polarized beam.

[0060] A second electro-optic modulator, a third cylindrical lens, a second scanning galvanometer, a second scanning lens, and a third half-wave plate are sequentially arranged on the reflected optical path of the first polarizing beam splitter. The second electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference fringes on the sample plane. The third cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and kept parallel in the x-direction. The second scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The second scanning lens is used to convert the linear beam into linearly polarized light that is compressed along the x-direction and kept parallel in the z-direction. The third half-wave plate is used to change the polarization state of the beam to a p-polarized beam.

[0061] In this invention, the galvanometer beam splitting system includes: a third polarization beam splitter for combining linearly polarized light emitted through the first scanning lens and the second scanning lens; a second quarter-wave plate for converting the linearly polarized light emitted through the first scanning lens and the second scanning lens into circularly polarized light; a second annular mask for filtering the two circularly polarized beams of the combined beam; and a tangential polarizer for converting the two circularly polarized beams into tangentially linearly polarized light.

[0062] In this invention, between the laser and the first polarizing beam splitter, the following components are sequentially placed: a collimating lens for collimating the laser beam; a polarizer for converting the laser beam into linearly polarized light; and a first half-wave plate for changing the rotation direction of the linearly polarized light and adjusting the intensity ratio of the two linearly polarized beams transmitted and reflected from the polarizing beam splitter.

[0063] A non-polarizing beam splitter, a z-scanning galvanometer, a third scanning lens, and a first tube mirror are sequentially placed between the galvanometer beam splitter system and the excitation objective. The non-polarizing mirror is used to combine the three linearly polarized beams into the subsequent system; the z-scanning galvanometer is used to control the beam to scan the sample under test along the z-direction; the scanning lens and the tube mirror constitute a 4f system, used to conjugate the beam from the z-scanning galvanometer to the back focal plane of the excitation objective.

[0064] The excitation objective is used to transmit three linearly polarized beams with the same vibration direction to the surface of the sample to be tested for interference, generating interference lattice fringes.

[0065] The imaging optical path module is arranged with a detector objective, a filter, a second tube mirror, and a camera in sequence. The detector objective is perpendicular to the excitation objective and is used to collect the fluorescence signal of the sample; the filter is used to filter out stray light in the fluorescence emitted by the sample; and the tube mirror is used to focus the fluorescence signal onto the camera.

[0066] Compared with the prior art, the present invention has the following beneficial technical effects:

[0067] (1) Using a galvanometer beam splitter system instead of a spatial light modulator and conventional grating devices to obtain the light field distribution at the back focal plane of the excitation objective and the interference pattern at the front focal plane improves the energy utilization of incident light.

[0068] (2) The phase of the image reflected from the front focal plane of the excitation objective lens is changed by using an electro-optic modulator to control the optical path of the reflection path. Compared with the traditional corner bevel prism, the displacement accuracy is higher and the modulation speed is faster.

[0069] (3) The distribution of the beam on the back focal plane of the excitation objective can be flexibly changed by using a scanning galvanometer, thereby changing the NA of the beam and obtaining lattice patterns of different modes on the sample surface.

[0070] (4) By combining three-dimensional super-resolution structured illumination microscopy with lattice light sheet microscopy, the imaging resolution in both x and z dimensions has been further improved, and super-resolution imaging under low incident light power conditions has been achieved.

[0071] (5) The device is simple, flexible and easy to operate; the incident light energy utilization rate is high and the interference lattice fringes have high contrast. It is compatible with structured light microscopy and can achieve a resolution exceeding the diffraction limit under low incident light power conditions. It is particularly suitable for imaging fluorescent samples in the life science field. Attached Figure Description

[0072] Figure 1 This is a schematic diagram of a lattice light sheet microscopy imaging device based on galvanometer scanning according to the present invention.

[0073] Figure 2The diagram shows the related devices for controlling the intensity distribution and polarization state of the light beam; (a) is a schematic diagram of the annular mask; (b) is a schematic diagram of the intensity distribution of the two beams after passing through (a) at the back focal plane of the excitation objective; and (c) is a schematic diagram of the tangential polarizer.

[0074] Figure 3 To illustrate the distribution of the excitation beam on the sample surface; where (a) is a two-dimensional schematic diagram of the lattice pattern generated by the lattice light sheet microscopy imaging system based on galvanometer scanning; (b) is a one-dimensional intensity distribution diagram corresponding to (a); and (c) is a spectrum diagram corresponding to (a).

