WO2024108954A1 - 一种单波长激发的荧光调制多色超分辨显微成像方法 - Google Patents

一种单波长激发的荧光调制多色超分辨显微成像方法 Download PDF

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WO2024108954A1
WO2024108954A1 PCT/CN2023/097044 CN2023097044W WO2024108954A1 WO 2024108954 A1 WO2024108954 A1 WO 2024108954A1 CN 2023097044 W CN2023097044 W CN 2023097044W WO 2024108954 A1 WO2024108954 A1 WO 2024108954A1
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fluorescence
laser
imaging
resolution
sample
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French (fr)
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陈越
王璐玮
郭嘉庆
宋军
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
    • G01N21/62—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 present invention belongs to the field of optical microscope imaging, and more specifically, relates to a nanometer microscope technology for realizing multi-color super-resolution imaging under a single-wavelength pulse laser and a fluorescence detector.
  • Optical microscopy for living cell research has the advantages of non-contact, non-destructive and specific. It can conduct in-situ, real-time and dynamic research on cells, and deeply understand the interactions and physiological processes between organelles, proteins and molecules, which has greatly promoted the progress and development of life sciences.
  • the resolution of optical microscopes is limited by optical diffraction, and it is impossible to clearly distinguish biological structures below 200nm in size.
  • multi-color super-resolution imaging basically uses spectral separation technology, which makes the microscope system
  • the system requires multiple lasers with different wavelengths as light sources and multiple detectors to collect fluorescence with different emission spectra.
  • the multicolor imaging system based on spectral separation technology not only has a complex optical path and poor imaging stability, but also has a high cost, which hinders the promotion and application of super-resolution technology. Therefore, a single-wavelength excitation fluorescence modulation multicolor super-resolution microscopy imaging method is proposed.
  • the main purpose of the present invention is to propose a nano-microscopy imaging technology that breaks through the optical diffraction limit and realizes multi-color super-resolution imaging in a simple optical imaging system based on a single-wavelength pulsed laser and a detector, reduces the complexity and cost of the multi-color super-resolution microscopy imaging system, improves the stability of multi-color super-resolution imaging, and provides technical support for low-cost super-resolution imaging of living cells and other living organisms and the study of the dynamic processes of subcellular structure interactions.
  • a single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging method comprises the following steps:
  • the intensity value of each pixel of the image is spatially modulated, from the Gaussian signal
  • the fluorescence signal photons of the ring image are subtracted from the photons, and the Gaussian excitation point spread function is compressed to achieve super-resolution imaging;
  • the light source of the optical imaging system is a single-wavelength picosecond pulse laser.
  • the two time series image stacks are fluorescence signals excited by Gaussian light and annular light respectively.
  • the fluorescence lifetime data includes three dimensions: x, y and t.
  • the phasor diagram analysis method obtains fluorescence lifetime information by converting the fitting in the time domain into mathematical calculation in the frequency domain.
  • the two-dimensional view of the distribution of fluorescence lifetime information enables an observer to quickly distinguish different lifetime populations in a fluorescence lifetime image.
  • the time difference between the Gaussian image and the annular image is in the order of nanoseconds, and the spatial position information of the Gaussian spot and the annular spot is recorded in real time.
  • a weight factor is introduced to enhance the intensity of the annular image, and the suppression effect on the luminous area around the Gaussian spot is adjusted through a spatial modulation process to remove the diffraction-limited low-frequency signal and background noise.
  • a single-wavelength excited fluorescence modulation multi-color super-resolution microscopic imaging system comprising
  • Pulsed laser using picosecond laser to generate 40MHz pulsed excitation light
  • Half-wave plate used to adjust the polarization direction of the laser
  • Glan laser prism used to separate lasers with different polarization directions
  • Beam splitters used for laser beam splitting and combining
  • a spiral phase plate is used to transform the laser wavefront from a Gaussian shape to a ring shape
  • a reflector used to change the transmission direction of the laser
  • a corner reflector the position of which is adjusted to change the optical path of the annular excitation light spot, and is used to control the pulse interval between the Gaussian laser and the annular laser in time;
  • a quarter glass slide converts linearly polarized laser light into right-handed circularly polarized light, improving the quality of the annular spot.
