WO2024087615A1 - 一种荧光发射比率三维超分辨成像方法 - Google Patents

一种荧光发射比率三维超分辨成像方法 Download PDF

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WO2024087615A1
WO2024087615A1 PCT/CN2023/096682 CN2023096682W WO2024087615A1 WO 2024087615 A1 WO2024087615 A1 WO 2024087615A1 CN 2023096682 W CN2023096682 W CN 2023096682W WO 2024087615 A1 WO2024087615 A1 WO 2024087615A1
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laser
reflector
imaging method
resolution imaging
fluorescence emission
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PCT/CN2023/096682
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French (fr)
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宋军
郭嘉庆
陈越
李灏
俞宪同
陈钰
王璐玮
屈军乐
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深圳大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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

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  • the invention belongs to the technical field of optical microscope imaging methods, and in particular relates to a fluorescence emission ratio three-dimensional super-resolution imaging method.
  • 3D super-resolution imaging of organelles and associated biomolecules can be used to observe living
  • the dynamic process of living samples and the interaction of substances between cells or within cells are crucial to revealing the structure and function of living biological samples.
  • the existing three-dimensional super-resolution imaging platform is unable to reveal the role of organelle interactions in material transport and utilization, organelle homeostasis regulation, and cannot provide technical support for the role of organelle interactions in disease occurrence.
  • the technical problem to be solved by the present invention is to provide a fluorescence emission ratio three-dimensional super-resolution imaging method in view of the shortcomings of the above-mentioned prior art, reduce the requirements of super-resolution technology for fluorescent dyes and sample preparation, broaden the selection range of dyes, provide technical support for low-cost in vivo three-dimensional super-resolution imaging research, and solve the problems raised in the above-mentioned background technology.
  • the technical solution adopted by the present invention is: a fluorescence emission ratio three-dimensional super-resolution imaging method, comprising a pulsed laser, wherein the laser light emitted by the pulsed laser passes through a half-wave plate, and then passes through a Glan laser prism, and is divided into two laser beams by the Glan laser prism, wherein one laser beam is transmitted to a high-speed photodiode detector, and the other laser beam is transmitted to a first beam splitter, and is again divided into two laser beams by the beam splitter, and respectively illuminates a first reflector and a 0/ ⁇ phase plate;
  • the laser light passing through the 0/ ⁇ phase plate is transmitted to the second reflector, and then to the corner reflector after passing through the second reflector;
  • the laser beam passing through the first reflector is transmitted to the second beam splitter, and is split into two beams again by the second beam splitter, and is transmitted to the corner reflector and the third reflector respectively;
  • the laser light passing through the third reflector passes through the dichroic mirror, galvanometer, scanning lens, tube lens and quarter glass slide in sequence, and is focused by the objective lens and irradiated onto the sample on the stage.
  • the high-speed photodiode detector is connected to a time-correlated single photon counter, and the time-correlated single photon counter is connected to a computer.
  • the sample on the stage generates fluorescence after being excited.
  • the fluorescence is collected by the objective lens and passes through a quarter glass slide, a tube lens, a scanning lens, a galvanometer and a dichroic mirror. After being reflected by the dichroic mirror, it reaches the photomultiplier tube through a filter.
  • the filter is used to remove stray light other than fluorescence and improve the image signal-to-noise ratio.
  • the time-correlated single-photon counter simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector and the photomultiplier tube, and transmits the data to a computer for storage and processing.
  • the pulse laser generates 40 MHz pulsed excitation light by a picosecond laser
  • the half-wave plate is used to adjust the polarization direction of the laser
  • the Glan laser prism is used to separate lasers with different polarization directions.
  • the 0/ ⁇ phase plate is used to generate an excitation light spot of a three-dimensional cage structure, and the corner reflector controls the pulse interval between the Gaussian laser and the ring laser in time by extending or shortening the optical path of the ring-shaped excitation light spot.
  • the dichroic mirror is used to transmit the excitation light and reflect the fluorescence signal
  • the galvanometer is used to synchronously scan the excitation light to achieve area array imaging of the sample
  • the scanning lens is used to collect the laser beam for area array scanning.
  • the objective lens is used to focus the laser onto a focal plane and collect the fluorescent signal reflected from the sample at the same time.
  • the tube lens and the objective lens are combined to form a microscope system.
  • the quarter glass is used to convert linearly polarized laser light into right-handed circularly polarized light.