[0075] Figure 4 This is a schematic diagram of a lattice interference light sheet microscopy imaging device based on galvanometer scanning according to the present invention. Detailed Implementation

[0076] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the invention is not limited to the specific embodiments disclosed below.

[0077] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0078] Example 1

[0079] like Figure 1The light sheet microscopy imaging device shown includes: a laser 1, a collimating lens 2, a polarizer 3, a half-wave plate 4, a first reflecting mirror 5, a first polarizing beam splitter 6, a first electro-optic modulator 7, a first lens 8, a second lens 9, a second reflecting mirror 10, a third reflecting mirror 11, a fourth reflecting mirror 12, a first cylindrical lens 13, a fifth reflecting mirror 14, a first annular mask 15, a first quarter-wave plate 16, a second polarizing beam splitter 17, a second quarter-wave plate 18, a sixth reflecting mirror 19, a third lens 20, a fourth lens 21, a second cylindrical lens 22, a first scanning galvanometer 23, and a fourth lens 24. The system includes a scanning lens 24, a seventh reflecting mirror 25, a second electro-optic modulator 26, a fifth lens 27, a sixth lens 28, a third cylindrical lens 29, a second scanning galvanometer 30, a second scanning lens 31, an eighth reflecting mirror 32, a triangular reflecting mirror 33, a second annular mask 34, a seventh lens 35, an eighth lens 36, a non-polarizing beam splitter 37, a ninth lens 38, a ninth reflecting mirror 39, a z-scanning galvanometer 40, a third scanning lens 41, a first tube mirror 42, an excitation objective lens 43, a sample to be tested 44, a detection objective lens 45, a filter 46, a second tube mirror 47, a camera 48, and a computer 49.

[0080] Laser 1 emits a laser beam. Collimating lens 2, polarizer 3, and waveplate 4 are placed sequentially on the optical axis of the laser beam path. Collimating lens 2 is used to collimate the laser beam to obtain a parallel beam. Polarizer 3 is used to convert the emitted laser into linearly polarized light. Waveplate 4 is used to change the rotation direction of the linearly polarized light and adjust the intensity ratio of the two linearly polarized beams transmitted and reflected from the polarizing beam splitter.

[0081] The circularly polarized light, after passing through the first reflecting mirror 5, enters the first polarizing beam splitter 6. The polarizing beam splitter 6 splits the beam into two paths. The s-polarized light from the first reflected path passes through the first electro-optic modulator 7 and enters the beam expanding system composed of the first lens 8 and the second lens 9 for beam expansion. The optical path length of this reflected path can be adjusted by changing the positions of the second reflecting mirror 10 and the third reflecting mirror 11. The first electro-optic modulator 7 can rapidly change the phase of the beam. The beam is reflected by the fourth reflecting mirror 12 and enters the first cylindrical lens 13, forming a linear s-polarized beam that is compressed along the x-direction and remains unchanged in the z-direction. After reflection by the fifth reflecting mirror 14, the linear beam is focused onto the first annular mask plate 15 for filtering. The p-polarized light transmitted in the second path is modulated into circularly polarized light after passing through the first quarter-wave plate 16, and then enters the second polarizing beam splitter 17, which further splits the beam into two paths.

[0082] The p-polarized light transmitted through the second polarizing beam splitter 17 passes through the second quarter-wave plate 18, is reflected perpendicularly by the sixth reflecting mirror 19, and then becomes s-polarized light after passing through the second quarter-wave plate 18 again. It is then reflected by the second polarizing beam splitter 17. The beam is expanded after passing through the third lens 20 and the fourth lens 21, and exits from the second cylindrical lens 22 as linearly polarized light compressed in the z-direction and parallel in the x-direction. This light then passes through the first scanning galvanometer 23, the first scanning lens 24, and the seventh reflecting mirror 25 before striking the triangular reflecting mirror 33. The s-polarized light reflected by the second polarizing beam splitter 17 enters the second electro-optic modulator 26 and exits. After being expanded by the fifth lens 27 and the sixth lens 28, it enters the third cylindrical lens 29, forming linearly polarized light compressed in the z-direction and parallel in the x-direction. This light then passes through the second scanning galvanometer 30, the second scanning lens 31, and the eighth reflecting mirror 32 before striking the triangular reflecting mirror 33. The first scanning mirror 23 and the second scanning mirror 30 can easily change the position of the emitted light on the back focal plane of the excitation objective 43, thereby flexibly changing the numerical aperture and rotation of the beam to illuminate the sample 44 with beams of different thicknesses and propagation lengths on the sample surface. The second electro-optic modulator 26 can quickly change the phase of the beam to achieve structured light illumination of the sample or to generate a uniform light sheet to illuminate the sample 44.

