WO2017049752A1 - 一种基于一阶贝塞尔光束的sted超分辨显微镜及调节方法 - Google Patents

一种基于一阶贝塞尔光束的sted超分辨显微镜及调节方法 Download PDF

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WO2017049752A1
WO2017049752A1 PCT/CN2015/095118 CN2015095118W WO2017049752A1 WO 2017049752 A1 WO2017049752 A1 WO 2017049752A1 CN 2015095118 W CN2015095118 W CN 2015095118W WO 2017049752 A1 WO2017049752 A1 WO 2017049752A1
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light
loss
excitation light
objective lens
bessel beam
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PCT/CN2015/095118
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French (fr)
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施可彬
席鹏
于文韬
龚旗煌
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北京大学
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
    • 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
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
    • 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
    • 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
    • G02B21/0036—Scanning details, e.g. scanning stages

Definitions

  • the invention relates to microscopy technology, in particular to a stimulated radiation loss super-resolution microscope (GB-STED) based on a first-order Bessel beam and a method for adjusting the same.
  • G-STED stimulated radiation loss super-resolution microscope
  • Super-resolution microscopy imaging has had a major impact in areas such as bioimaging, material characterization, and laser fine processing.
  • the stimulated radiation loss (STED) microscope is a microscopic technique that realizes super-resolution imaging or excitation by directly adjusting the signal generation area based on the co-focus scanning microscope, compared with other types of super-resolution.
  • Micro-technology its principle is relatively simple, and the imaging speed is fast, it can carry out real-time super-resolution imaging in vivo, which has great application in biomedical field, and provides a new feasible in the field of nanomaterial research, laser processing and optical storage. Methods.
  • the stimulated radiation loss microscopy requires two beams of illumination.
  • One of the unmodulated Gaussian light (excitation light) is focused by the objective lens to produce an excitation region similar to the Airy spot, and the fluorescent material at the focus emits fluorescence; the other Gaussian light of different wavelength passes through the 0-2Pi spiral phase.
  • the modulation of the bit plate forms a hollow bag-shaped spot at the focal plane of the objective lens, and its center light intensity is close to zero, so that the excited state fluorescent molecules in the region with strong light intensity on the ring It jumps back to the ground state in the form of stimulated radiation, and no longer emits fluorescence, so that only the region with weak light intensity near the center of the bagel can generate fluorescence, which reduces the area of the fluorescent light-emitting region and breaks the diffraction limit and improves the resolution.
  • this lossy light is very sensitive to the phase distribution. For high-impact samples, when the inside is imaged, the shape of the lossed light focused spot is distorted due to aberration and scattering, and the resolution is drastically reduced.
  • the present invention proposes to utilize the anti-scattering and self-healing properties of a Bessel beam to change the loss light into a first-order Bessel beam, thereby having A certain aberration and scattering self-compensation ability to achieve stable super-resolution imaging inside the sample.
  • An object of the present invention is to provide a stimulated radiation loss super-resolution microscope based on a first-order Bessel beam (GB-STED).
  • the stimulated radiation loss super-resolution microscope based on the first-order Bessel beam of the present invention comprises: an excitation light source (emission continuous laser or pulse laser), a loss light source (emission continuous laser or pulse laser), excitation beam expansion collimation System, lossy optical beam expanding collimation system, spiral phase plate, Bessel beam generating system, loss light focusing lens, beam combining system, objective lens, piezoelectric scanning system, filter, signal collecting system and single photon detector;
  • the excitation light output by the excitation light source is filled with the entrance lens after the excitation light beam expansion collimation system;
  • the linearly polarized loss light output by the loss light source is sequentially passed through the beam expansion collimation system, the spiral phase plate, Bessel
  • the beam generating system and the lossy light focusing lens are precisely combined by the combining system and the excitation light, and then focused by the objective lens onto the sample; the sample is placed on the piezoelectric scanning system, and the sample is scanned by the piezoelectric scanning system, and the signal
  • the first-order Bessel beam generated by the Bessel beam generating system is focused by the loss light focusing lens and the objective lens, and the focused spot is the axial line light; the excitation light is formed after the objective lens.
  • the focused spot is point light, and the distance between the Bessel beam generating system and the loss light focusing lens is adjusted such that the center of the spot light of the focused spot of the excitation light is located in the axial direction of the focused light of the Bessel beam. Center, and by adjusting the beam combining system, the loss light is precisely coincident with the lateral direction of the focused spot of the excitation light.
  • the loss light focusing lens and the objective lens satisfy the confocal condition to form a confocal system.
  • the back focus of the loss light focusing lens is located near the front focus of the objective lens, and the distance between the loss light focusing lens and the objective lens is adjusted, so that the axis of the focused spot is formed. To the longest.
  • the Bessel beam generating system adopts a pyramidal mirror.
