WO2022241672A1 - 共聚焦扫描式暗场显微成像方法与装置 - Google Patents

共聚焦扫描式暗场显微成像方法与装置 Download PDF

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WO2022241672A1
WO2022241672A1 PCT/CN2021/094648 CN2021094648W WO2022241672A1 WO 2022241672 A1 WO2022241672 A1 WO 2022241672A1 CN 2021094648 W CN2021094648 W CN 2021094648W WO 2022241672 A1 WO2022241672 A1 WO 2022241672A1
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light
spot
objective lens
modulator
confocal scanning
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PCT/CN2021/094648
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French (fr)
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匡翠方
邱宇轩
张宇森
刘旭
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浙江大学
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Priority to PCT/CN2021/094648 priority Critical patent/WO2022241672A1/zh
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Priority to US18/512,076 priority patent/US20240085683A1/en

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/10—Condensers affording dark-field illumination
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • 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
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0092—Polarisation microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/14—Condensers affording illumination for phase-contrast observation

Definitions

  • the invention relates to the field of optical microscopic imaging, in particular to a confocal scanning dark field microscopic imaging method and device.
  • Dark field microscopy is a microscopic technique based on the optical Tyndall effect.
  • the illuminating light passes through a condenser lens equipped with an annular diaphragm, forming a hollow annular light cone.
  • the numerical aperture of the objective lens is smaller than that of the condenser lens, the transmitted light from the sample cannot pass through the objective lens, and only the scattered light at a small angle is collected by the objective lens.
  • an image of a bright object in a dark field is formed, and the imaging contrast is improved.
  • dark-field microscopes are used to observe undyed transparent samples; in the fields of chemistry and materials science, spectrometers are used to analyze the scattered light collected by dark-field microscopes to study the scattering spectra of materials.
  • the invention provides a confocal scanning dark-field microscopic imaging method and device. Compared with the traditional wide-field dark-field microscope, the confocal design of the invention has the characteristics of low background noise, excellent tomographic ability and the like.
  • One aspect of the present invention provides a confocal scanning dark-field microscopy imaging method, the method comprising:
  • phase of the laser beam emitted by the laser is modulated into a 0-2n ⁇ vortex phase by a modulator, where n>3;
  • the modulated light beam is conjugated to the entrance pupil of the objective lens of the confocal scanning microscope, so that the focused spot of the objective lens is a hollow spot, and the radius of the inner ring of the hollow spot is larger than the radius of the solid spot without phase modulation;
  • the working process of the confocal scanning microscope is as follows: control the scanning mechanism so that the hollow spot scans on the sample surface; the scanning mechanism can be a scanning galvanometer or an electronically controlled mobile sample stage; the reflected light of the sample is focused on On the small hole, the reflected light is received by the detector, and the light intensity distribution under dark field illumination is obtained;
  • the point spread function PSF c (x, y) of the confocal scanning microscope is equal to the point spread function PSF e (x, y) of the illumination system composed of the laser and the modulator and the imaging system composed of the scanning mechanism, the tube mirror and the objective lens.
  • the product of the point spread function PSF f (x, y) of the system and the convolution of the aperture function p(x, y) of the small hole, the formula is as follows:
  • the point spread function of the illumination system is a hollow spot, and the higher the order of the vortex light, the larger the radius of the inner ring of the hollow spot; while the point spread function of the imaging system is determined by the aperture of the objective lens, It is a solid spot; for vortex light illumination higher than 3rd order, the radius of the inner ring of the hollow spot is larger than that of the solid spot, and the two spots are staggered from each other, realizing the lighting condition of the dark field.
  • the polarizer is used to convert the linearly polarized light of the P component into the liquid crystal spatial light modulator; when the applied electric field exceeds the threshold, the liquid crystal molecules appear electrically controlled birefringence effect, therefore, the liquid crystal spatial light modulation
  • the device only modulates linearly polarized light in one direction;
  • a half-wave plate is set behind the polarizer, and the laser beam passes through the polarizer and then passes through a half-wave plate to be converted into a linearly polarized light of the P component, so that the liquid crystal spatial light modulator can polarize the polarized light For pure phase modulation;
  • a D-shaped mirror is used to bend the optical path, so as to reduce the incident angle and improve the performance of the liquid crystal spatial light modulator.
