CN111337464A - High-resolution confocal imaging system and imaging method based on high-order Laguerre light beams - Google Patents
High-resolution confocal imaging system and imaging method based on high-order Laguerre light beams Download PDFInfo
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- CN111337464A CN111337464A CN202010146488.4A CN202010146488A CN111337464A CN 111337464 A CN111337464 A CN 111337464A CN 202010146488 A CN202010146488 A CN 202010146488A CN 111337464 A CN111337464 A CN 111337464A
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
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- G02B21/00—Microscopes
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- 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
Abstract
The invention provides a high-resolution confocal imaging system and an imaging method based on high-order Laguerre beams3,1The Laguerre Gaussian beam in the mode is subjected to confocal imaging to obtain a high-resolution confocal image based on the high-order Laguerre beam, and the spot size of the point spread function of the high-resolution differential confocal image based on the radial polarized light is obviously smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the high-resolution confocal image has high imaging resolution, and the defects of artifacts and the like caused by an image processing method are avoided.
Description
Technical Field
The invention relates to the technical field of optical microscopy, in particular to a high-resolution confocal imaging system and method based on high-order Laguerre light beams.
Background
The laser scanning confocal microscope imaging has the chromatographic characteristic, can carry out layered imaging on a sample, has higher imaging definition, and is widely applied to biology and basic medical research. However, the imaging resolution of the confocal microscope is generally 200nm-300nm due to the limitation of light wave diffraction. In order to improve the imaging resolution of a confocal microscope, various methods are provided, and it is common to adopt post-image processing methods such as image deconvolution, but the above-mentioned image processing methods can bring about some artifacts while improving the confocal imaging resolution.
Disclosure of Invention
In view of the foregoing, there is a need for a high-resolution confocal imaging system based on high-order laguerre beams with high resolution and without image artifacts.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high-resolution confocal imaging system based on a high-order laguerre beam, comprising: the device comprises a laser, a radial polarization converter, a liquid crystal spatial light modulator, a dichroic mirror, a scanning galvanometer, a scanning lens, a cylindrical lens, an objective lens, a fluorescent light filter, a focusing lens, a pinhole, a photomultiplier and a control unit, wherein the radial polarization converter, the liquid crystal spatial light modulator and the photomultiplier are all connected with the control unit, and the radial polarization converter, the liquid crystal spatial light modulator and the photomultiplier are connected with the control unit, wherein:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, and the radial polarized light forms LG after passing through the liquid crystal spatial light modulator3,1A Laguerre Gaussian beam of a mode, wherein 3 is the radial index of the Laguerre beam, and 1 is the angular index of the Laguerre beam;
the LG3,1The Laguerre Gaussian beam of the mode enters the dichroic mirror, enters the scanning galvanometer after being reflected by the dichroic mirror, is focused on a sample after sequentially passing through the scanning lens, the tube mirror and the objective lens, and excites the sample to generate a fluorescent beam; the fluorescent light beams sequentially pass through the objective lens, the barrel lens, the scanning lens and the scanning galvanometer and then enter the dichroic mirror, are transmitted by the dichroic mirror and then enter the fluorescent light filter, then pass through the focusing lens and enter a pinhole at the focus of the focusing lens, and are detected by the photomultiplier tube, and the photomultiplier tube converts the detected fluorescent light into an electric signal to form a high-resolution confocal image based on high-order Laguerre light beams.
In some preferred embodiments, the diameter of the pinhole is 0.2 times the image-wise airy disk diameter.
In addition, the invention also provides an imaging method of the high-resolution confocal imaging system based on the high-order Laguerre beam, which comprises the following steps:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, and the radial polarized light forms LG after passing through the liquid crystal spatial light modulator3,1A Laguerre Gaussian beam of a mode, wherein 3 is the radial index of the Laguerre beam, and 1 is the angular index of the Laguerre beam;
the LG3,1The Laguerre Gaussian beam of the mode enters the dichroic mirror, enters the scanning galvanometer after being reflected by the dichroic mirror, is focused on a sample after sequentially passing through the scanning lens, the tube mirror and the objective lens, and excites the sample to generate a fluorescent beam; the fluorescent lightThe beam sequentially passes through the objective lens, the cylindrical lens, the scanning lens and the scanning galvanometer, then enters the dichroic mirror, then enters the fluorescent light filter after being transmitted by the dichroic mirror, then enters a pinhole at the focus of the focusing lens after passing through the focusing lens, and is detected by the photomultiplier tube, and the photomultiplier tube converts the detected fluorescence into an electric signal to form a high-resolution confocal image based on high-order Laguerre light beams.
