CN116465868A - Crystal lattice light sheet microscopic imaging method and device based on galvanometer scanning - Google Patents
Crystal lattice light sheet microscopic imaging method and device based on galvanometer scanning Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0068—Optical details of the image generation arrangements using polarisation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/28—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising
- G02B27/283—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising used for beam splitting or combining
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
Abstract
The invention discloses a lattice polished section microscopic imaging method based on galvanometer scanning, which comprises the following steps: dividing the laser into three paths of linearly polarized light, and shaping the linearly polarized light through a cylindrical lens; the light is projected onto a fluorescent sample through an excitation objective lens to generate lattice fringe structural light illumination patterns by mutual interference; changing the phase of the interference lattice fringes by using an electro-optical modulator, realizing that the sample is subjected to structured light illumination by using the lattice fringes with step phase shift in a structured light mode, and enabling the lattice interference lattice fringes to rapidly shake in a shaking mode so as to realize that the sample is subjected to illumination by using a uniform light sheet; and collecting fluorescent signals emitted by the sample by the detection objective lens to obtain fluorescent intensity information, and reconstructing to obtain a high-resolution light sheet microscopic image. The invention also discloses a lattice light sheet microscopic imaging device based on galvanometer scanning. The invention has high utilization rate of the energy of the incident light and high contrast of the interference lattice fringes, and can obtain the resolution exceeding the diffraction limit under the condition of low incident light power.
Description
Technical Field
The invention belongs to the field of optical super-resolution microscopic imaging, and particularly relates to a lattice light sheet microscopic imaging method and device based on galvanometer scanning.
Background
Optical microscopy plays an important role in life sciences research. Among them, the light sheet fluorescence microscope is one of the most promising tools in the three-dimensional long-time imaging study of samples. The light sheet microscope adopts a side illumination strategy, samples are laterally illuminated, fluorescent signals of the samples are collected by utilizing a detection objective lens perpendicular to an excitation optical axis, off-axis excitation of the samples is avoided, the light sheet microscope naturally has optical layer cutting capability, images with good signal to noise ratio can be obtained under the irradiation of weak light illumination power, the premature photo-bleaching and photo-toxicity of the samples are avoided, and long-time living imaging can be carried out on the cell samples. In addition, the detection path adopts a wide-field detection mode, so that the imaging speed is high, and the dynamic process of capturing the biological sample is facilitated. However, the imaging resolution of the light sheet microscope is low, which is disadvantageous for detecting the fine structure of the sample.
In recent years, in order to improve imaging resolution of light sheet microscopes, researchers have proposed various resolution enhancement methods. The resolution can be effectively improved by producing a thinner gaussian light sheet using a higher Numerical Aperture (NA) excitation objective and detecting the sample with a high NA detection objective. However, this type of approach can cause rapid convergence and divergence of the light sheet, resulting in a smaller imaging field of view, compromising imaging speed. Subsequently, researchers replace gaussian beams with bessel beams to improve the axial resolution of the imaging field while ensuring the imaging field of view. However, the side lobe of Bessel light sheet can excite the sample off-axis, so that the optical layer cutting capability of the light sheet microscope is reduced, and more phototoxicity is brought to the sample.
In order to solve the problem caused by Bessel beam sidelobes, the patent application with the publication number of CN110220875A provides a lattice light sheet microscope, and the interference effect among Bessel beam arrays is utilized to successfully inhibit the Bessel beam sidelobes, so that the lattice light sheet with thin thickness and large field of view is obtained, and the lattice light sheet has the advantages of high imaging resolution and high imaging speed. However, in order to obtain the lattice light sheet, the original lattice light sheet microscopic imaging system adopts a binary spatial light modulator to modulate an incident light beam, and filters a diffracted light beam through a mask plate, so that only positive and negative first-order diffracted light is reserved to be applied to the generation of the lattice light sheet. Due to the spatial light modulator pixel gap, most of the incident light energy is concentrated in the zero-order diffracted light that is not modulated and utilized, resulting in very low light energy utilization of the system.
In order to solve the problem, researchers propose to pass the generated circular ring light beam through a customized mask plate with only 4 slits and light transmittance, so as to obtain accurate light field distribution of the back focal plane of the excitation objective lens, and form a lattice light sheet illumination sample. However, since the mask blocks most of the light sources, the light energy utilization of the system is still not high, and since the mask is fixed, the switching of the light beam NA at the entrance pupil of the excitation objective lens is not flexible enough. The patent application with publication number of CN110687670A provides an occasion light-forming sheet microscope, and the light beam is continuously or discretely scanned at the entrance pupil of the excitation objective lens by utilizing a scanning galvanometer, so that the light energy utilization rate of the system can be greatly improved. However, the light-imaging microscope cannot be compatible with the structured light obvious micro-imaging technology, so that the imaging resolution is difficult to improve, and the imaging speed is damaged to a certain extent because the entrance pupil needs to be scanned.
