WO2022111602A1 - 基于共路并行荧光辐射差分的超分辨显微成像方法和装置 - Google Patents

基于共路并行荧光辐射差分的超分辨显微成像方法和装置 Download PDF

Info

Publication number
WO2022111602A1
WO2022111602A1 PCT/CN2021/133299 CN2021133299W WO2022111602A1 WO 2022111602 A1 WO2022111602 A1 WO 2022111602A1 CN 2021133299 W CN2021133299 W CN 2021133299W WO 2022111602 A1 WO2022111602 A1 WO 2022111602A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
spot
super
liquid crystal
hollow
Prior art date
Application number
PCT/CN2021/133299
Other languages
English (en)
French (fr)
Inventor
匡翠方
黄宇然
张智敏
刘少聪
刘旭
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2022111602A1 publication Critical patent/WO2022111602A1/zh
Priority to US18/322,582 priority Critical patent/US20230296871A1/en

Links

Images

Classifications

    • 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
    • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
    • 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
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • 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
    • G02B21/002—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
    • 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

Definitions

  • the invention relates to the field of optical super-resolution microscopic imaging, in particular to a super-resolution microscopic imaging method and device based on common-path parallel fluorescence radiation difference.
  • optical microscope In biology, life science and other fields, optical microscope is an important means to observe cells, organelles, viruses, etc.
  • This limit is called the Abbe diffraction limit, and the value is 0.61 ⁇ /NA, where ⁇ is the wavelength of the light wave and NA is the numerical aperture of the objective lens.
  • FED Fluorescence emission difference microscopy
  • the FED microscope system includes two different modes of excitation light, one is confocal light, which appears as a solid spot on the focal plane; the other is negative confocal light, which appears as a doughnut-shaped hollow spot with an internal size smaller than the diffraction limit.
  • the FED microscopic image is obtained from the difference of these two images.
  • Parallel fluorescence emission difference super-resolution microscopy uses a beam deflection device to stagger the solid spot and the hollow spot on the object surface by a certain distance, The sample is thus scanned and detected simultaneously with both.
  • pFED Parallel fluorescence emission difference microscopy
  • the two excitation optical paths in pFED are susceptible to external disturbances such as drift and noise, respectively, which limit the imaging quality.
  • the present invention provides a super-resolution microscopy imaging method and device based on a common-path parallel fluorescence radiation difference. Compared with the pFED method, the present invention can better eliminate drift and noise by sharing the excitation light instead of passing through different devices. and other external disturbances.
  • One aspect of the present invention provides a super-resolution microscopy imaging method based on a common-path parallel fluorescence radiation difference, the method comprising the following steps:
  • the other half of the liquid crystal spatial light modulator is loaded as a blazed grating, so that the unmodulated component of linear polarization is modulated and tilted.
  • the tilt angle is calculated by adjusting the grating constant, so that the final solid spot and hollow spot are staggered on the surface;
  • the two circularly polarized lights present staggered solid and hollow light spots on the sample surface.
  • the solid and hollow light spots scan the sample at the same time, and the excited two fluorescent signals pass through their respective detection light paths and are received by the two detectors. , so as to obtain the confocal light intensity distribution and the negative confocal light intensity distribution;
  • the polarization direction is adjusted by a half-wave plate; the polarization direction should be determined by the ratio of the negative confocal light intensity to the confocal light intensity, that is, by adjusting The polarization direction matches the intensity of the two.
  • a D-shaped mirror is used to bend the light path before the linearly polarized light enters the liquid crystal spatial light modulator, so as to reduce the incident angle and improve the performance of the liquid crystal spatial light modulator.
  • the modulation of a component of linearly polarized light by the liquid crystal spatial light modulator is not limited to modulating it into vortex light to form a horizontal hollow light spot, but can also be formed on the object surface by 0/ ⁇ ring phase modulation.
  • Longitudinal hollow spot, this type of vertical hollow spot also has a weak hollow spot effect in the horizontal direction, so that three-dimensional fluorescence radiation differential super-resolution microscopy imaging can be realized, and its lateral resolution is relatively weaker than that of lateral hollow spot super-resolution microscopy imaging. resolution, but the longitudinal resolution can be greatly improved, allowing more detailed information about the sample to be obtained.
  • the dislocation parallel scanning of the light spot is obtained by loading the blazed grating with the liquid crystal spatial light modulator, which can reduce the difficulty of optical path calibration, and can perform Zernike aberration correction on the light spot to obtain the optimal solution. spot.