  • the center of the spot light formed by the excitation light behind the objective lens is located at the axial center of the line light of the Bessel beam.
  • the Bessel beam generating system uses a ring template and a collimating lens. This configuration requires the ring template to be at the front focal plane of the collimating lens by adjusting the distance between the collimating lens and the lossy light focusing lens.
  • the center of the spot light formed by the excitation light after the objective lens is located at the axial center of the line light of the Bessel beam;
  • the ring template includes a light-transmissive ring and the remaining opaque bottom plate, wherein the width of the light-transmitting ring is
  • the axial length of the line light generated after focusing by the objective lens is related, and the wider the width of the ring, the longer the length of the line light.
  • the Bessel beam generating system uses a spatial light modulator to adjust the distance between the spatial light modulator and the loss light focusing lens such that the center of the spot light formed by the excitation light behind the objective lens is located in the line light of the Bessel beam. Axial center.
  • the beam combining system employs first and second dichroic mirrors, the transmitted light of the two dichroic mirrors have overlapping bands, and the band of the signal light is located in the band of the transmitted light of the two dichroic mirrors.
  • the loss light is parallel to the excitation light, and the loss light and the excitation light are perpendicular to the signal light
  • the first dichroic mirror is totally reflected by the loss light, and is completely transmitted to the excitation light, and the signal light is completely Transmission
  • the second dichroic mirror is a total reflection of the excitation light, and is completely transmitted to the signal light; thus, the parallel loss light and the excitation light are respectively reflected by the first dichroic total reflection and the second dichroic mirror respectively, and the two optical paths are combined.
  • the signal After entering the objective lens together, the signal is incident on the sample, and the signal light is completely transmitted through the first and second dichroic mirrors, respectively, and collected by the signal collecting system.
  • the loss light is perpendicular to the excitation light and the loss light is parallel to the signal light
  • the first dichroic mirror is totally reflected to the loss light, and is completely transmitted to the excitation light and is totally transmitted to the signal light
  • the second dichroic mirror is the excitation
  • the light is totally transmissive and totally reflected by the signal light; thus, the loss light is totally reflected by the first dichroic mirror, and the excitation light that has passed through the first and second dichroic mirrors is completely transmitted, and the two optical paths are combined to enter the objective lens for focusing and incident.
  • the signal light is generated to the sample, and the signal light is completely transmitted through the first dichroic mirror, and then completely transmitted through the second dichroic mirror, and collected by the signal collecting system.
  • the signal collection system includes a signal collection lens and a multimode fiber as a confocal aperture to filter out signals outside the focused spot and improve longitudinal resolution.
  • the length of the line light formed in the axial direction is greater than 20 ⁇ m; the distance between the Bessel beam generating system and the loss light focusing lens is adjusted, so that the axis of the excitation light and the Sear beam The directions are coincident, and the beam combining system is adjusted such that the lateral coincidence accuracy of the two focused spots is within 10 nm.
  • a half slide is disposed between the spiral phase plate and the Bessel beam generating system, and a quarter slide is placed in front of the objective lens to adjust the loss light from linear polarization to left circular polarization. This can achieve a near-zero light intensity at the center of the loss-light focused spot, thereby increasing the imaging signal-to-noise ratio.
  • Another object of the present invention is to provide an adjustment method for the above-described stimulated radiation loss super-resolution microscope based on a first-order Bessel beam.
  • the invention relates to a method for adjusting a stimulated radiation loss super-resolution microscope based on a first-order Bessel beam, comprising the following steps:
  • the excitation light output from the excitation light source is filled with the entrance lens after the excitation light beam expansion collimation system; the loss of the linearly polarized loss light output by the loss light source is sequentially passed through the beam expansion collimation system, the spiral phase plate, Bessel
  • the beam generating system and the lossy light focusing lens are precisely combined by the combining system and the excitation light, and then focused by the objective lens onto the sample; the sample is placed on the piezoelectric scanning system, and the sample is scanned by the piezoelectric scanning system, and the signal generated by the sample is generated.
  • the first-order Bessel beam generated by the Bessel beam generating system is focused by the loss-light focusing lens and the objective lens to form the focal spot with the longest axial direction.
  • the stimulated radiation loss microscope based on the first-order Bessel beam of the present invention can be used as either a single photon fluorescence microscope or a multiphoton fluorescence microscope.
  • the advantages of the present invention over the conventional STED microscope are mainly:
  • Loss light is a first-order Bessel beam, which itself has anti-scattering and self-healing properties, and can maintain a good spot shape at a deep position of the sample, thereby improving the resolution of the deep region of the sample;
  • the experimental operation of the invention is relatively simple and does not require active adjustment; compared with the method using the adaptive optical system, the experimental device of the present invention is more Simple and cheap.
  • FIG. 1 is a schematic view of a GB-STED microscope according to Embodiment 1 of the present invention.