Disclosure of Invention
The invention provides a lattice light sheet microscopic imaging method and device based on galvanometer scanning, which utilize galvanometer scanning to flexibly switch light beams NA and utilize light beam interference effect to generate a structured light pattern on a sample surface.
In order to achieve the above purpose, the specific technical scheme adopted by the invention is as follows:
a lattice light sheet microscopic imaging method based on galvanometer scanning comprises the following steps:
1) Dividing the laser beam into three linearly polarized light with the same vibration direction;
2) Shaping three paths of linearly polarized light into linear light beams, and projecting the linear light beams onto a fluorescent sample through an excitation objective lens to generate lattice fringe structure light illumination patterns by mutual interference;
3) Changing the phase of the interference lattice fringes for a plurality of times, and collecting a plurality of fluorescence intensity images under the corresponding phase by the detection objective lens;
4) And (5) carrying out data processing by using a plurality of fluorescence intensity images, and reconstructing to obtain a super-resolution image.
In step 3), it is necessary to change the phase of the interference lattice fringes at least five times so that the light sheet illuminates the sample with a structured light fringe or a uniform light sheet.
In the present invention, the fluorescence intensity information obtained by projecting a single structured light pattern onto a sample contains five frequency components, and in order to separate the five frequency components, five equations are required to be obtained. The original lattice light sheet microscopic imaging system utilizes an x-scanning galvanometer conjugated with the back focal plane of an excitation objective lens to step the generated lattice light beam along the x direction to realize the phase shift of the lattice fringe structure light illumination pattern.
Preferably, the electro-optic modulator may control the fringe phase shifts of the interference pattern by 0 °, 72 °, 144 °, 216 °, and 288 °. The phase shift angle may be any value in theory, only to ensure that the sample is uniformly illuminated. In addition, the electro-optic modulator can also be made to change the phase of the light beam rapidly, thereby forming a uniform illumination sheet, which is matched to the dither pattern of a lattice light sheet microscope, with a faster imaging speed but a relatively lower imaging resolution than the structured light illumination pattern.
The invention is completely compatible with the image reconstruction algorithm applied by the traditional lattice light sheet microscopic imaging technology, and can be realized based on the existing algorithm in image data processing and reconstruction.
The invention provides a lattice light sheet microscopic imaging device based on galvanometer scanning, which comprises an excitation light path module and an imaging light path module;
the excitation light path module is provided with the following components:
the laser emits laser beams;
a center beam shaping system for shaping the beam into linear line vibration light and illuminating the sample from the center of the back focal plane of the excitation objective lens;
the vibrating mirror beam splitting system is used for splitting a laser beam into two linearly polarized lights which are symmetrical in propagation direction and same in vibration direction, and illuminating a sample from the edge of the back focal plane of the excitation objective lens;
the z scanning galvanometer is used for scanning the light beam and axially scanning the sample;
the excitation objective lens is used for focusing three linear beams on the surface of the sample to generate interference lattice fringe patterns by interference and exciting a fluorescent intensity signal of the sample;
the imaging light path module comprises:
a detection objective for collecting fluorescent signals of the sample;
a camera for recording the fluorescence intensity signal;
the computer is used for controlling the central beam shaping system and the galvanometer beam splitting system to change the phase, the numerical aperture and the rotation direction of the interference lattice pattern; the Z scanning galvanometer is used for controlling the Z scanning galvanometer to axially scan the sample; the fluorescent intensity signal is used for controlling the camera to acquire; and is used for data processing to obtain super-resolution images.
Preferably, the central beam shaping system comprises:
the first polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the first electro-optical modulator, the first cylindrical lens and the first annular mask plate are sequentially arranged on a reflection light path of the polarization beam splitter. The first electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the first cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction; the first annular mask plate is positioned at the focal plane of the first cylindrical lens and is used for filtering the light beam;
the transmission light path sequentially arranged on the first polarization beam splitter comprises a first quarter wave plate and a galvanometer beam splitting system. The first quarter wave plate is used for converting the transmitted p-polarized light into circularly polarized light.