  • stage is used to place and fix the sample and perform three-dimensional move.
  • first beam splitter and the second beam splitter are respectively used for laser beam splitting or beam combining
  • first reflector, the second reflector and the third reflector are respectively used for changing the transmission direction of the laser.
  • the present invention has the following advantages:
  • the fluorescence emission ratio three-dimensional nano-microscopy imaging method of the present invention can realize three-dimensional super-resolution imaging under a single-wavelength laser. Not only is the laser power low, but the imaging system is simple and low-cost. It can observe and study the dynamics and interaction processes of organelles in living cells from a three-dimensional super-resolution perspective.
  • FIG1 is a diagram of a fluorescence emission ratio three-dimensional nanomicroscopic imaging optical path system provided by an embodiment of the present invention
  • FIG2 is a schematic diagram of the principle of fluorescence emission ratio three-dimensional nanomicroscopy imaging implemented in the present invention
  • FIG3 is a method for determining the grayscale value of each pixel in a three-dimensional nano-microscopic image of a fluorescence emission ratio implemented in the present invention
  • FIG. 4 is a theoretical simulation result of the fluorescence emission ratio three-dimensional nano-microscopy imaging implemented in the present invention.
  • a fluorescence emission ratio three-dimensional super-resolution imaging method comprising a pulse laser 1, wherein the laser emitted by the pulse laser 1 passes through a half-wave plate 2 and then passes through a Glan laser prism 3, wherein the pulse laser 1 generates 40MHz pulsed excitation light by a picosecond laser, wherein the half-wave plate 2 is used to adjust the polarization direction of the laser, and the Glan laser prism 3 is used to separate lasers with different polarization directions;
  • One of the laser beams is transmitted to a high-speed photodiode detector 11, which is connected to a time-correlated single-photon counter 12, which is connected to a computer 13.
  • the time-correlated single-photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and processing.
  • Another laser beam is transmitted to the first beam splitter 4, and is split into two laser beams again by the beam splitter 4, which are respectively directed to the first reflector 5 and the 0/ ⁇ phase plate 6, wherein the 0/ ⁇ phase plate 6 is used to generate an excitation light spot of a three-dimensional cage structure.
  • the laser beam passing through the 0/ ⁇ phase plate 6 is transmitted to the second reflector 9.
  • the light is then transmitted to the corner reflector 10, which controls the pulse interval between the Gaussian laser and the ring laser in time by extending or shortening the optical path of the ring-shaped excitation light spot.
  • the laser beam passing through the first reflector 5 is transmitted to the second beam splitter 7, and is split into two beams again by the second beam splitter 7, and is transmitted to the corner reflector 10 and the third reflector 8 respectively;
  • the laser light passing through the third reflector 8 passes through the dichroic mirror 15 , the galvanometer mirror 17 , the scanning lens 18 , the tube lens 19 and the quarter glass slide 20 in sequence, and then is focused by the objective lens 21 and irradiated onto the sample on the stage 22 .
  • the dichroic mirror 15 is used to transmit the excitation light and reflect the fluorescence signal
  • the galvanometer 17 is used to synchronously scan the excitation light to achieve area array imaging of the sample;
  • the scanning lens 18 is used to collect the laser beam for area array scanning.
  • the quarter glass 20 is used to convert the linearly polarized laser light into right-handed circularly polarized light.
  • the objective lens 21 is used to focus the laser onto a focal plane and collect the fluorescent signal reflected by the sample.
  • the tube lens 19 and the objective lens 21 form a microscope system.
  • the stage 22 is used to place and fix the sample and to move the sample in three dimensions.
  • the sample on the stage 22 generates fluorescence after being excited.
  • the fluorescence is collected by the objective lens 21 and then passes through the quarter glass slide 20, the tube lens 19, the scanning lens 18, the galvanometer 17 and the dichroic mirror 15. After being reflected by the dichroic mirror 15, it reaches the photomultiplier tube 14 through the filter 16.
  • the filter 16 is used to remove stray light other than fluorescence and improve the image signal-to-noise ratio.
  • the time-correlated single photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and processing.
  • the first beam splitter 4 and the second beam splitter 7 are used for laser beam splitting or beam combining respectively.
  • the first reflector 5, the second reflector 9 and the third reflector 8 are respectively used to change the transmission direction of the laser.