  • FIG. 2 is a schematic view of a GB-STED microscope according to Embodiment 2 of the present invention.
  • FIG. 3 is a top view and a light intensity distribution curve of a focused spot of an excitation light and a loss light at a focal plane position of an objective lens according to a GB-STED microscope according to Embodiment 1 of the present invention, wherein: (a) is a focused spot of the excitation light. Morphology, (b) is the shape of the focused spot of the loss of light, and (c) is the intensity distribution curve of the two focused spots across the center in the Y direction;
  • Figure 4 is a comparison of resolutions at different depths in agar samples containing 40 nm fluorescent spheres using a STED microscope (represented by GB-STED in the figure) and a conventional STED microscope (represented by STED in the figure) according to the first embodiment of the present invention.
  • the stimulated radiation loss super-resolution microscope based on the first-order Bessel beam of the present embodiment includes: an excitation light source 1, a loss light source 2, an excitation light beam expanding collimation system 3-1, and loss light.
  • the loss light passes through the beam expanding collimation system 3-2, the spiral phase plate 4, the half slide 5, the Bessel beam generating system 6 and the loss light focusing lens 14, through the combining system 8 and the excitation light.
  • the objective lens 9 is focused onto the sample; the sample is placed on the piezoelectric scanning system 10, and the sample is scanned by the piezoelectric scanning system 10, and the signal light generated by the sample enters the single photon detector 13 through the signal collecting system 12. Thereby a super-resolution image of the sample is obtained.
  • the loss light is parallel to the excitation light, and the loss light and the excitation light are perpendicular to the signal light.
  • the first dichroic mirror 8-1 is totally reflected to the loss light, and is fully transmissive to the excitation light and totally transmitted to the signal light;
  • the second dichroic mirror 8-2 is a total reflection of the excitation light and a total transmission of the signal light; thus, the parallel loss light and the excitation light are totally reflected by the first dichroic mirror 8-1 and the second dichroic mirror 8-2 are respectively totally reflected, and the two optical paths are respectively
  • the objective lens 9 is focused, and then incident on the sample to generate signal light, which is completely transmitted through the first and second dichroic mirrors, respectively, and collected by the signal collecting system 12.
  • the Bessel beam generating system 6 employs a pyramidal mirror (appropriate angle of 176 degrees), the front focus of the loss light focusing lens 14 (focal length of 200 mm) and the center of the first-order Bessel beam produced by the pyramidal mirror.
  • the points are coincident; the excitation light is parallel to the loss light and perpendicular to the signal light, and the combining system 8 includes a first dichroic mirror 8-1 and a second dichroic mirror 8-2, and the first dichroic mirror 8-1 is totally reflected by the loss light. While the excitation light is totally transmitted and fully transmitted to the signal light, the second dichroic mirror 8-2 is totally reflected by the excitation light and is totally transmitted to the signal light.
  • the distance between the pyramidal lens and the loss-light focusing lens 14 is 285 mm, and the distance between the loss-light focusing lens and the objective lens 9 (60 times, numerical aperture 1.2) is 202 mm, and the first-order Bessel is obtained after the objective lens.
  • the loss beam length is the longest and the shape is optimal.
  • the excitation light source 1 is a 635 nm continuous optical semiconductor laser
  • the loss light source 2 is a 750 nm continuous light titanium sapphire laser.
  • the beam is expanded to a focused spot having a diameter of about 8 mm, and is combined.
  • the system 8 is precisely coincident behind the objective lens 9, the specific process is: the front focus of the loss light focusing lens 14 is located near the center point of the Bessel light generated by the pyramidal mirror, and the angle between the pyramid mirror 6 and the loss light focusing lens 14 is adjusted.
  • the axial length of the focused spot is as long as possible and the focused spot is optimal.
  • the shape of the focused spot of the excitation light and the focused spot of the loss light at the focal plane of the objective lens are as shown in Fig. 3, wherein (a) is the shape of the focused spot of the excitation light, and (b) is the focused spot of the loss light. Morphology, (c) is the intensity distribution curve of the two focused spots over the center in the Y direction, wherein the center intensity of the focused spot of the loss light in (c) is 3.7% of the maximum value, and the focused spot shape is better. And the focused spot of the excitation light and the focused spot of the loss light are exactly coincident.
  • GB-STED first-order Bessel beam-based STED super-resolution microscope
  • a conventional embodiment of the present embodiment A comparison of the resolution of STED super-resolution microscopy at different depths in agar samples containing 40 nm fluorescent spheres.
  • the resolution of the conventional STED microscope is deteriorating with the increase of the imaging depth, and the resolution of the STED super-resolution microscope based on the first-order Bessel beam of the present invention remains substantially unchanged within about 155 microns, which is about 110 nm. It can be seen that after the lossy light is changed from the modulated Gaussian beam to the first-order Bessel beam, the resolution of the deep imaging is significantly improved.