Preferably, the galvanometer beam splitting system includes:
the polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the second quarter wave plate, the reflecting mirror, the second lens, the first scanning galvanometer and the first scanning lens are sequentially arranged on a transmission light path of the polarization beam splitter; the second quarter wave plate is used for converting linearly polarized light into circularly polarized light, the reflecting mirror is used for reflecting the circularly polarized light to be changed into s polarized light through the second quarter wave plate again, and the s polarized light can be reflected again by the second polarization beam splitter; the second lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the first scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the first scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction;
the second electro-optical modulator, the second lens, the second scanning galvanometer and the second scanning lens are sequentially arranged on the reflecting light path of the second polarization beam splitter; the second electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the third cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the second scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the second scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and is kept parallel along the z direction;
in the invention, the galvanometer beam splitting system comprises: the triangular reflector is used for combining the two paths of linearly polarized light emitted from the first scanning lens and the second scanning lens;
preferably, the galvanometer beam splitting system includes: the second annular mask plate is used for filtering the two paths of linearly polarized light of the combined beam.
The laser and the central beam shaping system are sequentially arranged: the collimating lens is used for collimating the laser beam; a polarizer for converting the laser beam into linearly polarized light; and the half wave plate is used for changing the rotation direction of the linearly polarized light and adjusting the light intensity ratio of the two linearly polarized lights transmitted and reflected by the polarization beam splitter.
And a non-polarized beam splitter, a z-scanning galvanometer, a third scanning lens and a first tube lens are sequentially arranged between the galvanometer beam splitting system and the excitation objective lens. The non-polarized reflector is used for combining three linearly polarized lights into a subsequent system; the z scanning galvanometer is used for controlling the light beam to scan the sample to be detected along the z direction; the third scanning lens and the tube lens form a 4f system for conjugating the light beam on the z scanning galvanometer to the back focal plane of the excitation objective lens.
The excitation objective lens is used for transmitting three linearly polarized lights with the same vibration direction to the surface of the sample to be tested to interfere to generate interference lattice fringes.
The imaging light path module is sequentially provided with a detection objective lens, an optical filter, a second tube lens and a camera. The detection objective lens is perpendicular to the excitation objective lens and is used for collecting fluorescent signals of a sample; the optical filter is used for filtering stray light in fluorescence emitted by the sample to be detected; the tube mirror is used to focus the fluorescent signal to the camera.
In another technical scheme, the invention also provides a lattice light sheet microscopic imaging device based on galvanometer scanning, which comprises an excitation light path module and an imaging light path module;
the excitation light path module is provided with the following components:
the laser emits laser beams;
a center beam shaping system for shaping the beam into linear line vibration light and illuminating the sample from the center of the back focal plane of the excitation objective lens;
the vibrating mirror beam splitting system is used for splitting a laser beam into two linearly polarized lights which are symmetrical in propagation direction and same in vibration direction, and illuminating a sample from the edge of the back focal plane of the excitation objective lens;
the z scanning galvanometer is used for scanning the light beam and axially scanning the sample;
the excitation objective lens is used for focusing three linear beams on the surface of the sample to generate interference lattice fringe patterns by interference and exciting a fluorescent intensity signal of the sample;
the imaging light path module comprises:
a detection objective for collecting fluorescent signals of the sample;
a camera for recording the fluorescence intensity signal;
the computer is used for controlling the central beam shaping system and the galvanometer beam splitting system to change the phase, the numerical aperture and the rotation direction of the interference lattice pattern; the Z scanning galvanometer is used for controlling the Z scanning galvanometer to axially scan the sample; the fluorescent intensity signal is used for controlling the camera to acquire; and is used for data processing to obtain super-resolution images.
Preferably, the central beam shaping system comprises:
the first polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the first electro-optical modulator, the first cylindrical lens and the first annular mask plate are sequentially arranged on a reflection light path of the polarization beam splitter. The first electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the first cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction; the first annular mask plate is positioned at the focal plane of the first cylindrical lens and is used for filtering light beams.
The transmission light path sequentially arranged on the first polarization beam splitter comprises a first quarter wave plate and a galvanometer beam splitting system. The first quarter wave plate is used for converting the transmitted p-polarized light into circularly polarized light.
Preferably, the galvanometer beam splitting system includes:
the first polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the second lens, the first scanning galvanometer, the first scanning lens and the second half wave plate are sequentially arranged on a transmission light path of the first polarization beam splitter; the second lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the first scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the first scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction; the second half wave plate is used for changing the polarization state of the light beam into s polarized light beam.