In some preferred embodiments, the diameter of the pinhole is 0.2 times the image-wise airy disk diameter.
The invention adopts the technical scheme that the method has the advantages that:
the invention provides a high-resolution confocal imaging system and an imaging method based on high-order Laguerre beams3,1The Laguerre Gaussian beam in the mode is subjected to confocal imaging to obtain a high-resolution confocal image based on the high-order Laguerre beam, and the spot size of the point spread function of the high-resolution differential confocal image based on the radial polarized light is obviously smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the high-resolution confocal image has high imaging resolution, and the defects of artifacts and the like caused by an image processing method are avoided.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a high-resolution confocal imaging system based on a high-order laguerre beam according to embodiment 1 of the present invention;
FIG. 2 is a schematic representation of the invention provided in examples 1 and 2Liquid crystal spatial light modulator generation LG3,1Phase profile of a patterned laguerre gaussian beam.
FIG. 3 shows LG provided in examples 1 and 2 of the present invention3,1Schematic diagram of illumination point spread function under illumination of a laguerre gaussian beam of modes.
FIG. 4 is a diagram of the synthetic point spread function after pinhole filtering at 0.2 times the image-side Airy spot diameter provided in examples 1 and 2 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1, a schematic structural diagram of a high-resolution confocal imaging system 100 based on a high-order laguerre beam according to embodiment 1 of the present invention is shown, which includes: the device comprises a laser 110, a radial polarization converter 120, a liquid crystal spatial light modulator 130, a dichroic mirror 140, a scanning galvanometer 150, a scanning lens 160, a tube mirror 170, an objective lens 180, a fluorescence filter 190, a focusing lens 210, a pinhole 220, a photomultiplier 230 and a control unit 240.
Specifically, the radial polarization converter 120, the liquid crystal spatial light modulator 130 and the photomultiplier tube 230 are all connected to the control unit 240.
The high-resolution confocal imaging system 100 based on the high-order Laguerre beam provided by the invention has the following working principle:
the laser beam emitted from the laser 110 forms a radial polarized light through the radial polarization converter 120, and the radial polarized light forms an LG after passing through the liquid crystal spatial light modulator 1303,1A Laguerre Gaussian beam of a mode, wherein 3 is the radial index of the Laguerre beam, and 1 is the angular index of the Laguerre beam;
the LG3,1The laguerre gaussian beam of the mode enters the dichroic mirror 140, is reflected by the dichroic mirror 140, enters the scanning galvanometer 150, sequentially passes through the scanning lens 160, the barrel mirror 170 and the objective lens 180, is focused on a sample A, and excites the sample A to generate a fluorescent beam; the fluorescent light beam sequentially passes through the objective lens 180, the tube lens 170, the scanning lens 160 and the scanning galvanometer 150, then enters the dichroic mirror 140, is transmitted by the dichroic mirror 140, then enters the fluorescent light filter 190, then passes through the focusing lens 210, enters the pinhole 220 at the focus of the focusing lens 210, and is detected by the photomultiplier 230, and the photomultiplier 230 converts the detected fluorescent light into an electrical signal, so as to form a high-resolution confocal image based on a high-order Laguerre light beam.
In some preferred embodiments, the diameter of the pinhole is 0.2 times the image-wise airy disk diameter.
In the high-resolution confocal imaging system based on the high-order laguerre beam provided by the above embodiment, the optical imaging physical process is as follows:
i is image distribution, O is object distribution, H is imaging integrated point spread function,
is a convolution operator. The smaller the spot diameter of the point spread function H, the higher the resolution of the resulting image. H by the illumination point spread function HillAnd a probe point spread function hdetAnd pinhole formation, in relation to the following:
referring to FIG. 2, LG is generated for the liquid crystal spatial light modulator provided in embodiment 1 of the present invention3,1The phase profile of a patterned laguerre gaussian beam, represented by black to white, is from 0 to 2 pi.