  • liquid crystal spatial light modulator uses Zernike polynomials to correct aberrations, thereby improving the quality of hollow light spots.
  • a quarter-wave plate is set in front of the objective lens, and the light beam is converted into circularly polarized light by the quarter-wave plate and then enters the objective lens to improve the quality of the hollow spot for scanning the sample.
  • a confocal scanning dark-field microscopy imaging device which includes a laser, a modulator, a 4f system, and a scanning imaging module;
  • the scanning imaging module includes a scanning mechanism, a tube mirror, an objective lens, and a lens , pinholes and detectors;
  • the laser beam emitted by the laser is modulated into a 0-2n ⁇ vortex phase by a modulator, where n>3;
  • the 4f system adjusts the conjugate of the exit plane of the modulator and the entrance pupil of the objective lens, so that the focused spot of the objective lens is a hollow spot, and the radius of the inner ring of the hollow spot is greater than the radius of the solid spot when no phase modulation is performed;
  • the scanning mechanism makes the hollow spot scan on the surface of the sample, the reflected light of the sample is focused on the small hole conjugated to the object through the lens, the reflected light is received by the detector, and the light intensity distribution under dark field illumination is obtained;
  • the point spread function PSF c (x, y) of the confocal scanning dark-field microscopy imaging device is equal to the point spread function PSF e (x, y) of the illumination system composed of the laser, the modulator and the 4f system and is composed of the scanning
  • the product of the point spread function PSF f (x, y) of the imaging system composed of the mechanism, the tube lens and the objective lens is convolved with the aperture function p(x, y) of the small hole, and the formula is as follows:
  • the radius of the inner ring of the hollow spot is larger than that of the solid spot, and the two spots are staggered from each other to realize the lighting condition of the dark field.
  • the modulator is composed of a polarizer and a liquid crystal spatial light modulator
  • the polarizer is used to convert the linearly polarized light of the P component into the liquid crystal spatial light modulator;
  • the 4f system adjusts the conjugate of the exit plane of the liquid crystal spatial light modulator and the entrance pupil of the objective lens, and the liquid crystal spatial light modulator is loaded with a 0-2n ⁇ vortex phase.
  • the 4f system is composed of a lens 1 and a lens 2, the front focal plane of the lens 1 coincides with the exit plane of the modulator, the back focal plane of the lens 1 coincides with the front focal plane of the lens 2, and the back focal plane of the lens 2 Conjugate to the entrance pupil of the objective.
  • the scanning mechanism is a scanning galvanometer
  • the rear focal plane of the lens 2 coincides with the midpoint of the line connecting the centers of the two galvanometers, and the midpoint is conjugate to the entrance pupil of the objective lens.
  • a half-wave plate and a polarization beam splitting prism are arranged in front of the scanning mechanism, and the light beam is converted into P light through the half-wave plate, and the P light is used as the incident light of the scanning mechanism after completely passing through the polarization beam splitting prism ;
  • a quarter-wave plate is set in front of the objective lens, and the P light is converted into circularly polarized light by the quarter-wave plate, and then enters the objective lens, and the reflected light of the sample is converted into S light, and the reflected light is reflected by the polarizing beam splitter prism Focus on the small hole.
  • the small hole is used to eliminate out-of-focus stray light, which is realized by using a pinhole or a multimode optical fiber.
  • the present invention Compared with the prior art, the present invention has the following beneficial technical effects: the present invention adopts a confocal design, a small hole is placed in front of the detector, and the plane where the small hole is located is conjugate to the object plane, which prevents out-of-focus signals from entering detector.
  • This design improves the signal-to-noise ratio and resolution of imaging, and makes dark-field microscopy imaging have good tomographic capabilities.
