CN111257293A - Confocal imaging system and method based on radial polarized light - Google Patents

Confocal imaging system and method based on radial polarized light Download PDF

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CN111257293A
CN111257293A CN202010146494.XA CN202010146494A CN111257293A CN 111257293 A CN111257293 A CN 111257293A CN 202010146494 A CN202010146494 A CN 202010146494A CN 111257293 A CN111257293 A CN 111257293A
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polarized light
lens
radial
scanning
dichroic mirror
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缪佳
张运海
肖昀
葛群
王发民
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JIANGSU INSTITUTE OF MEDICAL DEVICE TESTING
Suzhou Institute of Biomedical Engineering and Technology of CAS
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JIANGSU INSTITUTE OF MEDICAL DEVICE TESTING
Suzhou Institute of Biomedical Engineering and Technology of CAS
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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
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • 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
    • 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
    • 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
    • G01N2021/6463—Optics

Abstract

The invention provides a confocal imaging system and an imaging method based on radial polarized light, wherein a radial polarization converter is introduced into a laser point scanning confocal imaging illumination light path, the radial polarized light is introduced into the illumination light path through the radial polarization converter, and a small-diameter pinhole is adopted to carry out confocal imaging once to obtain confocal imaging based on the radial polarized light.

Description

Confocal imaging system and method based on radial polarized light
Technical Field
The invention relates to the technical field of optical microscopy, in particular to a confocal imaging system and method based on radial polarized light.
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 providing a confocal imaging system based on radially polarized light with high resolution and without image artifacts.
In order to achieve the purpose, the invention adopts the following technical scheme:
a radially polarized light based confocal imaging system comprising: laser instrument, radial polarization converter, dichroscope, scanning galvanometer, scanning lens, tube mirror, objective, fluorescence filter, focusing lens, pinhole, photomultiplier and the control unit, the laser instrument reaches photomultiplier all connects the control unit, wherein:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, the radial polarized light enters the dichroic mirror, is reflected by the dichroic mirror and then enters the scanning vibrating mirror, and then sequentially passes through the scanning lens, the cylindrical mirror and the objective lens and is focused at a sample, and the sample is excited 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 then 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 confocal imaging based on radial polarized light.
In some preferred embodiments, the diameter of the pinhole is 0.7 times the image-wise airy disk diameter.
In addition, the invention also provides an imaging method of the confocal imaging system based on the radial polarized light, which comprises the following steps:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, the radial polarized light enters the dichroic mirror, is reflected by the dichroic mirror and then enters the scanning vibrating mirror, and then sequentially passes through the scanning lens, the cylindrical mirror and the objective lens and is focused at a sample, and the sample is excited 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 then 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 confocal imaging based on radial polarized light.
In some preferred embodiments, the diameter of the pinhole is 0.7 times the image-wise airy disk diameter.
The invention adopts the technical scheme that the method has the advantages that:
the invention provides a confocal imaging system and an imaging method based on radial polarized light, wherein a radial polarization converter is introduced into a laser point scanning confocal imaging illumination light path, the radial polarized light is introduced into the illumination light path through the radial polarization converter, and a small-diameter pinhole is adopted to carry out confocal imaging once to obtain confocal imaging based on the radial polarized light.
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 confocal imaging system based on radially polarized light according to embodiment 1 of the present invention;
FIG. 2 is a graphical representation of the spread function of the illumination point under radially polarized illumination provided by examples 1 and 2 of the present invention.
Fig. 3 is a schematic diagram of the illumination point spread function for the longitudinal component provided by embodiments 1 and 2 of the present invention.
Fig. 4 is a schematic diagram of the illumination point spread function for the lateral component provided by embodiments 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 confocal imaging system 100 based on radially polarized light according to embodiment 1 of the present invention includes: the laser 110, the radial polarization converter 120, the dichroic mirror 130, the scanning galvanometer 140, the scanning lens 150, the tube mirror 160, the objective lens 170, the fluorescence filter 180, the focusing lens 190, the pinhole 210, the photomultiplier 220, and the control unit 230.
Specifically, the laser 110 and the photomultiplier tube 220 are both connected to the control unit 230.