  • the solid spot and the hollow spot scan the sample at the same time, and after filtering out the defocused stray light through two pinholes, two detectors are used to obtain the fluorescence excited by the solid spot and the hollow spot, respectively. Signal.
  • the solid light spot and the hollow light spot scan the sample at the same time
  • four adjacent multimode optical fibers can be installed side by side, and the optical fiber end faces at both ends are used to receive the fluorescent signal, and a photomultiplier tube or avalanche photoelectricity is used. Diode detection; and the middle two optical fibers are not connected to the detector, which is used to more conveniently determine the interval between the two fluorescent signals.
  • the fluorescence radiation differential formula is as follows:
  • I(x,y) I 1 (x,y)- ⁇ I 2 (x+ ⁇ x,y+ ⁇ y)
  • I 1 (x,y) is the confocal light intensity distribution
  • I 2 (x+ ⁇ x,y+ ⁇ y) is the shifted negative confocal light intensity distribution
  • ⁇ is the difference coefficient
  • Another aspect of the present invention provides a super-resolution microscopy imaging device based on a common-path parallel fluorescence radiation difference, the device comprising:
  • Polarizers for generating linearly polarized light Polarizers for generating linearly polarized light
  • a liquid crystal spatial light modulator for separately modulating two components of linearly polarized light into vortex light and oblique light using halves;
  • Quarter-wave plate and half-wave plate for converting two excitation light into circularly polarized light
  • Beamsplitters used to reflect excitation light and transmit fluorescence
  • Pinhole or multimode fiber for eliminating out-of-focus stray light
  • Two detectors for detecting solid and hollow spots, respectively.
  • the present invention has the following beneficial technical effects: due to the common path of the two excitation lights, they pass through the same device and are simultaneously affected by the drift of each device and the noise introduced. The light spots are canceled when they are differentiated, thereby improving the signal-to-noise ratio of super-resolution images while achieving a faster imaging speed.
  • FIG. 1 is a flowchart of a super-resolution microscopy imaging method based on a common-path parallel fluorescence radiation difference according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of a super-resolution microscopy imaging device based on a common-path parallel fluorescence radiation difference according to an embodiment of the present invention.
  • the super-resolution microscopy imaging method based on the difference of common-path parallel fluorescence radiation includes the following steps:
  • Liquid crystal spatial light modulators have the property of modulating linearly polarized light in only one direction. When the applied electric field exceeds the threshold, the liquid crystal molecules exhibit an electronically controlled birefringence effect, causing the liquid crystal to produce birefringence for light in a corresponding direction. Taking advantage of this property, using half of the liquid crystal spatial light modulator, using 0-2 ⁇ vortex phase modulation, modulates the polarization component parallel to the modulation polarization direction in step 1 linear polarization, while the polarization component perpendicular to the adjusted polarization direction is not Do modulation.
  • the incident light is equivalent to passing through a half-wave plate, and the polarization direction is perpendicular to the original, while The original unmodulated polarization component is rotated to the modulated polarization direction, so that it can be modulated by the liquid crystal spatial light modulator, while the original modulated polarization component is perpendicular to the modulation polarization direction at this time, and is not modulated by the liquid crystal spatial light modulator. ;
  • the tilt angle can be calculated by adjusting the grating constant , so that the final solid spot and hollow spot are staggered on the object surface, and in this embodiment, the stagger is 2-3AU;
  • the two circularly polarized lights present staggered solid and hollow light spots on the sample surface.
  • the solid and hollow light spots scan the sample at the same time, and the excited two fluorescent signals pass through their respective detection light paths and are received by the two detectors. , so as to obtain the confocal light intensity distribution and the negative confocal light intensity distribution;
  • the polarization direction can be adjusted through a half-wave plate.
  • the polarization direction should be determined by the ratio of the negative confocal light intensity to the confocal light intensity, that is, by adjusting the polarization direction to match the two intensities.
  • a D-shaped mirror may be used to refract the light path to reduce the incident angle and improve the performance of the liquid crystal spatial light modulator.
  • the modulation of a component of linearly polarized light by the liquid crystal spatial light modulator is not limited to modulating it into vortex light to form a horizontal hollow light spot, but can also be formed on the object surface by 0/ ⁇ ring phase modulation.