  • the laser of the pulse laser 1 is emitted and then split by the Glan laser prism 2 and the first beam splitter 4 to form three light paths, one of which is collected by a high-speed photodiode detector and used as a reference signal for fluorescence lifetime imaging;
  • the other two beams of light are used to excite the sample, one is Gaussian light, and the other forms a 3D cage structure after passing through the 0/ ⁇ phase plate 6. Their pulse interval is half of the laser pulse period.
  • the two beams of light are combined after passing through the second beam splitter 7, and pass through the dichroic mirror 15, the galvanometer 17, the scanning lens 18, the tube lens 19 and the quarter glass slide 20 in sequence, and then are focused by the objective lens 21 to irradiate the sample.
  • the gold nanoparticle sample was imaged and the spot was adjusted through real-time imaging so that the focal planes of the Gaussian laser and the ring laser overlapped precisely in space.
  • the time-correlated single-photon counter 12 simultaneously receives the reference signal and fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and deal with.
  • Figure 2 is a schematic diagram of the present invention for realizing three-dimensional nano-microscopic imaging of fluorescence emission ratio. Fluorescence lifetime imaging is performed on the dye-labeled sample, and the fluorescence photons are collected by a time-correlated single photon counter 12 to obtain their spatiotemporal information, and then the fluorescence lifetime data is post-processed.
  • the fluorescence signal is divided into two parts based on the time channel where the cage laser pulse is located.
  • the fluorescence photons in the first half are excited by the Gaussian laser to form an image composed of the Gaussian light spot spread function, and the fluorescence photons in the remaining part are excited by the cage laser to form an image composed of the cage light spot spread function.
  • the time difference between the Gaussian image and the cage image is in the nanosecond range, which is equivalent to real-time recording of the spatial position information of the Gaussian spot and the cage spot.
  • the grayscale values of the corresponding coordinates of the two images are determined, and the grayscale values of each coordinate of the fluorescence emission ratio nanomicroscope image are obtained according to the determination scheme to achieve super-resolution imaging.
  • the Z axis of the sample stage is adjusted to perform fluorescence lifetime imaging at different focal planes or depths, and the obtained data is processed and stacked to synthesize a three-dimensional image.
  • Figure (b) shows the three-dimensional point spread function of a Gaussian laser, whose intensity distribution is ellipsoidal and the energy is maximum in the focal plane.
  • Figure (c) is the three-dimensional point spread function of the cage laser, whose intensity is strong at both ends and weak in the middle, and the intensity in the focal plane is distributed in a ring shape.
  • Figure 3 is a scheme for determining the grayscale value of a pixel in a fluorescence emission ratio nanomicroscope image according to the present invention. After signal processing of the collected fluorescence lifetime data, two images are obtained, and the grayscale value of each pixel in the image is represented by I Gaussian (x, y) and I 3D (x, y), respectively.
  • the grayscale value of the corresponding coordinate pixel in the I 3D-FERN (x, y) image is I Gaussian (x, y)/I 3D (x, y).
  • a fluorescence emission ratio nano-microscope image is obtained.
  • the image data at different depths on the Z axis are processed separately, and the images are stacked to obtain the final fluorescence emission ratio three-dimensional super-resolution result.
  • FIG. 4 is a theoretical simulation result of the present invention for realizing three-dimensional nano-microscopic imaging of fluorescence emission ratio.
  • Figure (a) shows the intensity distribution of Gaussian spot, cage spot and 3D-FERN spot in the XY and XZ planes respectively.
  • Figures (b) and (c) show the intensity distribution of the three light spot spread functions in the XY and XZ planes, respectively. Comparing the intensity curves in Figure (b) and Figure (c), it can be seen that the maximum half-height full width of the beam point spread function in the 3D-FERN image is smaller, so the resolution can be improved in both the lateral and axial directions.
  • the fluorescence emission ratio three-dimensional nanomicroscopic imaging method proposed by the present invention can achieve three-dimensional super-resolution imaging under a single-wavelength laser. Not only is the laser power low, but the imaging system is simple and low-cost, and the dynamics and interaction processes of organelles in living cells can be observed and studied from a three-dimensional super-resolution perspective.