[0083] The two beams, one above the other, are reflected by the triangular mirror 31 and filtered by the second annular mask 34. After passing through the seventh lens 35, the non-polarizing beam splitter 37, the ninth lens 38, and the ninth mirror 39, they strike the z-scanning mirror 40. The seventh lens 35 and the ninth lens 38 form a 4f imaging system. The second annular mask 34 is located at the front focal plane of the seventh lens 35, and the z-scanning mirror 40 is located at the rear focal plane of the ninth lens 38. Therefore, the image of the second annular mask 34 is conjugate onto the z-scanning mirror 40.

[0084] The eighth lens 36 and the ninth lens 38 form a 4f imaging system, with the first annular mask 15 located at the front focal plane of the eighth lens 36. Therefore, after the light is reflected by the unpolarized mirror 37 and the ninth mirror 39, the image of the first annular mask 15 is conjugate onto the z-scanning galvanometer 40.

[0085] The three beams are scanned by the z-scanning galvanometer 40, pass through the third scanning lens 41 and the first tube mirror 42, and then enter the excitation objective 43, generating an interference beam at the plane of the sample 44 under test. The third scanning lens 41 and the first tube mirror 42 form a 4f imaging system: the z-scanning galvanometer 40 is located at the front focal plane of the third scanning lens 41, and the rear focal plane of the excitation objective 43 coincides with the rear focal plane of the first tube mirror 42. Therefore, the first annular mask 15, the second annular mask 34, and the z-scanning galvanometer 40 are all conjugate to the rear focal plane of the excitation objective 43. The rotation angle of the z-scanning galvanometer 40 scans the beam, causing the illumination beam to scan the sample 44 under test along the z-axis. Figure 2The device shown controls the intensity distribution and polarization state of the light beam. Figure 2 (a) is a schematic diagram of a ring-shaped mask; Figure 2 (b) The image shows through Figure 2 (a) Schematic diagram of the light intensity distribution of the rear double beam at the rear focal plane of the excitation objective; Figure 2 (c) is a schematic diagram of a tangential polarizer.

[0086] The detector objective 45 and the excitation objective 43 are perpendicular to each other. The fluorescence excited by the sample is collected by the detector objective 45, passes through the filter 46 and the second tube mirror 47, and is then received and recorded by the camera 48. The filter 46 is used to filter out stray light from the fluorescence emitted by the sample, and the second tube mirror 47 is used to image the internal fluorescence intensity information of the sample onto the camera 48. Figure 3 As shown, Figure 3 (a) is a two-dimensional schematic diagram of the lattice pattern generated by the lattice light sheet microscopy imaging system based on galvanometer scanning; Figure 3 (b) is Figure 3 (a) Corresponding one-dimensional intensity distribution map; Figure 3 (c) is Figure 3 (a) The corresponding spectrum.

[0087] use Figure 1 The working method of the field interference lattice light sheet microscopy imaging device based on galvanometer scanning is as follows:

[0088] The laser beam emitted by laser 1 passes through polarizer 3 and the first half-wave plate 4 to form linearly polarized light, and then enters the first polarizing beam splitter 6 and the second polarizing beam splitter 17, splitting into three optical paths. The beam reflected from the first polarizing beam splitter 6 passes through the first cylindrical lens 13 to form linearly polarized light, which is then filtered by the first annular mask 15. The two linearly polarized beams transmitted from the first polarizing beam splitter 6 and split by the second polarizing beam splitter 17 are linearly polarized light emitted from the second cylindrical lens 22, the first scanning galvanometer 23, the third cylindrical lens 29, and the second scanning galvanometer 30, respectively. These two beams are parallel in the z-direction and converge in the x-direction, and are filtered by the second annular mask 34. The three beams are conjugated to the z-scanning galvanometer 40 and imaged onto the rear pupil plane of the excitation objective 43. After interference, the three laser beams form periodic illumination fringes, which excite the fluorescently labeled sample 44 to generate a fluorescence signal, which is received by the detector objective 45 perpendicular to the excitation objective and imaged onto the camera 48. By changing the rotation angle of the first scanning mirror 23 and the second scanning mirror 30, the position of the two linearly polarized beams at the entrance pupil is changed, thereby altering the NA of the beam.

[0089] In this embodiment, two methods can be used to excite the sample.