The confocal imaging system 100 based on radial polarized light provided by the invention has the following working principle:
the laser beam emitted from the laser 110 forms radial polarized light through the radial polarization converter 120, the radial polarized light enters the dichroic mirror 130, is reflected by the dichroic mirror 130, enters the scanning galvanometer 140, sequentially passes through the scanning lens 150, the cylindrical mirror 160 and the objective lens 170, is focused on the sample a, and excites the sample a to generate a fluorescent beam; the fluorescent light beam sequentially passes through the objective lens 170, the tube lens 160, the scanning lens 150 and the scanning galvanometer 140, then enters the dichroic mirror 130, is transmitted by the dichroic mirror 130, then enters the fluorescent light filter 180, then enters the pinhole 210 at the focus of the focusing lens 190 after passing through the focusing lens 190, and is detected by the photomultiplier tube 220, and the photomultiplier tube 220 converts the detected fluorescent light into an electrical signal, so as to form confocal imaging based on radial polarized light.
In some preferred embodiments, the diameter of the pinhole 210 is 0.7 times the image-wise airy disk diameter.
The physical process of the optical imaging comprises the following steps:
Figure BDA0002400930620000041
i is image distribution, O is object distribution, H is imaging integrated point spread function,
Figure BDA0002400930620000042
is a convolution operator. The smaller the spot diameter of the point spread function H, the resolution of the resulting imageThe higher the rate. H by the illumination point spread function HillAnd a probe point spread function hdetAnd pinhole formation, in relation to the following:
Figure BDA0002400930620000043
referring to fig. 2, in order to provide the illumination point spread function h under illumination of radial polarized light, radial polarized light is introduced into the illumination light path through the radial polarization converter provided in embodiment 1 of the present invention, and a pinhole with a diameter 0.7 times the diameter of the image airy disk is used to form the illumination point spread function h under illumination of radial polarized lightillWherein, the horizontal direction is X-axis coordinate of the point spread function, the vertical direction is Y-axis coordinate of the point spread function, the unit is μm, and the right brightness bar is a corresponding scale of the intensity of the point spread function.
It can be understood that hillCan be decomposed into a longitudinal component and a transverse component, see FIG. 3, as hillThe 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 mum, the right-side brightness bar is the corresponding scale of the intensity of the point spread function, as can be seen from figure 3, hillHas a smaller spot diameter than circularly polarized light used for conventional confocal imaging.
Please refer to fig. 4, which is hillThe illumination point spread function of the transverse component is the X-axis coordinate of the point spread function in the transverse direction, the Y-axis coordinate of the point spread function in the longitudinal direction, the unit is mum, the right-side brightness bar is a corresponding scale of the intensity of the point spread function, as can be seen from fig. 4, the intensity of the transverse component is far smaller than that of the longitudinal component, and the transverse component can be restrained by adopting a small-diameter pinhole, so that an effective point spread function with a small diameter is obtained.
In the confocal imaging system based on radial polarized light provided by the above embodiment of the present invention, the radial polarization converter is introduced into the laser point scanning confocal imaging illumination light path, the radial polarized light is introduced into the illumination light path through the radial polarization converter, and a small-diameter pinhole is adopted to perform one-time confocal imaging, so as to obtain confocal imaging based on radial polarized light, and the spot size of the point spread function of the confocal imaging based on radial polarized light is significantly smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the confocal imaging system based on radial polarized light has a higher imaging resolution, and avoids the disadvantages of artifacts and the like caused by an image processing method.
Example 2
In this embodiment, the present invention further provides a confocal imaging method based on radially polarized light, including the following steps:
the laser beam emitted from the laser 110 forms radial polarized light through the radial polarization converter 120, the radial polarized light enters the dichroic mirror 130, is reflected by the dichroic mirror 130, enters the scanning galvanometer 140, sequentially passes through the scanning lens 150, the cylindrical mirror 160 and the objective lens 170, is focused on the sample a, and excites the sample a to generate a fluorescent beam; the fluorescent light beam sequentially passes through the objective lens 170, the tube lens 160, the scanning lens 150 and the scanning galvanometer 140, then enters the dichroic mirror 130, is transmitted by the dichroic mirror 130, then enters the fluorescent light filter 180, then enters the pinhole 210 at the focus of the focusing lens 190 after passing through the focusing lens 190, and is detected by the photomultiplier tube 220, and the photomultiplier tube 220 converts the detected fluorescent light into an electrical signal, so as to form confocal imaging based on radial polarized light.
In some preferred embodiments, the diameter of the pinhole 210 is 0.7 times the image-wise airy disk diameter.