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Abstract

一种荧光发射比率三维超分辨成像方法，包括脉冲激光器（1）,脉冲激光器（1）发出的激光经过半波片（2）,再经过格兰激光棱镜（3）,经过格兰激光棱镜（3）其中一束激光传向高速光电二极管探测器（11）,另一束激光传向第一分束器（4）,分别照向第一反射镜（5）和0/π相位板（6）,经过0/π相位板（6）的激光传向第二反射镜（9）,经过第二反射镜（9）后传向角反射器（10）,经过第一反射镜（5）的激光传向第二分束器（7）,分别传向角反射器（10）和第三反射镜（8）,经过第三反射器（8）的激光依次通过双色镜（15）、振镜（17）、扫描透镜（18）、管镜（19）和四分之一玻片（20）后，通过物镜（21）后照射至样品上。成像方法可以实现三维超分辨成像，激光功率低，成像系统简易、成本低，能以三维超分辨的角度观测和研究活细胞中细胞器的动态和相互作用过程。

Description

一种荧光发射比率三维超分辨成像方法 技术领域
本发明属于光学显微镜成像方法技术领域，具体涉及一种荧光发射比率三维超分辨成像方法。
背景技术
细胞的生命活动离不开内部各种亚细胞结构的精确分工和协同合作，因此洞悉细胞器/蛋白分子的网络运转是一项重要的研究需求，为揭示生命体发育、疾病产生和消亡等过程的调控机制提供重要的参考。借助荧光显微镜技术，人们实现了对亚细胞结构的特异性观测，但是由于光的衍射，传统光学显微镜的横向分辨率极限约为250nm，轴向上约为600nm，因此无法清晰分辨尺寸小于上述数值的生物体结构，阻碍了对更精细结构的探究。近几十年，多种基于荧光“开/关”效应的超分辨成像方法被陆续提出，使清晰观测亚细胞结构成为可能，因此推动光学显微镜逐渐进入纳米成像时代。
然而，几乎所有超分辨成像模式在追求分辨率极限时都会产生新的问题，比如高激光功率导致样品损伤和染料漂白、复杂的成像系统导致实验成本增加和稳定性下降，以及需要特殊的荧光探针和样品制备流程极其繁琐等。总之，目前的超分辨成像技术在实现三维、多色和深层超分辨成像时仍然面临成像系统复杂、成本高、图像质量差和样品制备过程复杂，以及需要特殊荧光探针等一系列问题。
此外，对细胞器和相关生物分子进行三维超分辨成像可以观测活 体样本的动态过程，研究细胞间或细胞内物质的相互作用，对于揭示活体生物样本的结构和功能至关重要。现有的三维超分辨成像平台被不能揭示细胞器互作在物质转运和利用、细胞器稳态调控等方面的作用，不能在疾病发生中的作用等方面的研究提供技术支撑。
基于此，提出了一种荧光发射比率三维超分辨成像方法。
技术问题
本发明所要解决的技术问题在于针对上述现有技术的不足，提供一种荧光发射比率三维超分辨成像方法，降低超分辨技术对荧光染料和样品制备的要求，拓宽染料的选择范围，为低成本的活体三维超分辨成像研究提供技术支撑，解决上述背景技术中提出的问题。
技术解决方案
为解决上述技术问题，本发明采用的技术方案是：一种荧光发射比率三维超分辨成像方法，包括脉冲激光器，所述脉冲激光器发出的激光经过半波片，再经过格兰激光棱镜，经过格兰激光棱镜分成两束激光，其中一束激光传向高速光电二极管探测器，另一束激光传向第一分束器，经过分束器再次分成两束激光，分别照向第一反射镜和0/π相位板；
经过0/π相位板的激光传向第二反射镜，经过第二反射镜后传向角反射器；
经过第一反射镜的激光传向第二分束器，经过第二分束器再次分成两束，分别传向角反射器和第三反射镜；
经过第三反射器的激光依次通过双色镜、振镜、扫描透镜、管镜和四分之一玻片后，通过物镜聚焦后照射至载物台的样品上。
进一步的，所述高速光电二极管探测器连接有时间相关单光子计数器，所述时间相关单光子计数器连接有计算机。
进一步的，所述载物台的样品被激发后产生荧光，荧光被物镜收集后经四分之一玻片、管镜、扫描透镜、振镜和双色镜，被双色镜反射后经过滤光片到达光电倍增管，所述滤光片用于去除荧光以外的杂散光，提高图像信噪比，时间相关单光子计数器同时接收高速光电二极管探测器和光电倍增管采集的参考信号和荧光信号，并将数据传输至计算机进行存储和处理。
进一步的，所述脉冲激光器由皮秒激光器产生40MHz脉冲型激发光，所述半波片用于调节激光的偏振方向，所述格兰激光棱镜用于分离不同偏振方向的激光。