  • a dichroic mirror 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 placed behind the galvanometer and is used to collect the laser beam for area array scanning;
  • the tube lens together with the objective lens, forms a microscope system
  • the objective lens is used to focus the laser light to the focal plane and collect the fluorescence signal reflected from the sample;
  • a stage used to place and fix the sample and to move the sample in three dimensions
  • Filters are used to remove stray light other than fluorescence and improve the image signal-to-noise ratio
  • Multimode optical fiber used to transmit the fluorescence signal to the photomultiplier tube detector, and its core is used as the detection aperture of the confocal system;
  • Photomultiplier tube used to collect fluorescent photons, amplify fluorescent signals, and output electrical signals to a computer
  • High-speed photodiode detector used to detect the laser beam after the Glan laser prism splits it, as a reference signal for measuring the fluorescence lifetime
  • Time-correlated single-photon counter used to measure and record the spatiotemporal information of fluorescence photons
  • Computer used to control the software to acquire images, store data and process image data, etc.
  • the position of the corner reflector is adjusted to lengthen or shorten the optical path of the annular excitation light spot.
  • the present invention realizes super-resolution imaging in the spatial dimension by using a method that requires the Gaussian laser spot and the annular laser spot to be precisely overlapped in the focal plane.
  • Gold nanoparticle samples can be used to adjust the positions of the two laser spots in real time through scattering imaging so that they can be precisely overlapped in space.
  • the present invention selects multiple dyes with similar spectra but different fluorescence lifetimes and performs multi-target structural labeling on the sample.
  • the above principles and processes can be used to achieve three-color or more multi-color super-resolution imaging, thereby revealing the dynamic process of the interaction between organelles such as mitochondria, lysosomes and microtubules in living cells;
  • the method proposed in the present invention can achieve multi-color super-resolution imaging under the excitation of a single-wavelength pulsed laser and a fluorescence detector, as well as microwatt-level laser power, providing technical support for the study of the dynamic processes and interactions of subcellular structures in living cells.
  • FIG1 is an optical path diagram of a fluorescence modulation multi-color super-resolution microscopy imaging system based on single-wavelength excitation provided by an embodiment of the present invention
  • 2a-2b are schematic diagrams showing the effects of the excitation light in the time and space dimensions in the fluorescence modulation multi-color super-resolution microscopy imaging method based on single-wavelength excitation provided by an embodiment of the present invention
  • 3a to 3e are schematic diagrams of the process of a fluorescence modulation multi-color super-resolution microscopy imaging method based on single-wavelength excitation provided by an embodiment of the present invention
  • 4a to 4g are the imaging results of fluorescent bead samples based on single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy imaging provided by an embodiment of the present invention.
  • the present invention provides a fluorescence modulation multi-color super-resolution microscopic imaging method based on single-wavelength excitation, which is a new method that breaks through the optical diffraction limit on the basis of a laser scanning confocal imaging system, reduces the complexity and cost of the multi-color super-resolution imaging system, and improves the stability of the imaging system.
  • a single-wavelength excited fluorescence modulation multi-color super-resolution microscopic imaging method comprises the following steps:
  • a laser scanning confocal fluorescence lifetime imaging system comprising:
  • Pulsed laser a picosecond laser generates 40MHz pulsed excitation light.
  • Half-wave plate used to adjust the polarization direction of the laser
  • Glan-laser polarizer used to separate lasers with different polarization directions
  • Lens (Lens, L), converges the parallel transmitted laser beam
  • Beam splitter used for laser beam splitting and combining
  • VPP Vortex phase plate
  • Corner reflector (RR), whose position is adjusted to change (lengthen or shorten) the optical path of the annular excitation spot, is used to control the pulse interval between the Gaussian laser and the annular laser in time;
  • Quarter-wave plate converts linearly polarized laser light into right-handed circularly polarized light, improving the quality of the annular spot.