The physical process of the optical imaging comprises the following steps:
Figure BDA0002400930620000051
i is image distribution, O is object distribution, H is imaging integrated point spread function,
Figure BDA0002400930620000052
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 and closureIs represented by the following formula:
Figure BDA0002400930620000061
referring to fig. 2 again, for the illumination point spread function h under the illumination of the radial polarized light, which is provided by embodiment 2 of the present invention, the radial polarized light is introduced into the illumination light path through the radial polarization converter, and the pinhole with the diameter of 0.7 times the diameter of the image airy disk is used to form the illumination point spread function h under the illumination of the radial polarized lightill。
It can be understood that hillCan be decomposed into a longitudinal component and a transverse component, see FIG. 3, as hillOf the longitudinal component of the illumination point spread function, h being seen from fig. 3illHas a smaller spot diameter than circularly polarized light used for conventional confocal imaging.
Please refer to fig. 4, which is hillAs can be seen from fig. 4, the intensity of the transverse component is much smaller than that of the longitudinal component, and the transverse component can be suppressed by using the small-diameter pinhole, so that the effective point spread function with a small diameter is obtained.
In the confocal imaging method based on radial polarized light provided by the above embodiment of the present invention, the radial polarization converter is introduced into the laser point scanning confocal imaging illumination light path, the radial polarized light is introduced into the illumination light path through the radial polarization converter, and a small-diameter pinhole is adopted to perform one-time confocal imaging, so as to obtain confocal imaging based on radial polarized light, and the spot size of the point spread function of the confocal imaging based on radial polarized light is significantly smaller than that of the point spread function in conventional laser scanning confocal microscopic imaging, so that the confocal imaging method has a higher imaging resolution, and avoids the disadvantages of artifacts and the like caused by an image processing method.
Of course, the confocal imaging based on radially polarized light 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 confocal imaging system based on radially polarized light, comprising: laser instrument, radial polarization converter, dichroscope, scanning galvanometer, scanning lens, tube mirror, objective, fluorescence filter, focusing lens, pinhole, photomultiplier and the control unit, the laser instrument reaches photomultiplier all connects the control unit, wherein:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, the radial polarized light enters the dichroic mirror, is reflected by the dichroic mirror and then enters the scanning vibrating mirror, and then sequentially passes through the scanning lens, the cylindrical mirror and the objective lens and is focused at a sample, and the sample is excited 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 then 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 confocal imaging based on radial polarized light.
2. The confocal radial polarized light-based imaging system of claim 1, wherein the diameter of the pinhole is 0.7 image-wise airy disk diameter.
3. An imaging method of the confocal radially polarized-light-based imaging system according to claim 1, comprising the steps of:
the laser beam emitted by the laser forms radial polarized light through the radial polarization converter, the radial polarized light enters the dichroic mirror, is reflected by the dichroic mirror and then enters the scanning vibrating mirror, and then sequentially passes through the scanning lens, the cylindrical mirror and the objective lens and is focused at a sample, and the sample is excited 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 then 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 confocal imaging based on radial polarized light.
4. The imaging method of claim 3, wherein the diameter of the pinhole is 0.7 times the image-wise airy disk diameter.
CN202010146494.XA 2019-11-14 2020-03-05 Confocal imaging system and method based on radial polarized light Pending CN111257293A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872413A (en) * 2017-04-18 2017-06-20 北京航空航天大学 Coaxial interference surface plasma microscopic method and system based on pupil modulation
CN107907513A (en) * 2017-10-27 2018-04-13 浙江大学 A kind of spot scan super-resolution imaging method and device based on polarization demodulation
CN108020505A (en) * 2017-11-30 2018-05-11 哈尔滨工业大学 The burnt optical tweezer microscopic imaging device of zoom copolymerization and method
CN110118726A (en) * 2019-04-12 2019-08-13 浙江大学 A kind of method and apparatus of parallel detecting fluorescent emission difference micro-imaging

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106872413A (en) * 2017-04-18 2017-06-20 北京航空航天大学 Coaxial interference surface plasma microscopic method and system based on pupil modulation
CN107907513A (en) * 2017-10-27 2018-04-13 浙江大学 A kind of spot scan super-resolution imaging method and device based on polarization demodulation
CN108020505A (en) * 2017-11-30 2018-05-11 哈尔滨工业大学 The burnt optical tweezer microscopic imaging device of zoom copolymerization and method
CN110118726A (en) * 2019-04-12 2019-08-13 浙江大学 A kind of method and apparatus of parallel detecting fluorescent emission difference micro-imaging

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Application publication date: 20200609