进一步的，所述0/π相位板用于产生三维笼式结构的激发光斑，所述角反射器通过延长或缩短环型激发光斑的光程在时间上控制高斯型激光和环形激光之间的脉冲间隔。
进一步的，所述双色镜用于透射激发光，并反射荧光信号；
所述振镜用于对激发光进行同步扫描，实现对样品的面阵成像；
所述扫描透镜用于收集面阵扫描的激光光束。
进一步的，所述物镜用于将激光聚焦到焦平面，同时收集样品反射回来的荧光信号，所述管镜与所述物镜搭配构成显微镜系统。
进一步的，所述四分之一玻片用于将线偏振激光转换成右旋圆偏振光。
进一步的，所述载物台用于放置和固定样品，并对样品进行三维 移动。
进一步的，所述第一分束器和第二分束器分别用于激光分束或合束，所述第一反射镜、第二反射镜和第三反射镜分别用于改变激光的传输方向。
有益效果
本发明与现有技术相比具有以下优点：
本发明中的荧光发射比率三维纳米显微成像方法可以在一个单波长激光下实现三维超分辨成像，不仅激光功率低，而且成像系统简易、成本低，能以三维超分辨的角度观测和研究活细胞中细胞器的动态和相互作用过程。
附图说明
图1是本发明实施例提供的荧光发射比率三维纳米显微成像光路系统图；
图2是本发明实施的荧光发射比率三维纳米显微成像的原理图；
图3是本发明实施的荧光发射比率三维纳米显微图像各像素灰度值的判定方式；
图4是本发明实施的荧光发射比率三维纳米显微成像的理论模拟结果。
附图标记说明：
脉冲激光器1、半波片2、格兰激光棱镜3、第一分束器4、第一
反射镜5、0/π相位板6、第二分束器7、第三反射镜8、第二反射镜9、角反射器10、高速光电二极管探测器11、时间相关单光子计数器12、计算机13、光电倍增管14、双色镜15、滤光片16、振镜17、 扫描透镜18、管镜19、四分之一玻片20、物镜21和载物台22。
本发明的实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
如图1-4所示，本发明提供一种技术方案：一种荧光发射比率三维超分辨成像方法，包括脉冲激光器1，所述脉冲激光器1发出的激光经过半波片2，再经过格兰激光棱镜3，所述脉冲激光器1由皮秒激光器产生40MHz脉冲型激发光，所述半波片2用于调节激光的偏振方向，所述格兰激光棱镜3用于分离不同偏振方向的激光；
其中一束激光传向高速光电二极管探测器11，所述高速光电二极管探测器11连接有时间相关单光子计数器12，所述时间相关单光子计数器12连接有计算机13，时间相关单光子计数器12同时接收高速光电二极管探测器11和光电倍增管14采集的参考信号和荧光信号，并将数据传输至计算机13进行存储和处理。
另一束激光传向第一分束器4，经过分束器4再次分成两束激光，分别照向第一反射镜5和0/π相位板6，所述0/π相位板6用于产生三维笼式结构的激发光斑。
经过0/π相位板6的激光传向第二反射镜9，经过第二反射镜9 后传向角反射器10，，所述角反射器10通过延长或缩短环型激发光斑的光程在时间上控制高斯型激光和环形激光之间的脉冲间隔。
经过第一反射镜5的激光传向第二分束器7，经过第二分束器7再次分成两束，分别传向角反射器10和第三反射镜8；
经过第三反射器8的激光依次通过双色镜15、振镜17、扫描透镜18、管镜19和四分之一玻片20后，通过物镜21聚焦后照射至载物台22的样品上。
所述双色镜15用于透射激发光，并反射荧光信号；
所述振镜17用于对激发光进行同步扫描，实现对样品的面阵成像；
所述扫描透镜18用于收集面阵扫描的激光光束。
所述四分之一玻片20用于将线偏振激光转换成右旋圆偏振光。
所述物镜21用于将激光聚焦到焦平面，同时收集样品反射回来的荧光信号，所述管镜19与所述物镜21搭配构成显微镜系统。
所述载物台22用于放置和固定样品，并对样品进行三维移动。
所述载物台22的样品被激发后产生荧光，荧光被物镜21收集后经四分之一玻片20、管镜19、扫描透镜18、振镜17和双色镜15，被双色镜15反射后经过滤光片16到达光电倍增管14，所述滤光片16用于去除荧光以外的杂散光，提高图像信噪比，时间相关单光子计数器12同时接收高速光电二极管探测器11和光电倍增管14采集的参考信号和荧光信号，并将数据传输至计算机13进行存储和处理。
所述第一分束器4和第二分束器7分别用于激光分束或合束，所 述第一反射镜5、第二反射镜9和第三反射镜8分别用于改变激光的传输方向。