  • Fig. 1 is the implementation principle diagram of the confocal scanning dark field microscopy imaging method provided by the embodiment of the present invention
  • FIG. 2 is a schematic diagram of a confocal scanning dark-field microscopic imaging device provided by an embodiment of the present invention
  • Fig. 3 is a schematic diagram of a phase mask for generating high-order vortex beams.
  • phase of the laser beam emitted by the laser is modulated into a 0-2n ⁇ vortex phase by a modulator, where n>3;
  • the modulated light beam is conjugated to the entrance pupil of the objective lens of the confocal scanning microscope, so that the focused spot of the objective lens is a hollow spot, and the radius of the inner ring of the hollow spot is larger than the radius of the solid spot without phase modulation;
  • the working process of the confocal scanning microscope is as follows: control the scanning mechanism so that the hollow spot scans on the sample surface; the scanning mechanism can be a scanning galvanometer or an electronically controlled mobile sample stage; the reflected light of the sample is focused on On the small hole, the reflected light is received by the detector, and the light intensity distribution under dark field illumination is obtained;
  • the point spread function PSF c (x, y) of the confocal scanning microscope is equal to the point spread function PSF e (x, y) of the illumination system composed of the laser and the modulator and the imaging system composed of the scanning mechanism, the tube mirror and the objective lens.
  • the product of the point spread function PSF f (x, y) of the system and the convolution of the aperture function p(x, y) of the small hole, the formula is as follows:
  • the point spread function of the illumination system is a hollow spot, and the higher the order of the vortex light, the larger the radius of the inner ring of the hollow spot; while the point spread function of the imaging system is determined by the aperture of the objective lens, It is a solid spot; for vortex light illumination higher than 3rd order, the radius of the inner ring of the hollow spot is larger than that of the solid spot, and the two spots are staggered from each other, realizing the lighting condition of the dark field.
  • the polarizer is used to convert the linearly polarized light of the P component into the liquid crystal spatial light modulator; when the applied electric field exceeds the threshold, the liquid crystal molecules appear electrically controlled birefringence effect, therefore, the liquid crystal spatial light modulation
  • the device only modulates linearly polarized light in one direction;
  • a half-wave plate is set behind the polarizer, and the laser beam passes through the polarizer and then passes through a half-wave plate to be converted into a linearly polarized light of the P component, so that the liquid crystal spatial light modulator can polarize the polarized light For pure phase modulation;
  • a D-shaped mirror is used to bend the optical path, so as to reduce the incident angle and improve the performance of the liquid crystal spatial light modulator.
  • liquid crystal spatial light modulator uses Zernike polynomials to correct aberrations, thereby improving the quality of hollow light spots.
  • a quarter-wave plate is set in front of the objective lens, and the light beam is converted into circularly polarized light by the quarter-wave plate and then enters the objective lens to improve the quality of the hollow spot for scanning the sample.
  • the confocal scanning dark-field microscopic imaging device includes: a laser, a modulator, a 4f system, and a scanning imaging module;
  • the scanning imaging module includes a scanning mechanism, a tube mirror, an objective lens, a lens, a small holes and detectors;
  • the laser beam emitted by the laser is modulated into a 0-2n ⁇ vortex phase by a modulator, where n>3;
  • the 4f system adjusts the conjugate of the exit plane of the modulator and the entrance pupil of the objective lens, so that the focused spot of the objective lens is a hollow spot, and the radius of the inner ring of the hollow spot is greater than the radius of the solid spot when no phase modulation is performed;
  • the scanning mechanism makes the hollow spot scan on the surface of the sample, the reflected light of the sample is focused on the small hole conjugated to the object through the lens, the reflected light is received by the detector, and the light intensity distribution under dark field illumination is obtained;
  • the point spread function PSF c (x, y) of the confocal scanning dark-field microscopy imaging device is equal to the point spread function PSF e (x, y) of the illumination system composed of the laser, the modulator and the 4f system and is composed of the scanning
  • the product of the point spread function PSF f (x, y) of the imaging system composed of the mechanism, the tube lens and the objective lens is convolved with the aperture function p(x, y) of the small hole, and the formula is as follows:
  • the radius of the inner ring of the hollow spot is larger than that of the solid spot, and the two spots are staggered from each other to realize the lighting condition of the dark field.