  • Dichroic mirror used to transmit excitation light and reflect fluorescence signals
  • Galvanometer mirror used to synchronously scan the excitation light to achieve area array imaging of the sample
  • Scan lens placed behind the galvanometer, is used to collect the laser beam for area scanning
  • Objective used to focus the laser to the focal plane and collect the fluorescence signal reflected by the sample
  • the stage (3D Stage) is used to place and fix the sample and move the sample in three dimensions;
  • Filter used to remove stray light other than fluorescence and improve the image signal-to-noise ratio
  • Multimode fiber is used to transmit the fluorescence signal to the photomultiplier tube detector, and its core is used as the detection aperture of the confocal system;
  • Photomultiplier tube used to collect fluorescent photons, amplify fluorescent signals, and output electrical signals to a computer;
  • High speed photodiode detector used to detect the laser beam after Glan laser prism splitting, as a reference signal for measuring fluorescence lifetime
  • Time-correlated single photon counting used to measure and record the spatiotemporal information of fluorescence photons
  • Computer Used to control the software to collect images, store data and process image data
  • the red solid line in Figure 1 represents a Gaussian laser. After the laser is emitted, it passes through a half-wave plate and is split into two by a Glan laser prism. One beam is reflected and focused by a lens on a high-speed photodiode detector as a reference signal for fluorescence lifetime imaging. The other beam is transmitted and split again by a beam splitter (BS). The light transmitted by the beam splitter is still a Gaussian laser (red solid line). The reflected light beam is modulated by a 0-2 ⁇ spiral phase plate (VPP) to form a ring laser (red hollow line). The above two laser beams overlap at the second beam splitter. The green solid line represents the fluorescence signal reflected from the sample.
  • VPP 0-2 ⁇ spiral phase plate
  • the signal collected by the photomultiplier tube is transmitted to the TCSPC and the data is saved to the computer.
  • the pulse frequency is adjusted to 40MHz, and move the angle A reflector (RR) is used to adjust the pulse interval between the Gaussian laser and the ring laser.
  • FIG1 is an optical path diagram of a fluorescence modulation multicolor super-resolution microscopy imaging system based on single-wavelength excitation provided by an embodiment of the present invention. As can be seen from the figure, this system uses only one pulsed picosecond laser as a light source, and the detection end also uses only one photomultiplier tube detector to collect fluorescence.
  • the laser After the laser is emitted, it passes through a half-wave plate, and after being split by a Glan laser prism and a beam splitter, three optical paths are formed, one of which is collected by a high-speed photodiode detector as a reference signal for fluorescence lifetime imaging; the other two paths are used to excite the sample, one of which has a wavefront that is not modulated and is still Gaussian; the other light is modulated by a spiral phase plate, and the wavefront is distributed in a hollow ring.
  • the optical path of the ring laser is changed by adjusting the corner reflector, so that it produces a certain time delay relative to the Gaussian laser pulse, and the delay time is greater than the fluorescence lifetime of the dye being measured and less than half of the laser light source pulse period.
  • the above two excitation lights are combined after passing through the second beam splitter, and after passing through the ⁇ /4 glass slide, dichroic mirror, galvanometer, scanning lens and tube lens in sequence, they are focused on the sample through the objective lens.
  • the sample labeled with dye is excited by laser to produce fluorescence, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, it is focused on the core of the multimode optical fiber through the lens and filter.
  • the time-correlated single photon counter simultaneously receives the reference signal and fluorescence signal collected by the high-speed photodiode detector and the photomultiplier tube, and transmits the data to the computer for storage and processing.
  • Figure 2 is a schematic diagram of the effect of the excitation light in the time and space dimensions in the fluorescence modulation multicolor super-resolution microscopy imaging method based on single wavelength excitation provided by an embodiment of the present invention.
  • the first row in Figure 2a is the laser pulse sequence collected by the high-speed photodiode detector, which serves as the reference signal (starting point or end point) of the fluorescence lifetime imaging;
  • the second row in Figure 2a is the Gaussian laser pulse sequence, and the fluorescence decay curve (black solid line) generated under Gaussian laser excitation;
  • the third row in Figure 2a is a ring laser pulse sequence and a fluorescence decay curve (black dotted line) generated under ring laser excitation; there is a certain time interval between the two laser pulse sequences, and the specific value of the time interval is between the fluorescence lifetime ( ⁇ ) of the measured dye and the laser light source pulse period (T), where ⁇ T;
  • the last row in Figure 2a is the decay curve of the fluorescence e
  • the Gaussian laser pulse can be in front and the ring laser pulse can be in the back; or the ring laser pulse can be in front and the Gaussian laser pulse can be in the back.