  • the Bessel beam generating system 6 employs a ring template 6-1 and a lens 6-2 by adjusting the lens 6-2 of the Bessel beam generating system and the loss light focusing lens.
  • the distance between 14 is such that the center of the spot light formed by the excitation light behind the objective lens 9 is located at the axial center of the line light of the Bessel beam, and the lateral direction is exactly coincident.
  • the loss light is perpendicular to the excitation light, and the loss light is parallel to the signal light.
  • the first dichroic mirror 8-1 is totally reflective to the loss light, and is completely transmissive to the excitation light, and is completely transmissive to the signal light;
  • the two dichroic mirror 8-2 is a total transmission of the excitation light and is totally reflected by the signal light; thus, the loss light is totally reflected by the first dichroic mirror 8-1, and the excitation light is completely transmitted through the first and second dichroic mirrors.
  • the two optical paths are combined to enter the objective lens 9 for focusing, and the incident light is incident on the sample, and the signal light is completely transmitted through the first dichroic mirror 8-1, and then completely transmitted through the second dichroic mirror 8-2, and the signal collecting system 12 is used. collect.
  • the other structure is the same as in the first embodiment.

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Abstract

一种基于一阶贝塞尔光束的STED超分辨显微镜及其调节方法。STED显微镜包括：激发光光源（1）、损耗光光源（2）、激发光扩束准直系统（3-1）、损耗光扩束准直系统（3-2）、螺旋形相位板（4）、贝塞尔光束产生系统（6）、损耗光聚焦透镜（14）、合束系统（8）、物镜（9）、压电扫描系统（10）、滤波片（11）、信号收集系统（12）和单光子探测器（13）；损耗光为一阶贝塞尔光束，其本身具有抗散射和自愈特性，在样品较深的位置可以保持很好的光斑形貌，从而提高样品深层区域的分辨率；相比于调节物镜校正环来实现STED超分辨显微镜深层成像的方法，实验操作上较为简单，无需主动调节；相比于使用自适应光学系统的方法，实验装置上较为简单且廉价。

Description

一种基于一阶贝塞尔光束的STED超分辨显微镜及调节方法 技术领域
本发明涉及显微镜技术，具体涉及一种基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜(GB-STED)及其调节方法。
背景技术
超分辨显微成像技术在生物成像、材料表征以及激光精细加工等领域产生了重大的影响。其中受激辐射损耗(STED)显微镜是一种在共聚焦点扫描式显微镜的基础上，通过直接调节信号产生区域面积从而实现超分辨成像或者激发的显微技术，相较于其他类型的超分辨显微技术，其原理较为简单，且成像速度较快，可以进行活体实时超分辨成像，在生物医学领域有着巨大的应用，并且在纳米材料研究，激光加工以及光存储领域提供了一种新的可行的方法。