The second electrooptic modulator, the third cylindrical lens, the second scanning galvanometer, the second scanning lens and the third half wave plate are sequentially arranged on the reflecting light path of the first polarization beam splitter; the second electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of interference fringes on the sample plane; the third cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the second scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the second scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and is kept parallel along the z direction; the third half wave plate is used for changing the polarization state of the light beam into p polarized light beam.
In the invention, the galvanometer beam splitting system comprises: the third polarization beam splitter is used for combining linearly polarized light emitted by 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; the second annular mask plate is used for filtering the two paths of circularly polarized light of the combined beam; a tangential light polarizing plate for converting the two circularly polarized lights into tangential linearly polarized lights.
In the invention, the laser and the first polarization beam splitter are sequentially arranged between: the collimating lens is used 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 light intensity ratio of the two linearly polarized lights transmitted and reflected from the polarization beam splitter.
And a non-polarized beam splitter, a z-scanning galvanometer, a third scanning lens and a first tube lens are sequentially arranged between the galvanometer beam splitting system and the excitation objective lens. The non-polarized reflector is used for combining three linearly polarized lights into a subsequent system; the z scanning galvanometer is used for controlling the light beam to scan the sample to be detected along the z direction; the scanning lens and the tube lens form a 4f system for conjugating the light beam on the z scanning galvanometer to the back focal plane of the excitation objective lens.
The excitation objective lens is used for transmitting three linearly polarized lights with the same vibration direction to the surface of the sample to be tested to interfere to generate interference lattice fringes.
The imaging light path module is sequentially provided with a detection objective lens, an optical filter, a second tube lens and a camera. The detection objective lens is perpendicular to the excitation objective lens and is used for collecting fluorescent signals of a sample; the optical filter is used for filtering stray light in fluorescence emitted by the sample to be detected; the tube mirror is used to focus the fluorescent signal to the camera.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) The vibrating mirror beam splitting system is used for replacing a spatial light modulator and a conventional grating device to acquire light field distribution of a back focal plane and interference patterns of a front focal plane of an excitation objective lens, so that the energy utilization rate of incident light is improved;
(2) The electro-optical modulator is used for controlling the optical path of the reflecting path so as to change the phase of the reflected image of the front focal plane of the excitation objective lens, and compared with the traditional pyramid prism, the displacement accuracy is high and the modulation speed is high;
(3) The scanning galvanometer can flexibly change the distribution of the light beam on the back focal plane of the excitation objective lens, change the NA of the light beam and acquire lattice patterns of different modes on the sample surface;
(4) The three-dimensional super-resolution structure illumination microscopy is combined with the lattice light sheet microscopy, so that the imaging resolution of two dimensions xz is further improved, and super-resolution imaging under the condition of low incident light power is realized.
(5) The device is simple and flexible, and is convenient to operate; the method has the advantages of high energy utilization rate of incident light, high contrast of interference lattice fringes, compatibility with a structured light microscopic illumination technology, realization of resolution exceeding diffraction limit under the condition of low incident light power, and particular suitability for imaging fluorescent samples in the field of life science.
Drawings
FIG. 1 is a schematic diagram of a lattice light sheet microscopic imaging device based on galvanometer scanning according to the present invention.
FIG. 2 is a schematic diagram of a device for controlling the light intensity distribution and polarization state of a light beam; wherein (a) is a schematic diagram of an annular mask plate; (b) The diagram is a schematic diagram of the light intensity distribution of the dual light beams after the transmission of (a) and after the excitation of the objective lens; (c) is a schematic view of a tangential polarizer.
FIG. 3 is a distribution of an excitation beam over a sample plane; wherein (a) is a two-dimensional schematic diagram of a lattice pattern generated by a lattice light sheet microscopic imaging system based on galvanometer scanning; (b) is a one-dimensional intensity distribution map corresponding to the graph (a); (c) is a spectrogram corresponding to the graph (a).
FIG. 4 is a schematic diagram of a lattice interference light sheet microscopic imaging device based on galvanometer scanning according to the present invention.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those described herein, and therefore the present invention is not limited to the specific embodiments disclosed below.