  • this method requires the Gaussian laser spot and the ring laser spot to coincide precisely in the focal plane in order to achieve super-resolution imaging.
  • Gold nanoparticle samples can be used to adjust the positions of the two laser spots in real time through scattering imaging so that they coincide precisely in space, as shown in Figure 2b.
  • FIG3 is a flow chart of a fluorescence modulation multicolor super-resolution microscopy imaging method based on single-wavelength excitation provided by an embodiment of the present invention.
  • a fluorescence modulation microscopy imaging system is built.
  • the light source of the optical imaging system is a single-wavelength picosecond pulse laser, and the detection end uses only one photomultiplier tube detector to collect fluorescence signals.
  • the sample is marked with two dyes with similar spectra but different fluorescence lifetimes, and the prepared sample is imaged for fluorescence lifetime.
  • the time dimension (t) of the fluorescence lifetime data is separated, and it is divided into two time series stacks based on the time channel where the annular laser pulse is located, which contain fluorescence signals excited by Gaussian light and annular light, respectively, as shown in FIG3c.
  • the two time series stacks are converted to phasor space by sine and cosine transforms to form phasor diagrams of the two stacks.
  • Phasor plot analysis is a commonly used fluorescence lifetime data analysis method that obtains fluorescence lifetime information by converting the time domain fitting into the frequency domain mathematical calculation, so no fitting process is required.
  • the fluorescence lifetime data processing method based on phasor plot analysis is simple and direct. It provides a two-dimensional view of the lifetime distribution, allowing the observer to Rapidly distinguish different lifetime populations in a fluorescence lifetime image.
  • the phasor diagram analysis method is used to separate and extract the target photons of different labeling structures, and four fluorescence images (I g1 , I g2 , I d1 and I d2 ) produced by the two dyes under Gaussian and ring laser excitation are obtained, as shown in Figure 3d. Since the laser pulse period is 40MHz, the time difference between the Gaussian image and the ring image (I g and I d ) is in the nanosecond order ( ⁇ 10ns), which is equivalent to real-time recording of the spatial position information of the Gaussian spot and the ring spot.
  • the intensity value of each pixel of the image is spatially modulated, and the fluorescence signal photons of the ring image are subtracted from the Gaussian signal photons (I g -I d ), and the Gaussian excitation point spread function is compressed to achieve super-resolution imaging.
  • the weight factor ( ⁇ ) is introduced to enhance the intensity of the ring image.
  • the two super-resolution images (I s1 and I s2 ) are superimposed after applying different pseudo colors to obtain a two-color super-resolution image (I S ), as shown in Figure 3e.
  • I S two-color super-resolution image
  • Figure 4 is the imaging result of the fluorescent bead sample based on single-wavelength excitation fluorescence modulation multi-color super-resolution microscopy provided by an embodiment of the present invention.
  • Figure 4a is a confocal intensity image of two fluorescent bead particles (both with a diameter of 23nm) labeled with dyes STAR 635P and Alexa 647, respectively. The two fluorescent particles cannot be distinguished and identified from the image. Fluorescence lifetime imaging is performed under simultaneous irradiation with two laser spots (both with powers in the microwatt range) to obtain a fluorescence decay curve, as shown in Figure 4b.
  • the first complete fluorescence decay process is the autofluorescence signal generated under Gaussian light excitation
  • the second complete fluorescence decay process is the autofluorescence signal generated under annular light excitation.
  • the data is separated by time channels to obtain two time series stacks, and two phasor diagrams are obtained after sine and cosine transformation, as shown in Figure 4c.
  • Photon extraction is performed twice in the two phasor diagrams (STAR 635P is the blue solid line and dotted line area, Alexa 647 is the red solid line and dashed area) to form four images, as shown in Figure 4d.
  • the intensity value of each pixel of the image is spatially modulated.