[0090] Dithering mode: Computer 49 controls the first electro-optic modulator 7 and the second electro-optic modulator 26 to rapidly change the optical path difference of the reflected light path, causing the lattice fringe structured light pattern projected onto the test sample 44 to dither continuously and rapidly along the x-direction, forming a uniform illumination sheet. The excitation fluorescence signal of the test sample 44 is collected by the detector objective lens 45, filtered by the filter 46 to remove stray light from the collected fluorescence, and imaged onto the camera 48 by the second tube lens 47, thus obtaining the fluorescence intensity information of the test sample 44.

[0091] Structured light mode: The sample 44 is excited using a static structured light illumination pattern; the fluorescence signal emitted by the sample 44 is collected by the detection objective lens 45, and imaged onto the camera 48 through the filter 46 and the second tube lens 47. The fluorescence intensity information obtained from a single structured light pattern projected onto the sample contains five frequency components, so five image equations are needed to separate these five frequency components. The computer 49 controls the first electro-optic modulator 7 and the second electro-optic modulator 26 to change the phase of the light beam, so that the lattice fringe structured light pattern projected onto the sample 44 is discretely phase-shifted by 0°, 72°, 144°, 216°, and 288°, resulting in five phase-shifted fluorescence images, thereby extracting and shifting the frequency components. By combining the image reconstruction algorithm of structured light microscopy technology with the data processing of the five acquired images, a super-resolution image in the xz direction can be obtained.

[0092] Example 2

[0093] like Figure 4 As shown, the light sheet microscopy imaging device in this embodiment can also use a tangential polarizer to obtain linearly polarized light with the same vibration direction. Figure 4 and Figure 1 In contrast, in the transmission optical path of the second polarizing beam splitter in Embodiment 2, the original second quarter-wave plate 18 and sixth reflector 19 are replaced with tenth reflector 51 and eleventh reflector 52 to control the optical path of the transmission optical path; the original triangular reflector 33 is replaced with a third polarizing beam splitter 54, and a second half-wave plate 50 and a third half-wave plate 53 are added to control the polarization state of the beam, so that the linearly polarized light beams of the two optical paths can be combined by the third polarizing beam splitter 54; at the same time, a third quarter-wave plate 55 is added to convert the two linearly polarized lights into circularly polarized lights; and a tangential polarizer 56 is added to convert the two circularly polarized lights into linearly polarized lights with the same vibration direction.

[0094] Finally, the three linearly polarized beams with the same vibration direction are imaged onto the rear pupil surface of the excitation objective 43 and form an interference beam on the sample 44 to excite the fluorescence signal of the sample 44.

[0095] Everything else is the same as in Example 1.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lattice light sheet microscopic imaging device based on galvanometer scanning, comprising an excitation optical path module and an imaging optical path module, characterized in that: The excitation optical path module comprises, in sequence: A laser emits a laser beam; A central beam shaping system is used to shape the beam into a linear vibrating beam and illuminate the sample from the center of the back focal plane of the excitation objective. A galvanometer beam splitter system is used to split a laser beam into two linearly polarized beams that have symmetrical propagation directions and the same vibration direction and are incident on the sample from the edge of the back focal plane of the excitation objective. The aforementioned galvanometer beam splitting system includes: The second polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams. A second quarter-wave plate, a reflecting mirror, a second cylindrical lens, a first scanning galvanometer, and a first scanning lens are sequentially arranged in the transmission optical path of the polarizing beam splitter. The second quarter-wave plate is used to convert linearly polarized light into circularly polarized light. The reflecting mirror is used to reflect the circularly polarized light so that it passes through the second quarter-wave plate again and becomes s-polarized light, which can be reflected again by the second polarizing beam splitter. The second cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and remains parallel in the x-direction. The first scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The first scanning lens is used to transform the linear beam into linearly polarized light that is compressed along the x-direction and remains parallel in the z-direction. A second electro-optic modulator, a third cylindrical lens, a second scanning galvanometer, and a second scanning lens are sequentially arranged on the reflected optical path of the second polarizing beam splitter. The second electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes on the sample plane. The third cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and remains parallel in the x-direction. The second scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The second scanning lens is used to convert the linear beam into linearly polarized light that is compressed along the x-direction and remains parallel in the z-direction. A triangular reflector is used to combine two linearly polarized beams emitted from the first and second scanning mirrors. The second annular mask is used to filter the two line-polarized beams of the combined beam; z-scanning galvanometer is used to scan the beam and scan the sample axially; The excitation objective lens is used to focus three linear beams onto the sample surface to generate interference lattice fringe patterns and excite the sample fluorescence intensity signal. The imaging optical path module includes: The probe objective is used to collect the fluorescence signal of the sample; A camera is used to record the fluorescence intensity signal. A computer is used to control the central beam shaping system and the galvanometer beam splitting system to change the phase, numerical aperture, and rotation direction of the interference lattice pattern; and to control the z-scan galvanometer to perform axial scanning of the sample; and to obtain a super-resolution image by processing the fluorescence intensity signal acquired by the camera.