  • Longitudinal hollow spot, this type of vertical hollow spot also has a weak hollow spot effect in the horizontal direction, so that three-dimensional fluorescence radiation differential super-resolution microscopy imaging can be realized, and its lateral resolution is relatively weaker than that of lateral hollow spot super-resolution microscopy imaging. resolution, but the longitudinal resolution can be greatly improved, allowing more detailed information about the sample to be obtained.
  • the dislocation parallel scanning of the light spot is obtained by loading the blazed grating with the liquid crystal spatial light modulator, which can reduce the difficulty of optical path calibration, and can perform Zernike aberration correction on the light spot to obtain the optimal solution. spot.
  • the solid spot and the hollow spot scan the sample at the same time, and after filtering out the defocused stray light through two pinholes, two detectors are used to obtain the fluorescence excited by the solid spot and the hollow spot, respectively. Signal. Additionally, a filter can be placed in front of the pinhole to eliminate stray light. The sample face is conjugated to the pinhole plane, allowing oblique and hollow light to scan the sample and into the pinhole.
  • the pinhole can be replaced by the end face of the multimode optical fiber, four adjacent multimode optical fibers can be installed side by side, the optical fiber end faces at both ends are used to receive the fluorescent signal, and a photomultiplier tube (PMT) is used to receive the fluorescent signal. Or avalanche photodiode (APD) detection; and the middle two optical fibers are not connected to the detector, which is used to more conveniently determine the interval between the two fluorescent signals.
  • PMT photomultiplier tube
  • APD avalanche photodiode
  • the fluorescence radiation differential formula is as follows:
  • I(x,y) I 1 (x,y)- ⁇ I 2 (x+ ⁇ x,y+ ⁇ y)
  • I 1 (x,y) is the confocal light intensity distribution
  • I 2 (x+ ⁇ x,y+ ⁇ y) is the shifted negative confocal light intensity distribution
  • ⁇ is the difference coefficient
  • the imaging device provided by the present invention includes:
  • Polarizers for generating linearly polarized light Polarizers for generating linearly polarized light
  • a liquid crystal spatial light modulator for separately modulating two components of linearly polarized light into vortex light and oblique light using halves;
  • Quarter-wave plate and half-wave plate for converting two excitation light into circularly polarized light
  • Beamsplitters used to reflect excitation light and transmit fluorescence
  • Pinhole or multimode fiber for eliminating out-of-focus stray light
  • the two detectors are used to detect the solid light spot and the hollow light spot respectively.
  • the two detectors are separated by 2-3AU.
  • FIG. 2 A specific implementation example of the present invention is given below, but is not limited thereto.
  • the structure of the super-resolution microscopy imaging device based on common-channel real-time fluorescence radiation difference in this example is shown in Figure 2, including a laser generating and collimating device 1, a polarizer 2, a first mirror 3, a first 1/2 glass Plate 4, D-shaped mirror 5, liquid crystal spatial light modulator 6, first 1/4 glass 7, first lens 8, second reflecting mirror 9, third reflecting mirror 10, second 1/2 glass 11, The second 1/4 slide 12, the 4f system 13, the beam splitter 14, the scanning galvanometer and the microscope stand module 15, the filter 16, the second lens 17, the first multimode fiber 18, the second multimode fiber 19, The first avalanche diode 20 and the second avalanche diode 21 .
  • the laser generated by the laser generating and collimating device 1 becomes linearly polarized light after passing through the polarizer 2, and then reflected by the D-shaped mirror 5 to reach the lower half of the liquid crystal spatial light modulator 6 with a smaller incident angle.
  • the P component of linearly polarized light is modulated as vortex light here, while the S component is not modulated.
  • the outgoing light of the liquid crystal spatial light modulator 6 reaches the second mirror 9 after passing through the first 1/4 glass 7, and is reflected back to the first 1/4 glass 7 through the second mirror 9, and then reaches the liquid crystal spatial light modulator
  • the upper half of 6; the second mirror 9 is used to make the incident light pass through the first 1/4 glass 7 twice and return to the upper half of the liquid crystal spatial light modulator 6.
  • the incident light is equivalent to passing through a 1/2 glass, so that the original S component can be modulated and tilted in the upper half of the liquid crystal spatial light modulator 6, and the light is divided into two paths; the two paths of light are respectively incident on the second half glass 11 and the second 1
  • the /4 glass slide 12 becomes circularly polarized light, and after parallel scanning the sample through the 4f system 13 for relay conjugation, the beam splitter 14, the scanning galvanometer and the microscope stand module 15, it reaches the beam splitter 14 in the reverse direction.
  • the reflected and fluorescent signal passes through the beam splitter 14 and is then introduced into the two multimode fibers through the filter 16 and the second lens 17.
  • the end face of the multimode fiber plays the role of a pinhole in a common confocal microscope.
  • the spacing corresponds to the spot spacing.
  • the final fluorescence signal is detected by two avalanche diodes, and then processed by subsequent algorithms to achieve super-resolution imaging.

