WO2024087614A1 - 一种荧光发射比率超分辨成像方法 - Google Patents

一种荧光发射比率超分辨成像方法 Download PDF

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WO2024087614A1
WO2024087614A1 PCT/CN2023/096595 CN2023096595W WO2024087614A1 WO 2024087614 A1 WO2024087614 A1 WO 2024087614A1 CN 2023096595 W CN2023096595 W CN 2023096595W WO 2024087614 A1 WO2024087614 A1 WO 2024087614A1
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laser
reflector
resolution imaging
fluorescence emission
imaging method
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PCT/CN2023/096595
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French (fr)
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陈越
郭嘉庆
李灏
王璐玮
俞宪同
陈钰
宋军
屈军乐
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深圳大学
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Publication of WO2024087614A1 publication Critical patent/WO2024087614A1/zh

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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 super-resolution imaging method.
  • the technical problem to be solved by the present invention is to provide a fluorescence emission ratio 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 super-resolution imaging research, and solve the problems raised in the above-mentioned background technology.
  • a fluorescent emission A ratio 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 split 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 split into two laser beams again by the beam splitter, and is respectively irradiated to a first reflector and a spiral phase plate;
  • the laser light passing through the spiral phase plate is transmitted to the second reflector, and then transmitted 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 spiral phase plate is used to convert the wavefront of the laser from Gaussian to annular, and the corner reflector controls the pulse interval between the Gaussian laser and the annular laser in time by extending or shortening the optical path of the annular excitation 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 to move the sample in three dimensions.
  • 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 nano-microscopy imaging technology of the present invention breaks through the optical diffraction limit at low laser power, reduces the requirements of super-resolution technology on fluorescent dyes and sample preparation, broadens the selection range of dyes, and provides technical support for low-cost in vivo super-resolution imaging research.
  • FIG1 is a system optical path diagram of fluorescence emission ratio nanomicroscopy imaging provided by an embodiment of the present invention.
  • FIG2 is a schematic diagram of the fluorescence emission ratio nanomicroscopy imaging principle implemented in the present invention.
  • FIG3 is a method for determining the grayscale value of each pixel in a fluorescence emission ratio nanomicroscope image implemented in the present invention
  • FIG. 4 is a theoretical simulation result of fluorescence emission ratio nanomicroscopy imaging implemented in the present invention.
  • a fluorescence emission ratio super-resolution imaging method comprising a pulse laser 1, the laser emitted by the pulse laser 1 passes through a half-wave plate 2, and then passes through a Glan laser prism 3, the pulse laser 1 generates 40MHz pulsed excitation light by a picosecond laser, 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, and respectively illuminates the first reflector 5 and the spiral phase plate 6, wherein the spiral phase plate 6 is used to convert the wavefront of the laser from a Gaussian type to a ring type.
  • the laser light passing through the spiral phase plate 6 is transmitted to the second reflector 9, and then to the corner reflector 10.
  • the corner reflector 10 controls the pulse interval between the Gaussian laser light and the ring laser light 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 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 to the focal plane and collect the laser reflected from the sample.
  • the tube lens 19 and the objective lens 21 are matched to 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 used for changing the transmission direction of the laser, respectively.
  • the laser of the pulse laser 1 is emitted and split by the Glan laser prism 2 and the first beam splitter 4 to form three light paths, one of which is collected by the high-speed photodiode detector 11 and used as a reference signal for fluorescence lifetime imaging;
  • the other two beams are used to excite the sample. Their wavefronts are Gaussian and annular respectively, and the pulse interval is half of the laser pulse period.
  • the two beams are combined after passing through the second beam splitter 7, and then pass through the dichroic mirror 15, the galvanometer 17, the scanning lens 18, the tube lens 19 and the quarter-glass. After the film 20 is formed, it is focused by the objective lens 21 and illuminates 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 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.
  • FIG2 is a schematic diagram of the present invention for realizing fluorescence emission ratio nano-microscopic imaging.
  • Fluorescence lifetime imaging is performed on the dye-labeled sample, and the fluorescence photons are collected and their spatiotemporal information is obtained by the time-correlated single photon counter 12, and then the fluorescence lifetime data is post-processed.
  • the fluorescence signal is divided into two parts based on the time channel where the ring laser pulse is located.
  • the fluorescence photons in the first half are excited by Gaussian laser to form an image composed of Gaussian light spot expansion function, and the fluorescence photons in the remaining part are excited by ring laser to form an image composed of ring light spot expansion function.
  • the time difference between the Gaussian image and the annular image is in the nanosecond range, which is equivalent to recording the spatial position information of the Gaussian spot and the annular spot in real time. Then, 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.
  • FIG3 is a method for determining the pixel grayscale value of the fluorescence emission ratio nano-microscope image according to the present invention.
  • the grayscale value of each pixel in is represented by IGaussian(x,y) and IDount(x,y) respectively.
  • the grayscale value of the corresponding coordinate pixel in the IFERN(x,y) image is 0; if IGaussian(x,y) ⁇ 0, determine whether the grayscale value of the corresponding coordinate pixel in the IFERN(x,y) image is zero.
  • FIG4 is a theoretical simulation result of the fluorescence emission ratio nano-microscopy imaging achieved by the present invention.
  • FIG (a) is a fluorescence decay curve collected after two laser beams are irradiated.
  • the pulse interval between the Gaussian light and the annular light is half of the laser pulse period (T/2).
  • the power of the two laser beams is in the microwatt level and can be adjusted by rotating the angle of the half-wave plate in the system.
  • Figures (b) and (c) are the Gaussian image (IGaussian) and annular image (IDount) obtained after data processing, respectively.
  • the scheme shown in FIG3 is used to perform image processing, and the fluorescence emission ratio image (IFERN) shown in FIG3 is obtained. Comparing FIG3 and FIG3, it can be seen that the maximum half-height full width of the spot in FIG3 is smaller, so the resolution is higher. Therefore, the fluorescence emission ratio nanomicroscopy imaging method proposed in the present invention can achieve super-resolution imaging at microwatt laser power, broaden the selection range of fluorescent dyes, and provide technical support for the study of dynamic processes and interactions of subcellular structures in living cells.