受激辐射损耗显微技术需要两束光进行照明。其中一束未经调制的高斯光(激发光)经过物镜聚焦后产生一个近似于爱里斑的激发区域，焦点处的荧光物质发出荧光；另一束不同波长的高斯光通过0-2Pi螺旋形相位板的调制(损耗光)，在物镜焦平面位置形成一个中空的面包圈状的光斑，其中心光强接近于零，这样使处在圆环上光强较强区域内的激发态的荧光分子以受激辐射的方式跃迁回基态，不再发出荧光，这样只有面包圈中心附近损耗光光强较弱的区域才能产生荧光，减小了荧光发光区域的面积从而突破衍射极限，提高分辨率。但是，这束损耗光对于相位分布十分敏感，对于高像差的样品，在对其内部进行成像时，由于像差和散射导致损耗光聚焦光斑的形貌产生畸变，从而使分辨率急剧下降。
近年来，为了提高STED显微镜的成像深度，已经有人通过调节物镜校正环，在80-100微米的深度达到稳定的分辨率，但是这些方法通常操作较为复杂且对于高散射样品并不一定适用。还有人使用自适应光学系统对样品带来的像差进行补偿，但系统比较复杂且昂贵。
发明内容
针对以上现有技术中STED显微镜在深层成像时遇到的问题，本发明提出了一种利用贝塞尔光束的抗散射和自愈特性，将损耗光变为一阶贝塞尔光束，从而具有一定的像差和散射的自我补偿能力，以达到样品内部的稳定超分辨成像的实现方法。
本发明的一个目的在于提供一种基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜 (GB-STED)。
本发明的基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜包括：激发光光源(发射连续激光或脉冲激光)、损耗光光源(发射连续激光或脉冲激光)、激发光扩束准直系统、损耗光扩束准直系统、螺旋形相位板、贝塞尔光束产生系统、损耗光聚焦透镜、合束系统、物镜、压电扫描系统、滤波片、信号收集系统和单光子探测器；其中，激发光光源输出的激发光经过激发光扩束准直系统后充满物镜的入瞳；损耗光光源输出的线偏振的损耗光依次经过扩束准直系统、螺旋形相位板、贝塞尔光束产生系统和损耗光聚焦透镜，通过合束系统和激发光精确合束后，再由物镜聚焦到样品上；样品放置在压电扫描系统上，通过压电扫描系统扫描样品，样品产生的信号光通过信号收集系统进入单光子探测器，从而得到样品的超分辨图像；损耗光聚焦透镜与物镜满足共焦条件，构成共焦系统，经贝塞尔光束产生系统产生的一阶贝塞尔光束，通过损耗光聚焦透镜和物镜聚焦后，形成的聚焦光斑为轴向的线光；激发光在物镜后形成的聚焦光斑为点光，通过调节贝塞尔光束产生系统与损耗光聚焦透镜之间的距离，使得激发光的聚焦光斑的点光的中心位于贝塞尔光束的聚焦光斑的线光的轴向中心，并且通过调节合束系统使损耗光与激发光的聚焦光斑的横向精确重合。
损耗光聚焦透镜与物镜满足共焦条件，构成共焦系统，在损耗光聚焦透镜的后焦点位于物镜的前焦点附近，调节损耗光聚焦透镜与物镜之间的距离，使得形成的聚焦光斑的轴向最长。
贝塞尔光束产生系统采用角锥镜，通过调节角锥镜和损耗光聚焦透镜之间的距离，使得激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心；角锥镜的顶角越大，物镜后一阶贝塞尔光束的线光的轴向长度越长。或者，贝塞尔光束产生系统采用环形模板和一个准直透镜，这种配置下要求环形模板在准直透镜的前焦面处，通过调节准直透镜与损耗光聚焦透镜之间的距离，使得激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心；环形模板包括一个透光的圆环和其余不透光的底板，其中透光的圆环的宽度与经物镜聚焦后产生的线光的轴向长度有关，环的宽度越宽，线光的长度越长。或者， 贝塞尔光束产生系统采用空间光调制器，通过调节空间光调制器和损耗光聚焦透镜之间的距离使激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心。
合束系统采用第一和第二双色镜，两个双色镜的透过光具有重合的波段，并且信号光的波段位于两个双色镜的透过光重合的波段内。对于损耗光与激发光平行，且损耗光和激发光与信号光垂直的情况，第一双色镜为对损耗光全反射，而对激发光全透射，并且对信号光全 透射；第二双色镜为对激发光全反射，对信号光全透射；从而平行的损耗光和激发光分别经过第一双色镜全反射和第二双色镜全反射后，两路光路合束，共同进入物镜聚焦后，入射至样品产生信号光，信号光分别经第一和第二双色镜全透射，由信号收集系统收集。对于损耗光与激发光垂直，且损耗光与信号光平行的情况，第一双色镜为对损耗光全反射，而对激发光全透射，并且对信号光全透射；第二双色镜为对激发光全透射，对信号光全反射；从而损耗光经过第一双色镜全反射后，与经过第一和第二双色镜全透射后的激发光，两路光路合束，共同进入物镜聚焦，入射至样品产生信号光，信号光分别经第一双色镜全透射后，经第二双色镜全透射，由信号收集系统收集。