  • the modulator is composed of a polarizer and a liquid crystal spatial light modulator
  • the polarizer is used to convert the linearly polarized light of the P component into the liquid crystal spatial light modulator;
  • the 4f system adjusts the conjugate of the exit plane of the liquid crystal spatial light modulator and the entrance pupil of the objective lens, and the liquid crystal spatial light modulator is loaded with a 0-2n ⁇ vortex phase.
  • the 4f system is composed of a lens 1 and a lens 2, the front focal plane of the lens 1 coincides with the exit plane of the modulator, the back focal plane of the lens 1 coincides with the front focal plane of the lens 2, and the back focal plane of the lens 2 Conjugate to the entrance pupil of the objective.
  • the scanning mechanism is a scanning galvanometer
  • the rear focal plane of the lens 2 coincides with the midpoint of the line connecting the centers of the two galvanometers, and the midpoint is conjugate to the entrance pupil of the objective lens.
  • a half-wave plate and a polarization beam splitting prism are arranged in front of the scanning mechanism, and the light beam is converted into P light through the half-wave plate, and the P light is used as the incident light of the scanning mechanism after completely passing through the polarization beam splitting prism.
  • Light; a quarter-wave plate is set in front of the objective lens, the P light is converted into circularly polarized light by the quarter-wave plate, and then enters the objective lens, and the reflected light of the sample is converted into S light, and the reflected light is reflected by the polarizing beam splitter prism Then focus on the pinhole. Apertures are used to eliminate out-of-focus stray light and are implemented using pinholes or multimode fiber.
  • FIG. 2 The structure of the confocal scanning dark-field microscopy imaging device of this example is shown in Figure 2, including a laser generating and collimating device 1, a first D-shaped mirror 2, a polarizer 3, a first half-wave plate 4, and a liquid crystal space Light modulator 5, first lens 6, first mirror 7, second lens 8, third lens 9, fourth lens 10, second half-wave plate 11, polarization beam splitting prism 12, second D-shaped mirror 13 , scanning galvanometer module 14, second mirror 15, scanning lens 16, tube mirror 17, 1/4 wave plate 18, objective lens 19, sample stage 20, third mirror 21, fifth lens 22 and avalanche diode 23.
  • the phase mask for generating higher order vortex beams is shown in Figure 3.
  • the laser generating and collimating device 1 passes through the first D-shaped mirror 2, passes through the polarizer 3, becomes linearly polarized light, passes through the first half-wave plate 4, becomes P light, reaches the liquid crystal spatial light modulator 5, and modulates the linearly polarized light into high-order vortex light.
  • the outgoing light of the liquid crystal spatial light modulator 5 sequentially passes through the 4f system composed of the first lens 6 and the second lens 8, and the 4f system composed of the third lens 9 and the fourth lens 10, and passes through the second half-wave plate 11 to become a P light transmission system.
  • the scanning galvanometer module 14 After passing through the polarization beam splitter prism 12, it enters the scanning galvanometer module 14 through the second D-shaped mirror 13, wherein the midpoint of the line connecting the centers of the two galvanometers is conjugate to the exit plane of the spatial light modulator. After passing through the second reflection mirror 15, the scanning lens 16 and the tube mirror 17, it is adjusted into circularly polarized light by the 1/4 wave plate 18, passes through the objective lens 19, and scans on the surface of the sample. The reflected light passes through the objective lens 19 and is adjusted into S light by the 1/4 wave plate 18.
  • the reflected light passes through the tube mirror 17, the scanning lens 16, the second reflecting mirror 15, the scanning galvanometer module 14, and the second D-shaped mirror 13 in turn, is reflected at the polarization beam splitting prism 12, passes through the third reflecting mirror 21, the fifth The lens 22 focuses on the small hole, and the avalanche diode 23 behind the small hole collects the light signal.