2. The lattice light sheet microscopy imaging device as described in claim 1, characterized in that, The central beam shaping system includes: The first polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams. The polarizing beam splitter comprises a first electro-optic modulator, a first cylindrical lens, and a first annular mask, sequentially arranged in the reflected optical path. The first electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes on the sample plane. The first cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the x-direction and remains parallel in the z-direction. The first annular mask is located at the back focal plane of the first cylindrical lens and is used to filter the beam. The transmission optical path of the first polarizing beam splitter includes a first quarter-wave plate and a galvanometer beam splitting system; the first quarter-wave plate is used to convert the transmitted p-polarized light into circularly polarized light.

3. The lattice light sheet microscopy imaging device as described in claim 1, characterized in that, The laser and the galvanometer beam splitting system are sequentially positioned with the following components: Collimating lens, used to collimate laser beams; A polarizer is used to convert a laser beam into linearly polarized light; A half-wave plate is used to change the rotation direction of linearly polarized light and adjust the intensity ratio of the two linearly polarized light beams transmitted and reflected from the polarizing beam splitter.

4. The lattice light sheet microscopy imaging device as described in claim 1, characterized in that, Between the galvanometer beam splitter system and the excitation objective lens is placed: A non-polarizing mirror combines three linearly polarized beams of light. z-scanning galvanometer, controlling the beam to scan the sample under test along the z-direction; The scanning lens and tube mirror conjugate the two linearly polarized beams on the z-scanning galvanometer to the rear pupil plane of the excitation objective.

5. A lattice light sheet microscopy imaging device based on galvanometer scanning, comprising an excitation optical path module and an imaging optical path module, characterized in that: The excitation optical path module has the following components arranged in sequence: A laser emits a laser beam; A central beam shaping system is used to shape the beam into a linear vibrating beam and illuminate the sample from the center of the back focal plane of the excitation objective. A galvanometer beam splitter system is used to split a laser beam into two linearly polarized beams that have symmetrical propagation directions and the same vibration direction and are incident on the sample from the edge of the back focal plane of the excitation objective. The aforementioned galvanometer beam splitting system includes: The second polarizing beam splitter is used to split the incident circularly polarized light into two linearly polarized light beams. A second cylindrical lens, a first scanning galvanometer, and a second half-wave plate are sequentially arranged on the transmission optical path of the polarizing beam splitter. The second cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and kept parallel in the x-direction. The first scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The second half-wave plate is used to convert the p-polarized light in the transmission optical path into s-polarized light. A second electro-optic modulator, a third cylindrical lens, a second scanning galvanometer, and a third half-wave plate are sequentially arranged on the reflected optical path of the second polarizing beam splitter. The second electro-optic modulator is used to rapidly control the optical path of the reflected beam, thereby changing the phase of the interference lattice fringes on the sample plane. The third cylindrical lens is used to shape the circular laser beam into linearly polarized light that is compressed along the z-direction and kept parallel in the x-direction. The second scanning galvanometer is used to scan the beam along the x-direction at the entrance pupil of the excitation objective lens. The third half-wave plate is used to convert the s-linearly polarized light in the transmitted optical path into p-linearly polarized light. The third polarizing beam splitter is used to combine the two linearly polarized beams emitted from the first and second scanning mirrors. The third quarter-wave plate is used to convert the two linearly polarized light emitted from the third polarizing beam splitter into circularly polarized light. The second annular mask is used to filter the two line-polarized beams of the combined beam; A tangential polarizer is used to convert two circularly polarized lights into linearly polarized lights with the same vibration direction. z-scanning galvanometer is used to scan the beam and scan the sample axially; The excitation objective lens is used to focus two linearly polarized beams with the same vibration direction onto the sample surface to generate interference lattice fringe patterns and excite the sample fluorescence intensity signal. The imaging optical path module includes: The probe objective is used to collect the fluorescence signal of the sample; A camera is used to record the fluorescence intensity signal. A computer is used to control the beam splitting system of the galvanometer to change the phase, numerical aperture, and orientation of the interference lattice pattern; to control the z-scan galvanometer to perform axial scanning of the sample; and to obtain a super-resolution image by processing the fluorescence intensity signal acquired by the camera.

Images