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Example 1
The light sheet microscopic imaging device as shown in fig. 1 includes: the laser 1, the collimating lens 2, the polarizer 3, the half-wave plate 4, the first reflecting mirror 5, the first polarization beam splitter 6, the first electro-optic modulator 7, the first lens 8, the second lens 9, the second reflecting mirror 10, the third reflecting mirror 11, the fourth reflecting mirror 12, the first cylindrical lens 13, the fifth reflecting mirror 14, the first annular mask plate 15, the first quarter-wave plate 16, the second polarization beam splitter 17, the second quarter-wave plate 18, the sixth reflecting mirror 19, the third lens 20, the fourth lens 21, the second cylindrical lens 22, the first scanning galvanometer 23, the first scanning lens 24, the seventh reflecting mirror 25, the second electro-optic modulator 26, the fifth lens 27, the sixth lens 28, the third cylindrical lens 29, the second scanning galvanometer 30, the second scanning lens 31, the eighth reflecting mirror 32, the triangular reflecting mirror 33, the second annular mask plate 34, the seventh lens 35, the eighth lens 36, the non-polarization beam splitter 37, the ninth reflecting mirror 38, the ninth reflecting mirror 39, the z-scanning mirror 40, the third galvanometer lens 41, the objective lens 45, the objective lens 46, the lens 45, the lens 46, the lens to be measured sample to be measured, the lens 43, the lens to be measured and the lens to be measured 45.
The laser 1 emits a laser beam, and the collimator lens 2, the polarizer 3 and the half-wave plate 4 are placed in this order on the optical axis of the optical path of the laser beam. The collimating lens 2 is used for collimating laser beams to obtain parallel beams, the polarizer 3 is used for converting outgoing laser into linearly polarized light, the first half-wave plate 4 is used for changing the rotation direction of the linearly polarized light, and the light intensity ratio of the two linearly polarized light beams transmitted and reflected by the polarization beam splitter is adjusted.
The circularly polarized light after passing through the first reflecting mirror 5 enters the first polarization beam splitter 6. The polarization beam splitter 6 splits the light beam into two paths. The s polarized light of the first path of reflected light path enters a beam expanding system consisting of a first lens 8 and a second lens 9 through a first electro-optical modulator 7 to be expanded. The optical path length of the reflected light path can be adjusted by adjusting the positions of the second reflecting mirror 10 and the third reflecting mirror 11 back and forth. The first electro-optical modulator 7 can change the phase of the light beam rapidly. The light beam is reflected by the fourth reflecting mirror 12 and enters the first cylindrical lens 13 to form a linear s-polarized light beam which is compressed along the x direction and remains unchanged along the z direction. The line beam is reflected by the fifth mirror 14 and focused on the first annular mask 15 for filtering. The p polarized light transmitted by the second path is modulated into circular polarized light after passing through the first quarter wave plate 16, and then enters the second polarization beam splitter 17, and is further divided into two light paths.
The p-polarized light transmitted through the second polarization beam splitter 17 passes through the second quarter wave plate 18, is reflected after being perpendicularly incident on the sixth reflecting mirror 19, is changed into s-polarized light again through the second quarter wave plate 18, and is reflected by the second polarization beam splitter 17. The light beam is expanded after entering the third lens 20 and the fourth lens 21, is emitted from the second lens 22, forms linearly polarized light compressed along the z direction and parallel to the x direction, and passes through the first scanning galvanometer 23, the first scanning lens 24 and the seventh reflecting mirror 25, and is applied to the triangular reflecting mirror 33. The s-polarized light reflected by the second polarization beam splitter 17 enters the second electro-optical modulator 26, exits, expands the beam by the fifth lens 27 and the sixth lens 28, enters the third cylindrical lens 29, forms linear polarized light compressed along the z direction and parallel to the x direction, and passes through the second scanning vibrating mirror 30, the second scanning lens 31 and the eighth reflecting mirror 32 to strike the triangular reflecting mirror 33. The first scanning galvanometer 23 and the second scanning galvanometer 30 can easily change the position of the emergent light on the back focal plane of the excitation objective 43, thereby flexibly changing the numerical aperture and the rotation direction of the light beam and realizing illumination of the sample 44 to be measured on the sample surface by the light beams with different thicknesses and propagation lengths. The second electro-optic modulator 26 can rapidly change the phase of the light beam to achieve structured light illumination of the sample or to produce a uniform light sheet to illuminate the sample 44 to be measured.
The upper and lower light beams are reflected by the triangular reflector 31 and then filtered by the second annular mask plate 34; after passing through the seventh lens 35, the unpolarized beam splitter 37, the ninth lens 38, and the ninth mirror 39, it strikes the z-scanning galvanometer 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-scan galvanometer 40 is located at the back focal plane of the ninth lens 38. The image of the second annular mask 34 is therefore conjugated to the z-scan galvanometer 40.
The eighth lens 36 and the ninth lens 38 form a 4f imaging system, and the first annular mask 15 is located on the front focal plane of the eighth lens 36. The image of the first annular mask 15 is therefore conjugated to the z-scan galvanometer 40 after reflection of the light by the unpolarized mirror 37 and the ninth mirror 39.