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Abstract

一种共聚焦扫描式暗场显微成像方法与装置，将激光器发出的激光光束的相位调制成0-2nπ涡旋相位，其中n＞3；将调制后的光束与共聚焦扫描显微镜的物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；使共聚焦扫描显微镜工作，实现暗场显微成像。通过采用共聚焦设计，在探测器前放置一个小孔，小孔所在平面与物面共轭，阻挡了离焦信号进入探测器，提高了成像的信噪比和分辨率，使暗场显微成像具有良好的层析能力。

Description

共聚焦扫描式暗场显微成像方法与装置 技术领域
本发明涉及光学显微成像领域，具体地说，涉及一种共聚焦扫描式暗场显微成像方法与装置。
背景技术
暗场显微镜是一种基于光学丁达尔效应的显微技术。照明光通过装载环形光阑的聚光镜，形成中空环形光锥。因为物镜的数值孔径小于聚光镜的数值孔径，样品的透射光无法通过物镜，只有小角度的散射光被物镜收集。由此形成了暗视野下亮物体的像，提高了成像对比度。在生命科学领域，暗场显微镜用于观察未染色的透明样品；在化学、材料科学领域，使用光谱仪分析暗场显微镜收集的散射光研究材料的散射光谱。
发明内容
本发明提供了一种共聚焦扫描式暗场显微成像方法与装置，本发明与传统的宽场暗场显微镜相比，共聚焦式的设计具有低背景噪声、优秀的层析能力等特点。
本发明的目的是通过以下技术方案来实现的：
本发明一方面提供了一种共聚焦扫描式暗场显微成像方法，该方法包括：
通过调制器将激光器发出的激光光束的相位调制成0-2nπ涡旋相位，其中n＞3；
将调制后的光束与共聚焦扫描显微镜的物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
使共聚焦扫描显微镜工作，实现暗场显微成像；
所述共聚焦扫描显微镜的工作过程具体为：控制扫描机构使得空心光斑在样品表面扫描；扫描机构可以为扫描振镜或电控移动样品台；样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光 强度分布；
所述共聚焦扫描显微镜的点扩散函数PSF c(x,y)等于由激光器和调制器构成的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
Figure PCTCN2021094648-appb-000001
对于涡旋光束照明的照明系统，照明系统的点扩散函数为空心光斑，且涡旋光阶数越高，空心光斑的内环半径越大；而成像系统的点扩散函数由物镜的孔阑决定，是实心光斑；对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
进一步地，调制相位的具体方法为：
将激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；当外加电场超过阈值时，液晶分子出现电控双折射效应，因此，液晶空间光调制器只调制一个方向的线偏光；
调节液晶空间光调制器出射平面与扫描显微镜的物镜入瞳共轭；在液晶空间光调制器上加载0-2nπ涡旋相位。
进一步地，在起偏器后设置二分之一波片，激光光束通过起偏器后再通过一个二分之一波片转为P分量的线偏光，使液晶空间光调制器对该偏振光作纯相位调制；
所述P分量的线偏光入射液晶空间光调制器前使用一个D形镜转折光路，以减小入射角，改善液晶空间光调制器的性能。
进一步地，所述液晶空间光调制器使用泽尼克多项式校正像差，从而改善空心光斑的质量。
进一步地，在物镜前设置四分之一波片，将光束用四分之一波片转为圆偏光后入射到物镜，提高用于扫描样品的空心光斑质量。
本发明另一方面提供了一种共聚焦扫描式暗场显微成像装置，该装置包括激光器、调制器、4f系统和扫描成像模块；所述扫描成像模块包括扫描机构、管镜、物镜、透镜、小孔和探测器；
所述激光器发出的激光光束通过调制器将相位调制成0-2nπ涡旋相位，其中n＞3；
所述4f系统调节调制器的出射平面与物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
所述扫描机构使得空心光斑在样品表面扫描，样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光强度分布；