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

一种基于共路并行荧光辐射差分的超分辨显微成像方法和装置，利用液晶空间光调制器（6）对荧光辐射差分超分辨显微成像中的激发光进行调制，将液晶空间光调制器（6）两部分分别加载为0-2π涡旋相位调制和闪耀光栅，使得共路的激发光在样品面同时形成有一定距离的一个实心光斑和一个空心光斑，进行并行扫描，从而保证成像速度相较普通荧光辐射差分超分辨显微成像提高一倍的同时两激发光因共路而不易受到噪声、漂移干扰的影响。

Description

基于共路并行荧光辐射差分的超分辨显微成像方法和装置 技术领域
本发明涉及光学超分辨显微成像领域，具体地说，涉及一种基于共路并行荧光辐射差分的超分辨显微成像方法和装置。
背景技术
在生物学、生命科学等领域中，光学显微镜是观察细胞、细胞器、病毒等的重要手段。然而，由于衍射及成像系统孔径的存在，光学显微镜的分辨率存在限制，这一限制称为阿贝衍射极限，值为0.61λ/NA，其中λ是光波波长，NA是物镜的数值孔径。
为突破衍射极限，科研人员提出了多种超分辨显微技术，其中荧光辐射差分超分辨显微技术(Fluorescence emission difference microscopy,FED)具有较低的光漂白特性和较快的成像速度。FED显微系统包括两路不同模式的激发光，其一是共聚焦光，在焦面表现为实心光斑；另一路是负共聚焦光，表现为面包圈型的空心光斑，内部尺寸小于衍射极限。FED显微图像是由这两幅图像差分得到的。
考虑到FED需要两幅图像，为了进一步提高成像速度，并行荧光辐射差分超分辨显微技术(Parallel fluorescence emission difference microscopy,pFED)使用光束偏转装置在物面上将实心光斑和空心光斑错开一定距离，从而用二者同时对样品进行扫描和探测。然而，pFED中的两激发光路容易分别受到漂移、噪声等外界干扰，从而限制了其成像质量。
发明内容
本发明提供了一种基于共路并行荧光辐射差分的超分辨显微成像方法和装置，本发明与pFED方法相比，激发光共路而非经过不同的器件， 可以更好的消除漂移、噪声等外界干扰的影响。
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基于共路并行荧光辐射差分的超分辨显微成像方法，该方法包括以下步骤：
(1)将激光器发出的激光光束准直后利用起偏器转为线偏光，这一线偏光包含S分量和P分量；
(2)调节液晶空间光调制器的出射平面与显微物镜入瞳共轭；使用液晶空间光调制器的一半部分，利用0-2π涡旋相位调制，调制步骤1)线偏光中平行于调制偏振方向的偏振分量，而对垂直于调整偏振方向的偏振分量不做调制；
(3)液晶空间光调制器的出射光经四分之一波片后到达反射镜，通过反射镜反射回同一四分之一波片，之后到达液晶空间光调制器的另半部分；
(4)将液晶空间光调制器的另半部分加载为闪耀光栅，从而使线偏光未被调制的分量被调制而倾斜，根据光栅方程，通过调整光栅常数计算倾角，使得最终实心光斑和空心光斑在物面上错开；
(5)将空间光调制器的另半部分出射的两路光转为圆偏光；
(6)两路圆偏光在样品面上呈现错开的实心光斑和空心光斑，实心光斑和空心光斑同时对样品进行扫描，激发的两路荧光信号分别经过各自的探测光路，由两个探测器接收，从而获得共聚焦光强度分布和负共聚焦光强度分布；
(7)将负共聚焦光强度分布移位后与共聚焦光强度分布对应，根据荧光辐射差分公式，得到超分辨图像。
进一步地，所述步骤(1)中，光束通过起偏器后通过一个二分之一波片调整偏振方向；偏振方向应当通过负共聚焦光强度与共聚焦光强度之 比来确定，即通过调整偏振方向使此二者强度匹配。
进一步地，所述步骤(2)中，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，所述步骤(2)中，液晶空间光调制器对线偏光一个分量的调制不限于将其调制为涡旋光以形成横向空心光斑，也可通过0/π环形相位调制在物面形成纵向空心光斑，这类纵向空心光斑在横向同样具有较弱的空心光斑效果，从而可以实现三维荧光辐射差分超分辨显微成像，其横向分辨率相对弱于横向空心光斑超分辨显微成像的横向分辨率，但纵向分辨率可极大提高，从而能够获得更多样品的细节信息。
进一步地，所述步骤(6)中，光斑错位并行扫描是通过液晶空间光调制器加载闪耀光栅的方式获得的，能够降低光路校准难度，且可以对光斑进行泽尼克像差矫正，获得最优光斑。
进一步地，所述步骤(6)中，实心光斑和空心光斑同时对样品进行扫描，通过两个针孔滤除离焦杂散光后，分别利用两个探测器获得实心光斑和空心光斑激发的荧光信号。
进一步地，所述步骤(6)中，实心光斑和空心光斑同时对样品进行扫描，可以安装四个并排相邻的多模光纤，使用两端的光纤端面接收荧光信号，并用光电倍增管或雪崩光电二极管探测；而中间两光纤不接探测器，用于更方便的确定两荧光信号的间隔。
进一步地，所述步骤(7)中，荧光辐射差分公式如下：
I(x,y)＝I 1(x,y)-γI 2(x+Δx,y+Δy)
其中I 1(x,y)为共聚焦光强度分布，I 2(x+Δx,y+Δy)为移位后的负共聚焦光强度分布，γ为差值系数。
本发明另一方面提供了一种基于共路并行荧光辐射差分的超分辨显微成像装置，该装置包括：
用于产生激发光的激光器及准直器；
用于产生线偏光的起偏器；
用于调整偏振方向的二分之一波片；
用于使用各半部分分别将线偏光的两个分量调制为涡旋光和倾斜光的液晶空间光调制器；
用于使线偏光的一个分量偏振方向旋转90度的四分之一波片及透镜和反射镜；
用于将两激发光转为圆偏光的四分之一波片和二分之一波片；
用于反射激发光而透射荧光的分光镜；
用于扫描样品的扫描振镜及显微镜架模块；
用于消除杂散光的滤光片；
用于消除离焦杂散光的针孔或多模光纤；
用于分别探测实心光斑和空心光斑的两探测器。