The three beams are scanned by the z-scan galvanometer 40, pass through the third scan lens 41 and the first tube lens 42, enter the excitation objective lens 43, and generate interference beams at the plane of the sample 44 to be measured. The third scanning lens 41 and the first tube mirror 42 constitute a 4f imaging system: the z-scan beam 40 is located at the front focal plane of the third scan lens 41, and the back focal plane of the excitation objective lens 43 coincides with the back focal plane of the first tube lens 42. Therefore, the first annular mask 15, the second annular mask 34 and the z-scan galvanometer 40 are conjugated with the back focal plane of the excitation objective 43, and the z-scan galvanometer 40 rotates the angle scanning beam such that the illumination beam scans the sample 44 to be measured along the z-axis direction. FIG. 2 (a) is a schematic diagram of an annular mask plate, which is a related device for controlling the light intensity distribution and the polarization state of a light beam and is shown in FIG. 2; FIG. 2 (b) is a graph showing the intensity distribution of the dual beam after exciting the objective lens after passing through FIG. 2 (a); fig. 2 (c) is a schematic view of a tangential polarizer.
The detection objective 45 is perpendicular to the excitation objective 43. The fluorescence excited by the sample is collected by the detection objective 45, passed through the filter 46 and the second tube lens 47, and then received by the camera 48 for recording. The optical filter 46 is used for filtering stray light in fluorescence emitted by the sample to be detected, and the second tube mirror 47 is used for imaging the internal fluorescence intensity information of the sample to be detected onto the camera 48. FIG. 3 (a) is a two-dimensional schematic of a lattice pattern generated by a galvanometer scanning based lattice light sheet microscopy imaging system; FIG. 3 (b) is a one-dimensional intensity distribution corresponding to FIG. 3 (a); fig. 3 (c) is a spectrum diagram corresponding to fig. 3 (a).
The working method of the field interference lattice light sheet microscopic imaging device based on galvanometer scanning shown in fig. 1 is as follows:
the laser beam emitted by the laser 1 passes through the polarizer 3 and the first half wave plate 4 to form linearly polarized light, and then is incident on the first polarization beam splitter 6 and the second polarization beam splitter 17 to be split into three light paths in total. The light beam reflected from the first polarization beam splitter 6 passes through the first cylindrical lens 13 to form linear polarized light, and the linear polarized light is filtered by the first annular mask 15; the two linearly polarized lights transmitted from the first polarization beam splitter 6 and split by the second polarization beam splitter 17 are emitted from the second lens 22, the first scanning galvanometer 23, the third lens 29, and the second scanning galvanometer 30 as two linearly polarized lights converging in the x direction in parallel with the z direction, and filtered by the second annular mask 34. The three beams are conjugated to a z-scan galvanometer 40 and imaged to the rear pupil plane of excitation objective lens 43. The three laser beams interfere to form periodic illumination stripes to excite the sample 44 to be detected with fluorescent marks to generate fluorescent signals, and the fluorescent signals are received by a detection objective 45 perpendicular to the excitation objective and imaged on a camera 48. The positions of the two linearly polarized light beams at the entrance pupil are changed by changing the rotation angles of the first scanning galvanometer 23 and the second scanning galvanometer 30, thereby changing the NA of the light beams.
In this embodiment, two schemes may be employed to excite the sample.
Dither patterns: the first electro-optical modulator 7 and the second electro-optical modulator 26 are controlled by the computer 49 to quickly change the optical path difference of the reflected light path, so that the lattice fringe structure light pattern projected on the sample 44 to be measured continuously and quickly shakes along the x direction to form a uniform illumination light sheet. The excited fluorescent signal of the sample 44 to be detected is collected by the detecting objective 45, stray light in the collected fluorescent light is filtered by the filter 46, and is imaged on the camera 48 by the second tube lens 47, so that the fluorescent intensity information of the sample 44 to be detected can be obtained.
Structured light mode: exciting the sample 44 to be tested using a static structured light illumination pattern; the fluorescent signal emitted by the sample 44 to be measured is collected by the detection objective 45 and imaged on the camera 48 through the optical filter 46 and the second tube lens 47. The fluorescence intensity information obtained by projecting a single structured light pattern onto the sample contains five frequency components, so that five image equations are needed to separate the five frequency components, and the computer 49 controls the first electro-optical modulator 7 and the second electro-optical modulator 26 to change the phases of the light beams, so that the lattice fringe structured light pattern projected onto the sample 44 to be measured is discretely phase-shifted by 0 °, 72 °, 144 °, 216 °, 288 °, and five phase-shifted fluorescence images are obtained, thereby extracting and moving the frequency components. And carrying out data processing on the five acquired images by combining an image reconstruction algorithm of a structured light microscopic imaging technology, so as to obtain a super-resolution image in the xz direction.