所述共聚焦扫描式暗场显微成像装置的点扩散函数PSF c(x,y)等于由激光器、调制器和4f系统构成的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
Figure PCTCN2021094648-appb-000002
对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
进一步地，所述调制器由起偏器和液晶空间光调制器组成；
所述激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；
所述4f系统调节液晶空间光调制器的出射平面与物镜入瞳共轭，所述液晶空间光调制器上加载0-2nπ涡旋相位。
进一步地，所述4f系统由透镜1与透镜2组成，透镜1的前焦面与调制器的出射平面重合，透镜1的后焦面与透镜2的前焦面重合，透镜2的后焦面与物镜入瞳共轭。
进一步地，所述扫描机构为扫描振镜，透镜2的后焦面与两面振镜中心连线的中点重合，所述中点与物镜入瞳共轭。
进一步地，所述扫描机构前设置二分之一波片和偏振分束棱镜，通过二分之一波片将光束转为P光，P光完全透过偏振分束棱镜后作为扫描机构入射光；在物镜前设置四分之一波片，通过四分之一波片将P光转为圆偏光后入射物镜，并将样品的反射光转为S光，反射光被偏振分束棱镜反射后聚焦在小孔上。
进一步地，所述小孔用于消除离焦杂散光，采用针孔或多模光纤实现。
与现有技术相比，本发明具有以下有益的技术效果：本发明采用了共聚焦的设计，在探测器前放置一个小孔，小孔所在平面与物面共轭，阻挡了离焦信号进入探测器。这种设计提高了成像的信噪比和分辨率，使暗场显微成像具有良好的层析能力。
附图说明
图1为本发明实施例提供的共聚焦扫描式暗场显微成像方法实现原理图；
图2为本发明实施例提供的共聚焦扫描式暗场显微成像装置示意图；
图3为产生高阶涡旋光束的相位掩膜示意图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
如图1所示，本发明提供的一种共聚焦扫描式暗场显微成像方法，包括：
通过调制器将激光器发出的激光光束的相位调制成0-2nπ涡旋相位，其中n＞3；
将调制后的光束与共聚焦扫描显微镜的物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
使共聚焦扫描显微镜工作，实现暗场显微成像；
所述共聚焦扫描显微镜的工作过程具体为：控制扫描机构使得空心光斑在样品表面扫描；扫描机构可以为扫描振镜或电控移动样品台；样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光强度分布；
所述共聚焦扫描显微镜的点扩散函数PSF c(x,y)等于由激光器和调制器构成 的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
Figure PCTCN2021094648-appb-000003
对于涡旋光束照明的照明系统，照明系统的点扩散函数为空心光斑，且涡旋光阶数越高，空心光斑的内环半径越大；而成像系统的点扩散函数由物镜的孔阑决定，是实心光斑；对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
进一步地，调制相位的具体方法为：
将激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；当外加电场超过阈值时，液晶分子出现电控双折射效应，因此，液晶空间光调制器只调制一个方向的线偏光；
调节液晶空间光调制器出射平面与扫描显微镜的物镜入瞳共轭；在液晶空间光调制器上加载0-2nπ涡旋相位。
进一步地，在起偏器后设置二分之一波片，激光光束通过起偏器后再通过一个二分之一波片转为P分量的线偏光，使液晶空间光调制器对该偏振光作纯相位调制；
所述P分量的线偏光入射液晶空间光调制器前使用一个D形镜转折光路，以减小入射角，改善液晶空间光调制器的性能。
进一步地，所述液晶空间光调制器使用泽尼克多项式校正像差，从而改善空心光斑的质量。
进一步地，在物镜前设置四分之一波片，将光束用四分之一波片转为圆偏光后入射到物镜，提高用于扫描样品的空心光斑质量。