与现有技术相比，本发明具有以下有益的技术效果：由于两激发光共路，其经过相同的器件，同时受到各器件漂移及引入的噪声的影响，这些漂移和噪声在实心光斑与空心光斑进行差分时相消，从而在实现较快成像速度的同时改善了超分辨图像的信噪比。
附图说明
图1为本发明实施例的基于共路并行荧光辐射差分的超分辨显微成像方法流程图；
图2为本发明实施例的基于共路并行荧光辐射差分的超分辨显微成像装置示意图。
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，本发明提供的基于共路并行荧光辐射差分的超分辨显微成像方法，包括以下步骤：
(1)将激光器发出的激光光束准直后利用起偏器转为线偏光，这一线偏光包含S分量和P分量；
(2)调节液晶空间光调制器的出射平面与显微物镜入瞳共轭。液晶空间光调制器具有只调制一个方向的线偏光的性质。当外加电场超过阈值时，液晶分子出现电控双折射效应，使得液晶对一个对应方向的光产生双折射。利用这一性质，使用液晶空间光调制器的一半部分，利用0-2π涡旋相位调制，调制步骤1线偏光中平行于调制偏振方向的偏振分量，而对垂直于调整偏振方向的偏振分量不做调制。
(3)液晶空间光调制器的出射光经四分之一波片后到达反射镜，通过反射镜反射回同一四分之一波片，之后到达液晶空间光调制器的另半部分；利用反射镜转折光路使得入射光两次经过四分之一波片返回至液晶空间光调制器的另半部分，此时入射光相当于经过一个二分之一波片，偏振方向与原先垂直，而原来未被调制的偏振分量旋转到了可被调制的偏振方向，从而可以被液晶空间光调制器调制，而原来被调制的偏振分量此时处于垂直于调制偏振方向，未被液晶空间光调制器调制；
(4)将液晶空间光调制器的另半部分加载为闪耀光栅，从而使线偏光未被调制的分量被调制而倾斜，根据光栅方程(若是一级闪耀光栅)，可以通过调整光栅常数计算倾角，使得最终实心光斑和空心光斑在物面上错开，本实施例中错开2-3AU；
(5)将空间光调制器的另半部分出射的两路光转为圆偏光；例如，可以利用四分之一波片和二分之一波片使两路光转为圆偏光；
(6)两路圆偏光在样品面上呈现错开的实心光斑和空心光斑，实心光斑和空心光斑同时对样品进行扫描，激发的两路荧光信号分别经过各自的探测光路，由两个探测器接收，从而获得共聚焦光强度分布和负共聚焦光强度分布；
(7)将负共聚焦光强度分布移位后与共聚焦光强度分布对应，根据荧光辐射差分公式，得到超分辨图像。
进一步地，所述步骤(1)中，光束通过起偏器后可以通过一个二分之一波片调整偏振方向。偏振方向应当通过负共聚焦光强度与共聚焦光强度之比来确定，即通过调整偏振方向使此二者强度匹配。
进一步地，所述步骤(2)中，线偏光入射液晶空间光调制器前可以使用一个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，所述步骤(2)中，液晶空间光调制器对线偏光一个分量的调制不限于将其调制为涡旋光以形成横向空心光斑，也可通过0/π环形相位调制在物面形成纵向空心光斑，这类纵向空心光斑在横向同样具有较弱的空心光斑效果，从而可以实现三维荧光辐射差分超分辨显微成像，其横向分辨率相对弱于横向空心光斑超分辨显微成像的横向分辨率，但纵向分辨率可极大提高，从而能够获得更多样品的细节信息。
进一步地，所述步骤(6)中，光斑错位并行扫描是通过液晶空间光调制器加载闪耀光栅的方式获得的，能够降低光路校准难度，且可以对光斑进行泽尼克像差矫正，获得最优光斑。
进一步地，所述步骤(6)中，实心光斑和空心光斑同时对样品进行扫描，通过两个针孔滤除离焦杂散光后，分别利用两个探测器获得实心光斑和空心光斑激发的荧光信号。此外，在针孔前可以放置一滤光片以消除 杂散光。样品面与针孔平面共轭，从而使倾斜光和空心光扫描样品并进入针孔内。
进一步地，所述步骤(6)中，针孔可以用多模光纤的端面代替，可以安装四个并排相邻的多模光纤，使用两端的光纤端面接收荧光信号，并用光电倍增管(PMT)或雪崩光电二极管(APD)探测；而中间两光纤不接探测器，用于更方便的确定两荧光信号的间隔。
进一步地，所述步骤(7)中，荧光辐射差分公式如下：
I(x,y)＝I 1(x,y)-γI 2(x+Δx,y+Δy)
其中I 1(x,y)为共聚焦光强度分布，I 2(x+Δx,y+Δy)为移位后的负共聚焦光强度分布，γ为差值系数。
为实现上述方法，本发明提供的成像装置包括：
用于产生激发光的激光器及准直器；
用于产生线偏光的起偏器；
用于调整偏振方向的二分之一波片；
用于使用各半部分分别将线偏光的两个分量调制为涡旋光和倾斜光的液晶空间光调制器；
用于使线偏光的一个分量偏振方向旋转90度的四分之一波片及透镜和反射镜；
用于将两激发光转为圆偏光的四分之一波片和二分之一波片；
用于反射激发光而透射荧光的分光镜；
用于扫描样品的扫描振镜及显微镜架模块；
用于消除杂散光的滤光片；
用于消除离焦杂散光的针孔或多模光纤；
用于分别探测实心光斑和空心光斑的两探测器，本实施例中两探测器相距2-3AU。
以下给出本发明的一个具体实现示例，但不限于此。本示例的基于共路实时荧光辐射差分的超分辨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、起偏器2、第一反射镜3、第一1/2玻片4、D形镜5、液晶空间光调制器6、第一1/4玻片7、第一透镜8、第二反射镜9、第三反射镜10、第二1/2玻片11、第二1/4玻片12、4f系统13、分光镜14、扫描振镜及显微镜架模块15、滤光片16、第二透镜17、第一多模光纤18、第二多模光纤19、第一雪崩二极管20和第二雪崩二极管21。