脉冲激光器1、半波片2、格兰激光棱镜3、第一分束器4、第一反射镜5、0/π相位板6、第二分束器7、第三反射镜8、第二反射镜9、角反射器10、高速光电二极管探测器11、时间相关单光子计数器12、计算机13、光电倍增管14、双色镜15、滤光片16、振镜17、扫描透镜18、管镜19、四分之一玻片20、物镜21和载物台22
具体的，脉冲激光器1的激光出射后经格兰激光棱镜2和第一分束器4分束后形成三条光路，其中一路光被高速光电二极管探测器收集后作为荧光寿命成像时的参考信号；
另外两路光用于激发样品，一束为高斯光，另一束经过0/π相位板6后形成3D笼式结构，它们的脉冲间隔为激光脉冲周期的一半，这两路光经第二分束器7后合束，依次经过双色镜15、振镜17、扫描透镜18、管镜19和四分之一玻片20后，通过物镜21聚焦后照射样品。
对金纳米颗粒样品进行成像，通过实时成像进行光斑调节，使高斯激光和环形激光的焦平面在空间上精准重合。
样品被激发后产生荧光，荧光被同一物镜21收集后原路返回，经四分之一玻片20、管镜19、扫描透镜18、振镜17和双色镜15，被双色镜15反射后经过滤光片16到达光电倍增管14。时间相关单光子计数器12同时接收高速光电二极管探测器11和光电倍增管14采集的参考信号和荧光信号，并将数据传输至计算机13进行存储和 处理。
图2为本发明实现荧光发射比率三维纳米显微成像的原理图。对染料标记后的样品进行荧光寿命成像，通过时间相关单光子计数器12采集荧光光子并获取其时空信息，然后对荧光寿命数据进行后期处理。
首先，以笼式激光脉冲所在的时间通道为界限将荧光信号分为两部分，前半部分的荧光光子由高斯型激光激发，形成由高斯光斑点扩展函数组成的图像，剩余部分的荧光光子由笼式激光激发，形成由笼式光斑点扩展函数组成图像；
如图(a)所示。当激光脉冲频率为40MHz时，高斯图像和笼式图像之间时间差为纳秒量级，相当于实时记录了高斯光斑和笼式光斑的空间位置信息。然后，对这两幅图像对应坐标的灰度值进行判定，根据判定方案得到荧光发射比率纳米显微图像各坐标的灰度值，实现超分辨成像。调节样品台的Z轴，在不同焦平面或深度分别进行荧光寿命成像，对得到的数据处理后堆叠合成三维图像。
图(b)为高斯激光的三维点扩展函数，其强度分布呈椭球状，且能量在焦平面最大。
图(c)为笼式激光的三维点扩展函数，其强度呈两端强中间弱，且焦平面强度呈环形分布。
图3是本发明实现荧光发射比率纳米显微图像像素灰度值的判定方案。对采集到的荧光寿命数据进行信号处理后得到两幅图，图像中各像素的灰度值分别由IGaussian(x,y)和I3D(x,y)表示。
首先，判断IGaussian(x,y)各个坐标位置像素的灰度值是否为零，
若IGaussian(x,y)＝0，则I3D-FERN(x,y)图像中对应坐标像素的灰度值为0；
若IGaussian(x,y)≠0，则判断I3D(x,y)对应坐标位置像素的灰度值是否为零。此时，若I3D(x,y)＝0，则I3D-FERN(x,y)图像中对应坐标像素的灰度值与IGaussian(x,y)相同；
若I3D(x,y)≠0，则I3D-FERN(x,y)图像中对应坐标像素的灰度值为IGaussian(x,y)/I3D(x,y)。
根据上述判定，得到荧光发射比率纳米显微图像。对Z轴上不同深度的图像数据分别进行处理，将图像堆叠后得到最终的荧光发射比率三维超分辨结果
图4是本发明实现荧光发射比率三维纳米显微成像的理论模拟结果。
图(a)所示分别为高斯光斑、笼式光斑和3D-FERN光斑在XY和XZ平面的强度分布。经过理论分析和模拟，当0/π相位板6的中心π相位直径为整个相位板直径约0.7倍的时候，激光的大部分能量分布在非焦平面的位置，但焦平面仍然形成一个中心强度为零的强度分布。因此，仅用一束激光光斑通过0/π相位板6即可产生3D笼式光斑，且其在焦平面仍然形成一个中心强度为零的环形光斑。由于两束光斑波长相同，它们在轴向上聚焦于同一平面，因此三维立体光斑可以精准重合。
图(b)和图(c)为三个光斑点扩展函数分别在XY和XZ平面上的强 度分布曲线。对比图(b)和图(c)中的强度曲线可以看出，3D-FERN图像中光束点扩展函数的最大半高全宽更小，因此在横向和轴向上均可以实现分辨率的提升。