为实现上述方法，本发明提供的共聚焦扫描式暗场显微成像装置包括：激光器、调制器、4f系统和扫描成像模块；所述扫描成像模块包括扫描机构、管镜、物镜、透镜、小孔和探测器；
所述激光器发出的激光光束通过调制器将相位调制成0-2nπ涡旋相位，其中n＞3；
所述4f系统调节调制器的出射平面与物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
所述扫描机构使得空心光斑在样品表面扫描，样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光强度分布；
所述共聚焦扫描式暗场显微成像装置的点扩散函数PSF c(x,y)等于由激光器、调制器和4f系统构成的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
Figure PCTCN2021094648-appb-000004
对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
进一步地，所述调制器由起偏器和液晶空间光调制器组成；
所述激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；
所述4f系统调节液晶空间光调制器的出射平面与物镜入瞳共轭，所述液晶空间光调制器上加载0-2nπ涡旋相位。
进一步地，所述4f系统由透镜1与透镜2组成，透镜1的前焦面与调制器的出射平面重合，透镜1的后焦面与透镜2的前焦面重合，透镜2的后焦面与物镜入瞳共轭。
进一步地，所述扫描机构为扫描振镜，透镜2的后焦面与两面振镜中心连线的中点重合，所述中点与物镜入瞳共轭。
进一步地，所述扫描机构前设置二分之一波片和偏振分束棱镜，通过二分之一波片将光束转为P光，P光完全透过偏振分束棱镜后作为扫描机构的入射光；在物镜前设置四分之一波片，通过四分之一波片将P光转为圆偏光后入射物镜，并将样品的反射光转为S光，反射光被偏振分束棱镜反射后聚焦在小孔上。小孔用于消除离焦杂散光，采用针孔或多模光纤实现。
以下给出本发明的一个具体实现示例，但不限于此。本示例的共聚焦扫描 式暗场显微成像装置结构如图2所示，包括激光发生和准直装置1、第一D形镜2、起偏器3、第一半波片4、液晶空间光调制器5、第一透镜6、第一反射镜7、第二透镜8、第三透镜9、第四透镜10、第二半波片11、偏振分束棱镜12、第二D形镜13、扫描振镜模块14、第二反射镜15、扫描透镜16、管镜17、1/4波片18、物镜19、样品台20、第三反射镜21、第五透镜22和雪崩二极管23。产生高阶涡旋光束的相位掩膜如图3所示。
装置工作时，激光发生和准直装置1经过第一D形镜2，经过起偏器3后成为线偏光，经过第一半波片4成为P光到达液晶空间光调制器5将线偏光调制成高阶涡旋光。液晶空间光调制器5的出射光依次经过第一透镜6与第二透镜8组成的4f系统，第三透镜9与第四透镜10组成的4f系统，经过第二半波片11成为P光透过偏振分束棱镜12，经过第二D形镜13进入扫描振镜模块14，其中两面振镜中心连线的中点与空间光调制器的出射平面共轭。经过第二反射镜15、扫描透镜16、管镜17，被1/4波片18调成圆偏光，经过物镜19，在样品表面扫描。反射光经过物镜19，被1/4波片18调成S光。反射光依次经过管镜17、扫描透镜16、第二反射镜15、扫描振镜模块14、第二D形镜13，在偏振分束棱镜12处被反射，经过第三反射镜21、第五透镜22聚焦在小孔处，小孔后的雪崩二极管23收集光信号。
以上所述仅是本发明的优选实施方式，虽然本发明已以较佳实施例披露如上，然而并非用以限定本发明。任何熟悉本领域的技术人员，在不脱离本发明技术方案范围情况下，都可利用上述揭示的方法和技术内容对本发明技术方案做出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何的简单修改、等同变化及修饰，均仍属于本发明技术方案保护的范围内。

Claims (8)

  1. 一种共聚焦扫描式暗场显微成像方法，其特征在于，该方法包括：
    通过调制器将激光器发出的激光光束的相位调制成0-2nπ涡旋相位，其中n＞3；