装置工作时，激光发生和准直装置1产生的激光经过起偏器2后成为线偏光，再由D形镜5反射后以较小的入射角到达液晶空间光调制器6的下半部分。利用液晶空间光调制器仅可调制一个方向偏振光的性质，线偏光的P分量在此处被调制为涡旋光，而S分量未被调制。液晶空间光调制器6的出射光经第一1/4玻片7后到达第二反射镜9，通过第二反射镜9反射回第一1/4玻片7，之后到达液晶空间光调制器6的上半部分；利用第二反射镜9使得入射光两次经过第一1/4玻片7返回至液晶空间光调制器6的上半部分，此时入射光相当于经过一个1/2玻片，从而使得原S分量得以在液晶空间光调制器6的上半部分被调制而倾斜，将光分为两路；两路光分别入射至第二1/2玻片11和第二1/4玻片12成为圆偏光，通过用于中继共轭的4f系统13、分光镜14，扫描振镜及显微镜架模块15并行扫描样品后，反向到达分光镜14，此时激发光被反射而荧光信号通过分光镜14后经滤光片16及第二透镜17后导入两多模光纤中，此处多模光纤的端面起到了普通共聚焦显微镜中针孔的作用，两光纤端面的间距和光斑间距相对应。最终荧光信号被两个雪崩二极管探测，再经后续算法处理实现超分辨成像。
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. 一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，包括以下步骤：
    (1)将激光器发出的激光光束准直后利用起偏器转为线偏光，这一线偏光包含S分量和P分量；
    (2)调节液晶空间光调制器的出射平面与显微物镜入瞳共轭；使用液晶空间光调制器的一半部分，利用0-2π涡旋相位调制，调制步骤(1)线偏光中平行于调制偏振方向的偏振分量，而对垂直于调整偏振方向的偏振分量不做调制；
    (3)液晶空间光调制器的出射光经四分之一波片后到达反射镜，通过反射镜反射回同一四分之一波片，之后到达液晶空间光调制器的另半部分；
    (4)将液晶空间光调制器的另半部分加载为闪耀光栅，从而使线偏光未被调制的分量被调制而倾斜，根据光栅方程，通过调整光栅常数计算倾角，使得最终实心光斑和空心光斑在物面上错开；
    (5)将空间光调制器的另半部分出射的两路光转为圆偏光；
    (6)两路圆偏光在样品面上呈现错开的实心光斑和空心光斑，实心光斑和空心光斑同时对样品进行扫描，激发的两路荧光信号分别经过各自的探测光路，由两个探测器接收，从而获得共聚焦光强度分布和负共聚焦光强度分布；
    (7)将负共聚焦光强度分布移位后与共聚焦光强度分布对应，根据荧光辐射差分公式，得到超分辨图像。
  2. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(1)中，光束通过起偏器后通过一个二分之一波片调整偏振方向；偏振方向应当通过负共聚焦光强度与共 聚焦光强度之比来确定，即通过调整偏振方向使此二者强度匹配。
  3. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(2)中，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1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(2)中，液晶空间光调制器对线偏光一个分量的调制不限于将其调制为涡旋光以形成横向空心光斑，也可通过0/π环形相位调制在物面形成纵向空心光斑，这类纵向空心光斑在横向同样具有较弱的空心光斑效果，从而可以实现三维荧光辐射差分超分辨显微成像，其横向分辨率相对弱于横向空心光斑超分辨显微成像的横向分辨率，但纵向分辨率可极大提高，从而能够获得更多样品的细节信息。
  5. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(6)中，实心光斑和空心光斑同时对样品进行扫描，通过两个针孔滤除离焦杂散光后，分别利用两个探测器获得实心光斑和空心光斑激发的荧光信号。
  6. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(6)中，光斑错位并行扫描是通过液晶空间光调制器加载闪耀光栅的方式获得的，能够降低光路校准难度，且可以对光斑进行泽尼克像差矫正，获得最优光斑。
  7. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(6)中，实心光斑和空心光斑同时对样品进行扫描，可以安装四个并排相邻的多模光纤，使用两端的光纤端面接收荧光信号，并用光电倍增管或雪崩光电二极管探测；而中间两光纤不接探测器，用于更方便的确定两荧光信号的间隔。
  8. 根据权利要求1所述的一种基于共路并行荧光辐射差分的超分辨显微成像方法，其特征在于，所述步骤(7)中，荧光辐射差分公式如下：
    I(x,y)＝I 1(x,y)-γI 2(x+Δx,y+Δy)
    其中I 1(x,y)为共聚焦光强度分布，I 2(x+Δx,y+Δy)为移位后的负共聚焦光强度分布，γ为差值系数。
PCT/CN2021/133299 2020-11-27 2021-11-25 基于共路并行荧光辐射差分的超分辨显微成像方法和装置 WO2022111602A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/322,582 US20230296871A1 (en) 2020-11-27 2023-05-23 Super-resolution microscopic imaging method and apparatus based on common-path parallel fluorescence emission difference microscopy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011357705.0 2020-11-27
CN202011357705.0A CN112649405B (zh) 2020-11-27 2020-11-27 基于共路并行荧光辐射差分的超分辨显微成像方法和装置