由于本发明的方法只涉及受激吸收过程，因此只需要微瓦级的激光功率。所以，本发明提出的荧光发射比率三维纳米显微成像方法可以在一个单波长激光下实现三维超分辨成像，不仅激光功率低，而且成像系统简易、成本低，能以三维超分辨的角度观测和研究活细胞中细胞器的动态和相互作用过程。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (10)

  1. 一种荧光发射比率三维超分辨成像方法，其特征在于：包括脉冲激光器(1)，所述脉冲激光器(1)发出的激光经过半波片(2)，再经过格兰激光棱镜(3)，经过格兰激光棱镜(3)分成两束激光，其中一束激光传向高速光电二极管探测器(11)，另一束激光传向第一分束器(4)，经过分束器(4)再次分成两束激光，分别照向第一反射镜(5)和0/π相位板(6)；
    经过0/π相位板(6)的激光传向第二反射镜(9)，经过第二反射镜(9)后传向角反射器(10)；
    经过第一反射镜(5)的激光传向第二分束器(7)，经过第二分束器(7)再次分成两束，分别传向角反射器(10)和第三反射镜(8)；
    经过第三反射器(8)的激光依次通过双色镜(15)、振镜(17)、扫描透镜(18)、管镜(19)和四分之一玻片(20)后，通过物镜(21)聚焦后照射至载物台(22)的样品上。
  2. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述高速光电二极管探测器(11)连接有时间相关单光子计数器(12)，所述时间相关单光子计数器(12)连接有计算机(13)。
  3. 根据权利要求2所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述载物台(22)的样品被激发后产生荧光，荧光被物镜(21)收集后经四分之一玻片(20)、管镜(19)、扫描透镜(18)、振镜(17)和双色镜(15)，被双色镜(15)反射后经过滤光片(16)到达光电倍增管(14)，所述滤光片(16)用于去除荧光以外的杂散光，提高图像信噪比，时间相关单光子计数器(12)同时接收高速光电二极管探测器(11)和光电倍增管(14)采集的参考信号和荧光信号，并将数据传输至计算机(13)进行存储和处理。
  4. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述脉冲激光器(1)由皮秒激光器产生40MHz脉冲型激发光，所述半波片(2)用于调节激光的偏振方向，所述格兰激光棱镜(3)用于分离不同偏振方向 的激光。
  5. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述0/π相位板(6)用于产生三维笼式结构的激发光斑，所述角反射器(10)通过延长或缩短环型激发光斑的光程在时间上控制高斯型激光和环形激光之间的脉冲间隔。
  6. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述双色镜(15)用于透射激发光，并反射荧光信号；
    所述振镜(17)用于对激发光进行同步扫描，实现对样品的面阵成像；
    所述扫描透镜(18)用于收集面阵扫描的激光光束。
  7. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述物镜(21)用于将激光聚焦到焦平面，同时收集样品反射回来的荧光信号，所述管镜(19)与所述物镜(21)搭配构成显微镜系统。
  8. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述四分之一玻片(20)用于将线偏振激光转换成右旋圆偏振光。
  9. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述载物台(22)用于放置和固定样品，并对样品进行三维移动。
  10. 根据权利要求1所述的一种荧光发射比率三维超分辨成像方法，其特征在于，所述第一分束器(4)和第二分束器(7)分别用于激光分束或合束，所述第一反射镜(5)、第二反射镜(9)和第三反射镜(8)分别用于改变激光的传输方向。
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