信号收集系统包括信号收集透镜和一个作为共聚焦小孔的多模光纤，从而滤去聚焦光斑之外的信号，提高纵向分辨率。
损耗光经损耗光聚焦透镜和物镜聚焦后，在轴向形成线光的长度大于20微米；调节贝塞尔光束产生系统与损耗光聚焦透镜之间的距离，使得激发光与塞尔光束的轴向重合，并且调节合束系统使得两个聚焦光斑的横向重合精度达到10纳米以内。
进一步，在螺旋形相位板与贝塞尔光束产生系统之间设置二分之一玻片，并且在物镜前放置四分之一玻片，从而将损耗光从线偏振调整为左旋圆偏振光，这样可以在损耗光聚焦光斑中心获得接近于零的光强，从而提高成像信噪比。
本发明的另一个目的在于提供上述基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜的调节方法。
本发明的一种基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜的调节方法，包括以下步骤：
1) 激发光光源输出的激发光经过激发光扩束准直系统后充满物镜的入瞳；损耗光光源输出的线偏振的损耗光依次经过扩束准直系统、螺旋形相位板、贝塞尔光束产生系统和损耗光聚焦透镜，通过合束系统和激发光精确合束后，再由物镜聚焦到样品上；样品放置在压电扫描系统上，通过压电扫描系统扫描样品，样品产生的信号光通过信号收集系统进入单光子探测器，从而得到样品的超分辨图像；
2) 通过调节贝塞尔光束产生系统与损耗光聚焦透镜之间的距离，使得激发光的聚焦光斑的点光的中心位于贝塞尔光束的聚焦光斑的线光的轴向中心；
3) 通过调节合束系统使损耗光与激发光的聚焦光斑的横向精确重合：
a)损耗光与激发光平行的情况，通过分别调节第一和第二双色镜的角度，分别改变 损耗光和激发光的偏转角，从而使得损耗光与激发光的聚焦光斑的横向精确重合；
b)损耗光与激发光垂直的情况，通过调节第一双色镜的角度，改变损耗光的偏转，从而使得损耗光与激发光的聚焦光斑的横向精确重合；
4) 调节损耗光聚焦透镜与物镜之间的距离，经贝塞尔光束产生系统产生的一阶贝塞尔光束，通过损耗光聚焦透镜和物镜聚焦后，形成的聚焦光斑的轴向最长。
本发明的基于一阶贝塞尔光束的受激辐射损耗显微镜既可以作为单光子荧光显微镜，也可以作为多光子荧光显微镜。与传统STED显微镜相比，本发明的优点主要在于：
(1)损耗光为一阶贝塞尔光束，其本身具有抗散射和自愈特性，在样品较深的位置可以保持很好的光斑形貌，从而提高样品深层区域的分辨率；
(2)相比于调节物镜校正环来实现STED超分辨显微镜深层成像的方法，本发明实验操作上较为简单，无需主动调节；相比于使用自适应光学系统的方法，本发明实验装置上较为简单且廉价。
附图说明
图1为本发明实施例一所述GB-STED显微镜的示意图；
图2为本发明实施例二所述GB-STED显微镜的示意图；
图3为本发明实施例一所述GB-STED显微镜在物镜的焦平面位置激发光和损耗光的聚焦光斑的形貌图和光强分布曲线，其中：(a)为激发光的聚焦光斑的形貌，(b)为损耗光的聚焦光斑的形貌，(c)为两个聚焦光斑过中心沿Y方向的光强分布曲线；
图4为利用本发明实施例一所述STED显微镜(图中用GB-STED代表)与传统STED显微镜(图中用STED代表)在含有40nm荧光球的琼脂样品中不同深度的分辨率对比图。
具体实施方式
下面结合附图，通过实施例对本发明做进一步说明。
实施例一
如图1所示，本实施例的基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜包括：激发光光源1、损耗光光源2、激发光扩束准直系统3-1、损耗光扩束准直系统3-2、螺旋形相位板4、二分之一玻片5、贝塞尔光束产生系统6、损耗光聚焦透镜14、合束系统8、物镜9、压电扫描系统10、滤波片11、信号收集系统12和单光子探测器13；其中，激发光光源1输出的激发光经过激发光扩束准直系统3-1后充满物镜9的入瞳；损耗光光源2输出的线偏振 的损耗光依次经过扩束准直系统3-2、螺旋形相位板4、二分之一玻片5、贝塞尔光束产生系统6和损耗光聚焦透镜14，通过合束系统8和激发光精确合束后，再由物镜9聚焦到样品上；样品放置在压电扫描系统10上，通过压电扫描系统10扫描样品，样品产生的信号光通过信号收集系统12进入单光子探测器13，从而得到样品的超分辨图像。损耗光与激发光平行，且损耗光和激发光与信号光垂直，第一双色镜8-1为对损耗光全反射，而对激发光全透射，并且对信号光全透射；第二双色镜8-2为对激发光全反射，对信号光全透射；从而平行的损耗光和激发光分别经过第一双色镜8-1全反射和第二双色镜8-2全反射后，两路光路合束，共同进入物镜9聚焦后，入射至样品产生信号光，信号光分别经第一和第二双色镜全透射，由信号收集系统12收集。