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/322,582 Continuation US20230296871A1 (en) 2020-11-27 2023-05-23 Super-resolution microscopic imaging method and apparatus based on common-path parallel fluorescence emission difference microscopy

Publications (1)

Publication Number Publication Date
WO2022111602A1 true WO2022111602A1 (zh) 2022-06-02

Family

ID=75349515

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/133299 WO2022111602A1 (zh) 2020-11-27 2021-11-25 基于共路并行荧光辐射差分的超分辨显微成像方法和装置

Country Status (3)

Country Link
US (1) US20230296871A1 (zh)
CN (1) CN112649405B (zh)
WO (1) WO2022111602A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112649405B (zh) * 2020-11-27 2022-04-19 浙江大学 基于共路并行荧光辐射差分的超分辨显微成像方法和装置
CN113281891B (zh) * 2021-05-19 2022-06-10 浙江大学 共聚焦扫描式暗场显微成像方法与装置
CN114355621B (zh) * 2022-03-17 2022-07-08 之江实验室 一种多点非标记差分超分辨成像方法与装置

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090250632A1 (en) * 2006-10-06 2009-10-08 Michael Kempe Method and Arrangement for Collimated Microscopic Imaging
CN103411941A (zh) * 2013-08-21 2013-11-27 北京信息科技大学 基于高级次轴对称偏振光的并行共焦显微成像方法及装置
CN108120702A (zh) * 2017-11-30 2018-06-05 浙江大学 一种基于并行探测的超分辨荧光寿命成像方法和装置
CN109632756A (zh) * 2019-01-18 2019-04-16 浙江大学 一种基于并行光斑扫描的实时荧光辐射微分超分辨显微方法与装置
CN110118726A (zh) * 2019-04-12 2019-08-13 浙江大学 一种并行探测荧光发射差分显微成像的方法和装置
CN110632045A (zh) * 2019-09-10 2019-12-31 之江实验室 一种产生并行超分辨焦斑的方法和装置
CN111781173A (zh) * 2020-06-11 2020-10-16 浙江大学 基于并行sted和超临界角成像的三维超分辨显微成像方法和装置
CN112649405A (zh) * 2020-11-27 2021-04-13 浙江大学 基于共路并行荧光辐射差分的超分辨显微成像方法和装置