    将调制后的光束与共聚焦扫描显微镜的物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
    使共聚焦扫描显微镜工作，实现暗场显微成像；
    所述共聚焦扫描显微镜的工作过程具体为：控制扫描机构使得空心光斑在样品表面扫描；样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光强度分布；
    所述共聚焦扫描显微镜的点扩散函数PSF c(x,y)等于由激光器和调制器构成的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
    Figure PCTCN2021094648-appb-100001
    对于涡旋光束照明的照明系统，照明系统的点扩散函数为空心光斑，且涡旋光阶数越高，空心光斑的内环半径越大；而成像系统的点扩散函数由物镜孔阑决定，是实心光斑；对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
  2. 根据权利要求1所述的一种共聚焦扫描式暗场显微成像方法，其特征在于，调制相位的具体方法为：
    将激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；
    调节液晶空间光调制器出射平面与共聚焦扫描显微镜的物镜入瞳共轭；在液晶空间光调制器上加载0-2nπ涡旋相位。
  3. 根据权利要求2所述的一种共聚焦扫描式暗场显微成像方法，其特征在于，在起偏器后设置二分之一波片，激光光束通过起偏器后再通过一个二分之 一波片转为P分量的线偏光，使液晶空间光调制器对该偏振光作纯相位调制；
    所述P分量的线偏光入射液晶空间光调制器前使用一个D形镜转折光路，以减小入射角，改善液晶空间光调制器的性能。
  4. 根据权利要求2所述的一种共聚焦扫描式暗场显微成像方法，其特征在于，所述液晶空间光调制器使用泽尼克多项式校正像差，从而改善空心光斑的质量。
  5. 根据权利要求1所述的一种共聚焦扫描式暗场显微成像方法，其特征在于，在物镜前设置四分之一波片，将光束用四分之一波片转为圆偏光后入射到物镜，提高用于扫描样品的空心光斑质量。
  6. 一种共聚焦扫描式暗场显微成像装置，其特征在于，该装置包括激光器、调制器、4f系统和扫描成像模块；所述扫描成像模块包括扫描机构、管镜、物镜、透镜、小孔和探测器；
    所述激光器发出的激光光束通过调制器将相位调制成0-2nπ涡旋相位，其中n＞3；
    所述4f系统调节调制器的出射平面与物镜入瞳共轭，使得物镜的聚焦光斑为空心光斑，且空心光斑内环半径大于不进行相位调制时的实心光斑半径；
    所述扫描机构使得空心光斑在样品表面扫描，样品的反射光经过透镜聚焦在与物共轭的小孔上，反射光被探测器接收，获得暗场照明下的光强度分布；
    所述共聚焦扫描式暗场显微成像装置的点扩散函数PSF c(x,y)等于由激光器、调制器和4f系统构成的照明系统的点扩散函数PSF e(x,y)和由扫描机构、管镜和物镜构成的成像系统的点扩散函数PSF f(x,y)的乘积与小孔孔阑函数p(x,y)的卷积，公式如下：
    Figure PCTCN2021094648-appb-100002
    对高于3阶的涡旋光照明，空心光斑内环半径大于实心光斑半径，两光斑相互错开，实现了暗场的照明条件。
  7. 根据权利要求6所述的一种共聚焦扫描式暗场显微成像装置，其特征在于，所述调制器由起偏器和液晶空间光调制器组成；
    所述激光器发出的激光光束准直后用起偏器转为P分量的线偏光入射到液晶空间光调制器；
    所述4f系统调节液晶空间光调制器的出射平面与物镜入瞳共轭，所述液晶空间光调制器上加载0-2nπ涡旋相位。
  8. 根据权利要求6所述的一种共聚焦扫描式暗场显微成像装置，其特征在于，所述扫描机构前设置二分之一波片和偏振分束棱镜，通过二分之一波片将光束转为P光，P光完全透过偏振分束棱镜后作为扫描机构的入射光；在物镜前设置四分之一波片，通过四分之一波片将P光转为圆偏光后入射物镜，并将样品的反射光转为S光，反射光被偏振分束棱镜反射后聚焦在小孔上。
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