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Abstract

一种荧光发射比率超分辨成像方法，包括：脉冲激光器(1)发出的激光经过半波片(2)、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)，经过第一分束器(4)再次分成两束激光，分别照向第一反射镜(5)和螺旋相位板(6)；经过螺旋相位板(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)、物镜(21)后照射至载物台(22)的样品上。降低了对荧光染料和样品制备的要求，为低成本的活体超分辨成像研究提供技术支撑。

Description

一种荧光发射比率超分辨成像方法 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超分辨成像方法。
背景技术
人类对信息的摄取主要是通过眼睛，但是人类眼睛的敏感性与光的波长息息相关。由于只能识别波长为400～760nm范围内的光波，正常视力的人眼只能在明视距离处25cm分辨出亚毫米级的物体。如果要观察更小的物体，则需要借助于显微镜。光学显微镜的发明让人类第一次观察到了由细胞作为基本结构和功能单元的活体生物，如今已广泛的应用于生活和科学研究的各个领域。面向活细胞研究的光学显微成像技术具有非接触、无损伤和特异性的优点，可以对细胞进行原位、实时和动态研究，深入理解细胞器、蛋白质以及分子之间的相互作用和生理过程，极大地推动了生命科学的进步和发展。但是，光学显微镜的分辨率受光学衍射的限制，无法清晰辨别尺寸在200nm以下的生物结构。为了研究和揭示亚细胞结构及相关分子的相互作用和作用规律，迫切需要突破光学衍射极限的成像技术。
为了解决纳米级分辨率的成像需求，超分辨显微成像技术应运而生。在近三十年的时间里，基于不同原理的超分辨成像方法被陆续提出，使光学显微镜逐渐进入纳米成像时代。近年来，超分辨光学成像技术的不断发展让光学显微镜与生物医学等领域的联系更加紧密。从 光学工程的角度来讲，显微镜技术的性能由一些硬性指标决定，比如成像分辨率、成像深度、定位精度和成像速率等。此外，有效光子数、标记特异性和样品状态等都是在实际的生物应用中获得最佳成像效果的限制因素。因此，一套先进的光学成像系统不仅需要保持被观测样品自身的生物特性，还应该最大程度地保障获取信息的真实性和有效性。
然而，几乎所有超分辨成像模式在追求超分辨率极限时都会产生新的问题，比如高的激光功率、低的成像速度、复杂的成像系统和昂贵的实验成本等。受激辐射损耗技术损耗激光功率极高，导致严重的样品损伤和染料光漂白，因此不适合细胞等活体样品的长时间超分辨成像；单分子定位显微镜技术需要图像重构，因此时间分辨率低，且成像深度有限，限制了在生物样品动态成像中的应用。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、图像质量低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。
基于此，提出了一种荧光发射比率超分辨成像方法。
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螺旋相位板；
经过螺旋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，所述螺旋相位板用于将激光的波前由高斯型转换成环形，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：
本发明中的在低激光功率下突破光学衍射极限的荧光发射比率纳米显微成像技术，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，为低成本的活体超分辨成像研究提供技术支撑。
附图说明
图1是本发明实施例提供的荧光发射比率纳米显微成像的系统光路图；
图2是本发明实施的荧光发射比率纳米显微成像原理图；
图3是本发明实施的荧光发射比率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、螺旋相位板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和螺旋相位板6，所述螺旋相位板6用于将激光的波前由高斯型转换成环形。
经过螺旋相位板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、螺旋相位板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11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，它们的波前分别为高斯型和环形，且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，以环形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环形激光激发，形成由环形光斑点扩展函数组成图像。
当激光脉冲频率为40MHz时，高斯图像和环形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环形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。
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IDount(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IFERN(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IDount(x,y)对应坐标位置像素的灰度值是否为零。此时，若IDount(x,y)＝0，则IFERN(x,y)图像中对应坐标像素的灰度值与IGaussian(x,y)相同；若IDount(x,y)≠0，则IFERN(x,y)图像中对应坐标像素的灰度值为IGaussian(x,y)/IDount(x,y)。根据上述判定，最终得到荧光发射比率纳米显微图像。
图4是本发明实现荧光发射比率纳米显微成像的理论模拟结果。图(a)为两束激光照射后采集到的荧光衰减曲线。高斯光和环形光之间的脉冲间隔为激光脉冲周期的一半(T/2)，两束激光的功率均为微瓦量级，且可以通过旋转系统中半波片的角度进行调节。
以3×3均匀分布的9个点物体作为成像对象，图(b)和(c)分别为经过数据处理后得到的高斯图像(IGaussian)和环形图像(IDount)。
利用图3所示的方案进行图像处理，得到图(d)所示的荧光发射比率图像(IFERN)。对比图(b)和图(d)可以看出，图(d)中光斑的最大半高全宽更小，因此分辨率更高。所以，本发明提出的荧光发射比率纳米显微成像方法可以在微瓦级激光功率下实现超分辨成像，拓宽了荧光染料的选择范围，为活细胞中亚细胞结构的动态过程和相互作用研究提供技术支撑。
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)和螺旋相位板(6)；
    经过螺旋相位板(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超分辨成像方法，其特征在于，所述螺旋相位板(6)用于将激光的波前由高斯型转换成环形，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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