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6134010A (en) * 1997-11-07 2000-10-17 Lucid, Inc. Imaging system using polarization effects to enhance image quality
US7038848B2 (en) * 2002-12-27 2006-05-02 Olympus Corporation Confocal microscope
CN106970055B (zh) * 2017-04-28 2019-08-27 浙江大学 一种三维荧光差分超分辨显微方法及装置
CN107941763B (zh) * 2017-10-27 2020-06-30 浙江大学 一种共轴三维受激辐射损耗超分辨显微成像方法和装置
CN109283674A (zh) * 2018-10-08 2019-01-29 西北大学 一种荧光差分显微镜光路装置
CN111257295A (zh) * 2019-11-14 2020-06-09 江苏省医疗器械检验所 一种基于径向偏振光的高分辨差分共聚焦成像系统及成像方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090250632A1 (en) * 2006-10-06 2009-10-08 Michael Kempe Method and Arrangement for Collimated Microscopic Imaging
CN103411941A (zh) * 2013-08-21 2013-11-27 北京信息科技大学 基于高级次轴对称偏振光的并行共焦显微成像方法及装置
CN108120702A (zh) * 2017-11-30 2018-06-05 浙江大学 一种基于并行探测的超分辨荧光寿命成像方法和装置
CN109632756A (zh) * 2019-01-18 2019-04-16 浙江大学 一种基于并行光斑扫描的实时荧光辐射微分超分辨显微方法与装置
CN110118726A (zh) * 2019-04-12 2019-08-13 浙江大学 一种并行探测荧光发射差分显微成像的方法和装置
CN110632045A (zh) * 2019-09-10 2019-12-31 之江实验室 一种产生并行超分辨焦斑的方法和装置
CN111781173A (zh) * 2020-06-11 2020-10-16 浙江大学 基于并行sted和超临界角成像的三维超分辨显微成像方法和装置
CN112649405A (zh) * 2020-11-27 2021-04-13 浙江大学 基于共路并行荧光辐射差分的超分辨显微成像方法和装置

Also Published As

Publication number Publication date
US20230296871A1 (en) 2023-09-21
CN112649405A (zh) 2021-04-13
CN112649405B (zh) 2022-04-19

Similar Documents

Publication Publication Date Title
WO2022111602A1 (zh) 基于共路并行荧光辐射差分的超分辨显微成像方法和装置
CN102735617B (zh) 一种超分辨显微方法和装置
CN105973853B (zh) 一种基于双模式竞争激发的超分辨显微方法和装置
CN107941763B (zh) 一种共轴三维受激辐射损耗超分辨显微成像方法和装置
US10156710B2 (en) Microscope and microscopy techniques
CN105487214B (zh) 一种快速三维超分辨率显微方法和装置
CN109632756B (zh) 一种基于并行光斑扫描的实时荧光辐射微分超分辨显微方法与装置
US11409092B2 (en) Parallel multi-region imaging device
US7701632B2 (en) Method and arrangement for changing the spectral composition and/or intensity of illumination light and/or specimen light in an adjustable manner
US7038848B2 (en) Confocal microscope
CN110632045B (zh) 一种产生并行超分辨焦斑的方法和装置
CN104634766B (zh) 一种基于泵浦‑探针技术的超分辨装置和方法
US6449039B1 (en) Laser scanning fluorescence microscopy with compensation for spatial dispersion of fast laser pulses
US7872799B2 (en) Device for controlling light radiation
Ghosh et al. Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy
US7746470B2 (en) Optical scanning device and method of deriving same
KR100980306B1 (ko) 하이컨텐트 스크리닝을 위한 공초점 형광 현미경
JP2003227796A (ja) 深部分解による試料把握のための方法および配置
CN106770095A (zh) 一种基于非线性光斑调制的超分辨显微成像方法和装置
CN104614318A (zh) 一种快速的超分辨显微成像方法和装置
CN102798622A (zh) 一种基于强度差分的三维超分辨显微方法和装置
CN113281891B (zh) 共聚焦扫描式暗场显微成像方法与装置
WO2022241672A1 (zh) 共聚焦扫描式暗场显微成像方法与装置
CN102866137B (zh) 一种二维超分辨显微方法和装置
KR102105814B1 (ko) 레이저 공간 변조 초고분해능 광학 현미경

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21897102

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21897102

Country of ref document: EP

Kind code of ref document: A1