Example 2
As shown in fig. 4, the light sheet microscopic imaging device of this embodiment may also use a tangential polarizer to obtain linearly polarized light with the same vibration direction. Fig. 4 is a view showing that, compared with fig. 1, in the transmission optical path of the second polarization beam splitter in embodiment 2, the original second quarter-wave plate 18 and sixth mirror 19 are replaced with tenth mirror 51 and eleventh mirror 52 to control the optical path of the transmission optical path; the original triangular reflector 33 is replaced by a third polarization beam splitter 54, and a second half wave plate 50 and a third half wave plate 53 are added to control the polarization states of the light beams, so that linearly polarized light beams of the two light paths can be combined by the third polarization beam splitter 54; a third quarter wave plate 55 is added to convert the two paths of linearly polarized light into circularly polarized light; the addition of the tangential polarizer 56 converts the two circularly polarized light into linearly polarized light having the same vibration direction.
Finally, three linearly polarized lights with the same vibration direction are imaged on the rear pupil surface of the excitation objective lens 43, and an interference light beam is formed on the sample 44 to be detected to excite the fluorescent signal of the sample 44 to be detected.
The remainder was the same as in example 1.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. The lattice polished section microscopic imaging method based on galvanometer scanning is characterized by comprising the following steps of:
1) Dividing the laser beam into three linearly polarized light with the same vibration direction;
2) Shaping three paths of linearly polarized light into linear light beams, and projecting the linear light beams onto a fluorescent sample through an excitation objective lens to generate lattice fringe structure light illumination patterns by mutual interference;
3) Changing the phase of the interference lattice fringes for a plurality of times, and collecting a plurality of fluorescence intensity images under the corresponding phase by using a detection objective lens;
4) And (5) carrying out data processing by using a plurality of fluorescence intensity images, and reconstructing to obtain a super-resolution image.
2. The method of microscopic imaging of an optical sheet of claim 1 wherein in step 3), at least five changes in the phase of the interference lattice fringes are required to illuminate the optical sheet with structured light fringes or a uniform optical sheet.
3. The optical sheet microscopy imaging method of claim 1, wherein the excitation objective lens and the detection objective lens are disposed perpendicular to each other.
4. The utility model provides a lattice polished section microscopic imaging device based on galvanometer scanning, includes excitation light path module and formation of image light path module, its characterized in that:
the excitation light path module comprises the following components in sequence:
the laser emits laser beams;
a center beam shaping system for shaping the beam into linear line vibration light and illuminating the sample from the center of the back focal plane of the excitation objective lens;
the vibrating mirror beam splitting system is used for splitting a laser beam into two linearly polarized lights which are symmetrical in propagation direction and same in vibration direction, and illuminating a sample from the edge of the back focal plane of the excitation objective lens;
the z scanning galvanometer is used for scanning the light beam and axially scanning the sample;
the excitation objective lens is used for focusing three linear beams on the surface of the sample to generate interference lattice fringe patterns by interference and exciting a fluorescent intensity signal of the sample;
the imaging light path module comprises:
a detection objective for collecting fluorescent signals of the sample;
a camera for recording the fluorescence intensity signal;
the computer is used for controlling the central beam shaping system and the galvanometer beam splitting system to change the phase, the numerical aperture and the rotation direction of the interference lattice pattern; and controlling the z scanning galvanometer to axially scan the sample; and according to the fluorescence intensity signals acquired by the camera, obtaining a super-resolution image through data processing.
5. The patterned light sheet microimaging device of claim 4, wherein the central beam shaping system comprises:
the first polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the first electro-optic modulator, the first cylindrical lens and the first annular mask plate are sequentially arranged on a reflection light path of the polarization beam splitter; the first electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the first cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction; the first annular mask plate is positioned at the rear focal plane of the first cylindrical lens and is used for filtering light beams;
the transmission light path sequentially arranged on the first polarization beam splitter comprises a first quarter wave plate and a galvanometer beam splitting system; the first quarter wave plate is used for converting the transmitted p-polarized light into circularly polarized light.