在本实施例中，贝塞尔光束产生系统6采用角锥镜(顶角176度)，损耗光聚焦透镜14(焦距200mm)的前焦点与角锥镜产生的一阶贝塞尔光束的中心点重合；激发光与损耗光平行，与信号光垂直，合束系统8包括第一双色镜8-1和第二双色镜8-2，第一双色镜8-1为对损耗光全反射，而对激发光全透射，并且对信号光全透射，第二双色镜8-2为对激发光全反射，对信号光全透射。角锥镜与损耗光聚焦透镜14之间的距离为285毫米，损耗光聚焦透镜与物镜9(60倍，数值孔径1.2)之间的距离为202毫米，此时得到物镜后一阶贝塞尔损耗光束长度最长且形貌最优。
激发光光源1为635纳米连续光半导体激光器，损耗光光源2为750纳米连续光钛宝石激光器，经过各自个扩束准直系统后均扩束到直径约8毫米的聚焦光斑，并通过合束系统8在物镜9后精确重合，具体过程是：损耗光聚焦透镜14的前焦点位于角锥镜产生的贝塞尔光的中心点附近，调节角锥镜6和损耗光聚焦透镜14之间的距离，使高斯型的激发光的聚焦光斑的点光在损耗光的一阶贝塞尔光束的聚焦光斑的线光的轴向中间位置；调节合束系统8中的第一和第二双色镜的角度，使两个聚焦光斑在横向上精确重合；在物镜的前焦点位于损耗光聚焦透镜的后焦点附近，调节损耗光聚焦透镜14和物镜9之间的距离使一阶贝塞尔损耗光的聚焦光斑的轴向长度尽可能长，且聚焦光斑最优。
激发光的聚焦光斑和损耗光的聚焦光斑在物镜焦平面位置的形貌如图3所示，其中，(a)为激发光的聚焦光斑的形貌，(b)为损耗光的聚焦光斑的形貌，(c)为两个聚焦光斑过中心沿Y方向的光强分布曲线，其中，(c)中损耗光的聚焦光斑的中心光强为最大值的3.7％，聚焦光斑形貌较好，且激发光的聚焦光斑和损耗光的聚焦光斑精确重合。
图4是本实施例的基于一阶贝塞尔光束的STED超分辨显微镜(GB-STED)以及传统 STED超分辨显微镜在含有40nm荧光球的琼脂样品中不同深度的分辨率的对比图。传统STED显微镜的分辨率随着成像深度的增加不断恶化，而本发明的基于一阶贝塞尔光束的STED超分辨显微镜在155微米以内其分辨率保持基本不变，约在110纳米左右。可见，将损耗光由调制的高斯光束改为一阶贝塞尔光束后，其在深层成像的分辨率得到了明显改善。
实施例二
如图2所示，在本实施例中，贝塞尔光束产生系统6采用环形模板6-1和一个透镜6-2，通过调节贝塞尔光束产生系统的透镜6-2与损耗光聚焦透镜14之间的距离，使得激发光在物镜9后形成的点光的中心位于贝塞尔光束的线光的轴向中心，并且横向精确重合。
在本实施例中，损耗光与激发光垂直，且损耗光与信号光平行，第一双色镜8-1为对损耗光全反射，而对激发光全透射，并且对信号光全透射；第二双色镜8-2为对激发光全透射，对信号光全反射；从而损耗光经过第一双色镜8-1全反射后，与经过第一和第二双色镜全透射后的激发光，两路光路合束，共同进入物镜9聚焦，入射至样品产生信号光，信号光分别经第一双色镜8-1全透射后，经第二双色镜8-2全透射，由信号收集系统12收集。其他结构同实施例一。
最后需要注意的是，公布实施方式的目的在于帮助进一步理解本发明，但是本领域的技术人员可以理解：在不脱离本发明及所附的权利要求的精神和范围内，各种替换和修改都是可能的。因此，本发明不应局限于实施例所公开的内容，本发明要求保护的范围以权利要求书界定的范围为准。

Claims (10)

  1. 一种基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜，包括：激发光光源、损耗光光源、激发光扩束准直系统、损耗光扩束准直系统、螺旋形相位板、贝塞尔光束产生系统、损耗光聚焦透镜、合束系统、物镜、压电扫描系统、滤波片、信号收集系统和单光子探测器；其中，所述激发光光源输出的激发光经过激发光扩束准直系统后充满物镜的入瞳；所述损耗光光源输出的线偏振的损耗光依次经过扩束准直系统、螺旋形相位板、贝塞尔光束产生系统和损耗光聚焦透镜，通过合束系统和激发光精确合束后，再由物镜聚焦到样品上；样品放置在压电扫描系统上，通过压电扫描系统扫描样品，样品产生的信号光通过信号收集系统进入单光子探测器，从而得到样品的超分辨图像；所述损耗光聚焦透镜与物镜满足共焦条件，构成共焦系统，经贝塞尔光束产生系统产生的一阶贝塞尔光束，通过损耗光聚焦透镜和物镜聚焦后，形成的聚焦光斑为轴向的线光；激发光在物镜后形成的聚焦光斑为点光，通过调节贝塞尔光束产生系统与损耗光聚焦透镜之间的距离，使得激发光的聚焦光斑的点光的中心位于贝塞尔光束的聚焦光斑的线光的轴向中心，并且通过调节合束系统使损耗光与激发光的聚焦光斑的横向精确重合。
  2. 如权利要求1所述的受激辐射损耗超分辨显微镜，其特征在于，所述贝塞尔光束产生系统采用角锥镜，通过调节角锥镜和损耗光聚焦透镜之间的距离，使得激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心；所述角锥镜的顶角越大，物镜后一阶贝塞尔光束的线光的轴向长度越长。