Please refer to fig. 3, which illustrates LG provided in embodiment 1 of the present invention3,1Illumination point spread function h under modal laguerre gaussian beam illuminationillFig. 4 is a comprehensive point spread function after pinhole filtering of 0.2 times of image airy disk diameter, in fig. 3 and 4, the horizontal direction is the X-axis coordinate of the point spread function, the vertical direction is the Y-axis coordinate of the point spread function, the unit is μm, the right-side brightness bar is the corresponding scale of the intensity of the point spread function, and it can be seen from fig. 4 that the spot size of the point spread function is significantly smaller than that of the point spread function in the conventional laser scanning confocal microscopy, thus having higher imaging resolution.
The invention provides a high-resolution confocal imaging system based on high-order Laguerre beams, which introduces a liquid crystal spatial light modulator and a radial polarization converter into a laser point scanning confocal imaging illumination light path, introduces radial polarized light into the illumination light path through the radial polarization converter, and forms LG on the liquid crystal spatial light modulator3,1The Laguerre Gaussian beam in the mode is subjected to confocal imaging to obtain a high-resolution confocal image based on the high-order Laguerre beam, and the spot size of the point spread function of the high-resolution differential confocal image based on the radial polarized light is obviously smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the high-resolution confocal image has high imaging resolution, and the defects of artifacts and the like caused by an image processing method are avoided.
Example 2
This embodiment 2 provides a high-resolution confocal imaging method based on a high-order laguerre beam, which includes the following steps:
the laser beam emitted from the laser 110 forms a radial polarized light through the radial polarization converter 120, and the radial polarized light forms an LG after passing through the liquid crystal spatial light modulator 1303,1A Laguerre Gaussian beam of a mode, wherein 3 is the radial index of the Laguerre beam, and 1 is the angular index of the Laguerre beam;
the LG3,1The laguerre gaussian beam enters the dichroic mirror 140, is reflected by the dichroic mirror 140, enters the scanning galvanometer 150, and then sequentially passes through the scanning lens 160, the barrel mirror 170, and the objectThe mirror 180 is focused on the sample A, and excites the sample A to generate a fluorescent light beam; the fluorescent light beam sequentially passes through the objective lens 180, the tube lens 170, the scanning lens 160 and the scanning galvanometer 150, then enters the dichroic mirror 140, is transmitted by the dichroic mirror 140, then enters the fluorescent light filter 190, then passes through the focusing lens 210, enters the pinhole 220 at the focus of the focusing lens 210, and is detected by the photomultiplier 230, and the photomultiplier 230 converts the detected fluorescent light into an electrical signal, so as to form a high-resolution confocal image based on a high-order Laguerre light beam.
In some preferred embodiments, the diameter of the pinhole is 0.2 times the image-wise airy disk diameter.
In the high-resolution confocal imaging system based on the high-order laguerre beam provided by the embodiment 2, the optical imaging physical process is as follows:
i is image distribution, O is object distribution, H is imaging integrated point spread function,
is a convolution operator. The smaller the spot diameter of the point spread function H, the higher the resolution of the resulting image. H by the illumination point spread function HillAnd a probe point spread function hdetAnd pinhole formation, in relation to the following:
referring to fig. 2 again, LG is generated for the liquid crystal spatial light modulator provided in embodiment 2 of the present invention3,1The phase profile of a patterned laguerre gaussian beam, represented by black to white, is from 0 to 2 pi.
Please refer to fig. 3, which illustrates LG in example 2 of the present invention3,1Illumination point spread function h under modal laguerre gaussian beam illuminationillFIG. 4 is a 0.2-fold process provided for example 2 of the present inventionThe comprehensive point spread function after the pinhole filtering of the image-side airy disk diameter can be seen from the graph of fig. 4 that the spot size of the point spread function is obviously smaller than that of the point spread function in the conventional laser scanning confocal microscopic imaging, so that the point spread function has higher imaging resolution.