6. The patterned light sheet microscopic imaging device of claim 4, wherein the galvanometer beam splitting system comprises:
the second polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the second quarter wave plate, the reflecting mirror, the second lens, the first scanning galvanometer and the first scanning lens are sequentially arranged on a transmission light path of the polarization beam splitter; the second quarter wave plate is used for converting linearly polarized light into circularly polarized light, the reflecting mirror is used for reflecting the circularly polarized light to be changed into s polarized light through the second quarter wave plate again, and the s polarized light can be reflected again by the second polarization beam splitter; the second lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the first scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the first scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction;
the second electrooptic modulator, the third cylindrical lens, the second scanning galvanometer and the second scanning lens are sequentially arranged on the reflecting light path of the second polarization beam splitter; the second electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the third cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the second scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the second scanning lens is used for converting the linear light beam into linear linearly polarized light which is compressed along the x direction and kept parallel along the z direction;
the triangular reflector is used for combining two paths of linearly polarized light emitted from the first scanning galvanometer and the second scanning galvanometer;
the second annular mask plate is used for filtering the two paths of linearly polarized light of the combined beam.
7. The patterned light sheet microscopic imaging device according to claim 4, wherein the laser and the galvanometer beam splitting system are sequentially arranged between:
the collimating lens is used for collimating the laser beam;
a polarizer for converting the laser beam into linearly polarized light;
the half wave plate is used for changing the rotation direction of the linearly polarized light and adjusting the light intensity ratio of the two linearly polarized lights transmitted and reflected by the polarization beam splitter.
8. The lattice light sheet microscopic imaging device of claim 4, wherein the galvanometer beam splitting system and the excitation objective lens are arranged between:
a non-polarizing mirror for combining the three linearly polarized lights;
a z scanning galvanometer, which controls the light beam to scan the sample to be detected along the z direction;
and the scanning lens and the tube mirror conjugate the two linearly polarized lights on the z scanning galvanometer to the rear pupil surface of the excitation objective lens.
9. The utility model provides a lattice polished section microscopic imaging device based on galvanometer scanning, includes excitation light path module and formation of image light path module, its characterized in that:
the excitation light path module is provided with the following components:
the laser emits laser beams;
a center beam shaping system for shaping the beam into linear line vibration light and illuminating the sample from the center of the back focal plane of the excitation objective lens;
the vibrating mirror beam splitting system is used for splitting a laser beam into two linearly polarized lights which are symmetrical in propagation direction and same in vibration direction, and illuminating a sample from the edge of the back focal plane of the excitation objective lens;
the z scanning galvanometer is used for scanning the light beam and axially scanning the sample;
the excitation objective lens is used for focusing two linearly polarized lights with the same vibration direction on the surface of the sample to interfere to generate an interference lattice fringe pattern and excite a fluorescence intensity signal of the sample;
the imaging light path module comprises:
a detection objective for collecting fluorescent signals of the sample;
a camera for recording the fluorescence intensity signal;
the computer is used for controlling the galvanometer beam splitting system to change the phase, the numerical aperture and the direction of the interference lattice pattern; the Z scanning galvanometer is used for controlling the Z scanning galvanometer to axially scan the sample; and according to the fluorescence intensity signals acquired by the camera, obtaining a super-resolution image through data processing.
10. The patterned light sheet microscopic imaging device of claim 9, wherein the galvanometer beam splitting system comprises:
the second polarization beam splitter is used for splitting incident circularly polarized light into two paths of linearly polarized light beams;
the second lens, the first scanning galvanometer and the second half wave plate are sequentially arranged on a transmission light path of the polarization beam splitter; the second lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the first scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the second half wave plate is used for converting p linearly polarized light of the transmission light path into s linearly polarized light;
the second electro-optical modulator, the second lens, the second scanning galvanometer and the third half wave plate are sequentially arranged on the reflecting light path of the second polarization beam splitter; the second electro-optical modulator is used for rapidly controlling the optical path of the reflected light path beam so as to change the phase of the interference lattice fringes on the sample plane; the third cylindrical lens is used for shaping the circular laser beam to form linear linearly polarized light which is compressed along the z direction and is kept parallel along the x direction; the second scanning galvanometer is used for scanning the light beam along the x direction at the entrance pupil of the excitation objective lens; the third half wave plate is used for converting the s-linear polarized light of the transmission light path into p-linear polarized light;
the third polarization beam splitter is used for combining two paths of linearly polarized light emitted from the first scanning galvanometer and the second scanning galvanometer;
the third quarter wave plate is used for converting the two paths of linearly polarized light emitted from the third polarization beam splitter into circularly polarized light;
the second annular mask plate is used for filtering the two paths of linearly polarized light of the combined beam;
and the tangential polaroid is used for converting the two paths of circularly polarized light into linearly polarized light with the same vibration direction.
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