  3. 如权利要求1所述的受激辐射损耗超分辨显微镜，其特征在于，所述贝塞尔光束产生系统采用环形模板和一个准直透镜，所述环形模板在准直透镜的前焦面处，通过调节准直透镜与损耗光聚焦透镜之间的距离，使得激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心。
  4. 如权利要求3所述的受激辐射损耗超分辨显微镜，其特征在于，所述环形模板包括一个透光的圆环和其余不透光的底板，其中透光的圆环的宽度与经物镜聚焦后产生的线光的轴向长度有关，环的宽度越宽，线光的长度越长。
  5. 如权利要求1所述的受激辐射损耗超分辨显微镜，其特征在于，所述贝塞尔光束产生系统采用空间光调制器，通过调节空间光调制器和损耗光聚焦透镜之间的距离使激发光在物镜后形成的点光的中心位于贝塞尔光束的线光的轴向中心。
  6. 如权利要求1所述的受激辐射损耗超分辨显微镜，其特征在于，所述合束系统采用第一和 第二双色镜，两个双色镜的透过光具有重合的波段，并且信号光的波段位于两个双色镜的透过光重合的波段内。
  7. 如权利要求6所述的受激辐射损耗超分辨显微镜，其特征在于，对于损耗光与激发光平行，且损耗光和激发光与信号光垂直的情况，第一双色镜为对损耗光全反射，而对激发光全透射，并且对信号光全透射；第二双色镜为对激发光全反射，对信号光全透射；从而平行的损耗光和激发光分别经过第一双色镜全反射和第二双色镜全反射后，两路光路合束，共同进入物镜聚焦后，入射至样品产生信号光，信号光分别经第一和第二双色镜全透射，由信号收集系统收集。
  8. 如权利要求6所述的受激辐射损耗超分辨显微镜，其特征在于，对于损耗光与激发光垂直，且损耗光与信号光平行的情况，第一双色镜为对损耗光全反射，而对激发光全透射，并且对信号光全透射；第二双色镜为对激发光全透射，对信号光全反射；从而损耗光经过第一双色镜全反射后，与经过第一和第二双色镜全透射后的激发光，两路光路合束，共同进入物镜聚焦，入射至样品产生信号光，信号光分别经第一双色镜全透射后，经第二双色镜全透射，由信号收集系统收集。
  9. 如权利要求1所述的受激辐射损耗超分辨显微镜，其特征在于，在螺旋形相位板与贝塞尔光束产生系统之间设置二分之一玻片，并且在物镜前放置四分之一玻片，从而将损耗光从线偏振调整为左旋圆偏振光。
  10. 权利要求1所述基于一阶贝塞尔光束的受激辐射损耗超分辨显微镜的调节方法，包括以下步骤：
    5)激发光光源输出的激发光经过激发光扩束准直系统后充满物镜的入瞳；损耗光光源输出的线偏振的损耗光依次经过扩束准直系统、螺旋形相位板、贝塞尔光束产生系统和损耗光聚焦透镜，通过合束系统和激发光精确合束后，再由物镜聚焦到样品上；样品放置在压电扫描系统上，通过压电扫描系统扫描样品，样品产生的信号光通过信号收集系统进入单光子探测器，从而得到样品的超分辨图像；
    6)通过调节贝塞尔光束产生系统与损耗光聚焦透镜之间的距离，使得激发光的聚焦光斑的点光的中心位于贝塞尔光束的聚焦光斑的线光的轴向中心；
    7)通过调节合束系统使损耗光与激发光的聚焦光斑的横向精确重合：
    a)损耗光与激发光平行的情况，通过分别调节第一和第二双色镜的角度，分别改变损耗光和激发光的偏转角，从而使得损耗光与激发光的聚焦光斑的横向精确重合；
    b)损耗光与激发光垂直的情况，通过调节第一双色镜的角度，改变损耗光的偏转，从而使得损耗光与激发光的聚焦光斑的横向精确重合；
    8)调节损耗光聚焦透镜与物镜之间的距离，使得经贝塞尔光束产生系统产生的一阶贝塞尔光束，通过损耗光聚焦透镜和物镜聚焦后，形成的聚焦光斑的轴向最长。
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CN108519329B (zh) * 2018-03-26 2021-01-15 华中科技大学 一种多路扫描与探测的线共聚焦成像装置
CN112255210A (zh) * 2020-10-13 2021-01-22 鲁东大学 一种钙钛矿薄膜畴边界激子动力学的超分辨系统
CN112255210B (zh) * 2020-10-13 2022-09-23 鲁东大学 一种钙钛矿薄膜畴边界激子动力学的超分辨系统
CN113484320A (zh) * 2021-07-01 2021-10-08 西北大学 一种远场光学超薄片层成像系统及方法
CN117270184A (zh) * 2023-11-22 2023-12-22 国科大杭州高等研究院 一种突破衍射极限分辨率的多模光纤显微成像系统和方法
CN117270184B (zh) * 2023-11-22 2024-03-29 国科大杭州高等研究院 一种突破衍射极限分辨率的多模光纤显微成像系统和方法

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