The invention provides a high-resolution confocal imaging method based on high-order Laguerre beams, which is characterized in that a liquid crystal spatial light modulator and a radial polarization converter are introduced into a laser point scanning confocal imaging illumination light path, radial polarized light is introduced into the illumination light path through the radial polarization converter, and LG is formed on the liquid crystal spatial light modulator3,1The Laguerre Gaussian beam in the mode is subjected to confocal imaging to obtain a high-resolution confocal image based on the high-order Laguerre beam, and the spot size of the point spread function of the high-resolution differential confocal image based on the radial polarized light is obviously smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the high-resolution confocal image has high imaging resolution, and the defects of artifacts and the like caused by an image processing method are avoided.
Of course, the high-resolution confocal imaging based on the high-order laguerre beam of the present invention can also have various changes and modifications, and is not limited to the specific structure of the above-mentioned embodiments. In conclusion, the scope of the present invention should include those changes or substitutions and modifications which are obvious to those of ordinary skill in the art.
Claims (4)
1. A high-resolution confocal imaging system based on a high-order laguerre beam, comprising: the device comprises a laser, a radial polarization converter, a liquid crystal spatial light modulator, a dichroic mirror, a scanning galvanometer, a scanning lens, a cylindrical lens, an objective lens, a fluorescent light filter, a focusing lens, a pinhole, a photomultiplier and a control unit, wherein the radial polarization converter, the liquid crystal spatial light modulator and the photomultiplier are all connected with the control unit, and the radial polarization converter, the liquid crystal spatial light modulator and the photomultiplier are connected with the control unit, wherein:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, and the radial polarized light forms LG after passing through the liquid crystal spatial light modulator3,1A patterned Laguerre Gaussian beam of which 3 is LaguerreThe radial index of the beam, 1 being its angular index;
the LG3,1The Laguerre Gaussian beam of the mode enters the dichroic mirror, enters the scanning galvanometer after being reflected by the dichroic mirror, is focused on a sample after sequentially passing through the scanning lens, the tube mirror and the objective lens, and excites the sample to generate a fluorescent beam; the fluorescent light beams sequentially pass through the objective lens, the barrel lens, the scanning lens and the scanning galvanometer and then enter the dichroic mirror, are transmitted by the dichroic mirror and then enter the fluorescent light filter, then pass through the focusing lens and enter a pinhole at the focus of the focusing lens, and are detected by the photomultiplier tube, and the photomultiplier tube converts the detected fluorescent light into an electric signal to form a high-resolution confocal image based on high-order Laguerre light beams.
2. The high-resolution confocal imaging system based on a higher-order laguerre beam of claim 1, wherein the diameter of the pinhole is 0.2 times the image-wise airy disk diameter.
3. An imaging method of a high-resolution confocal imaging system based on a high-order Laguerre beam as claimed in claim 1, comprising the following steps:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, and the radial polarized light forms LG after passing through the liquid crystal spatial light modulator3,1A Laguerre Gaussian beam of a mode, wherein 3 is the radial index of the Laguerre beam, and 1 is the angular index of the Laguerre beam;
the LG3,1The Laguerre Gaussian beam of the mode enters the dichroic mirror, enters the scanning galvanometer after being reflected by the dichroic mirror, is focused on a sample after sequentially passing through the scanning lens, the tube mirror and the objective lens, and excites the sample to generate a fluorescent beam; the fluorescent light beam sequentially passes through the objective lens, the tube lens, the scanning lens and the scanning vibrating mirror, then enters the dichroic mirror, is transmitted by the dichroic mirror, then enters the fluorescent light filter, and then passes through the fluorescent light filterThe focusing lens enters a pinhole at the focus of the focusing lens and is detected by the photomultiplier tube, and the photomultiplier tube converts detected fluorescence into an electric signal to form a high-resolution confocal image based on a high-order Laguerre beam.
4. The imaging method of claim 3, wherein the pinhole has a diameter of 0.2 times the image-side airy disk diameter.
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| CN202083830U (en) * | 2011-05-20 | 2011-12-21 | 上海理工大学 | Device for capturing high-refractive index particulate |
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Application publication date: 20200626 |