  • Figure 4f not only has a higher resolution, but also can distinguish between two fluorescent beads labeled with different dyes, realizing two-color super-resolution imaging. Therefore, the method proposed in the present invention can realize multi-color super-resolution imaging under the excitation of a single-wavelength pulsed laser and a fluorescence detector, as well as microwatt-level laser power, providing technical support for the study of dynamic processes and interactions of subcellular structures in living cells.

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Abstract

一种单波长激发的荧光调制多色超分辨显微成像方法，包括以下步骤：S1、首先，根据激光扫描共聚焦成像和荧光寿命成像原理，搭建一套荧光调制显微成像系统；S2、用两种光谱相似但荧光寿命不同的染料对样品进行标记，对制备好的样品进行荧光寿命成像，此时由光源分出的两束激光光斑同时照射样品，采集荧光信号并保存荧光寿命数据,在空间维度上，该方法实现超分辨成像需要高斯激光光斑和环形激光光斑在焦平面精准重合。可以使用金纳米颗粒样品，通过散射成像实时调节两束激光光斑的位置，使其在空间上精准重合。

Description

一种单波长激发的荧光调制多色超分辨显微成像方法 技术领域
本发明属于光学显微镜成像领域，更具体地，涉及一种在一个单波长脉冲激光和一个荧光探测器下实现多色超分辨成像的纳米显微镜技术。
背景技术
光学显微镜出现于16世纪晚期，人类第一次通过透镜观察到由细胞作为基本结构和功能单元的活体生物。经过数百年的发展，显微镜已经遍布于生活和科学研究的各个领域。由于全球显微镜市场的稳步发展，显微镜的产量和用户量都在逐渐增加，相关产品也在快速升级换代。自上世纪七八十年代以来，中国逐渐承接了来自欧洲和日本关于显微镜制造的产业转移，能生产95％的教育类和普及类显微镜。作为世界显微镜生产大国，我国有几十家专业生产显微镜的厂家，但是产品基本面向中低端。根据中国仪器仪表行业协会统计，2015～2017年我国显微镜出口量在220～300万台之间，年均进口5万台左右，出口数量远高于进口数量，但出口金额却远低于进口金额，这反映了中国进口的光学显微镜单台平均价格远高于出口显微镜，国内高端显微镜市场依赖于进口产品。
我国显微镜行业发展缺乏技术沉淀，20年以上经营积累的企业十分稀缺，高精密制造及光学核心部件设计及工艺严重制约了产业升级。目前，国内具备生产高端显微镜能力的企业主要是舜宇光学科技、麦克奥迪、永新光学等企业，但是高端显微镜中，如系统显微镜、共聚焦扫描和超分辨显微镜等主要集中在国外企业，它们占据着世界显微镜市场50％以上的市场份额，其发展战略左右着显微镜市场的走向。最重要的是，国内三甲医院所使用的显微镜几乎被上述国外企业垄断。目前世界市场对高端显微镜的需求迅速增长，中国市场在这方面的需 求增长更快，其中超分辨显微镜的增长超过20％。但是，国内显微镜行业研发技术水平相较于国际先进水平仍有较大差距，行业技术水平亟待提升。因此，若能打破技术壁垒，切入高端显微镜市场，制造出高性能、高可靠性的高端光学显微镜，无疑是一个极大的市场机遇。
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方案，因此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。2014年，诺贝尔化学奖授予Eric Betzig、Stefan W.Hell和W.E.Moerner三位科学家，以表彰他们在超分辨光学显微镜领域的巨大贡献。
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。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、需要特殊的荧光染料，以及复杂的成像系统和昂贵的实验成本等。其中，受激辐射损耗(Stimulated emission depletion,STED)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(Single-molecule localization microscopy,SMLM)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。最重要的是，多色超分辨成像方面基本采用光谱分离技术，使得显微镜系 统需要多个波长不同的激光器作为光源，以及多个探测器收集不同发射光谱的荧光。基于光谱分离技术的多色成像系统不仅光路复杂、成像稳定性差，而且成本很高，进而阻碍了超分辨技术的推广和应用，为此，提出一种单波长激发的荧光调制多色超分辨显微成像方法。
技术问题
有鉴于此，本发明的主要目的在于提出一种在基于一个单波长脉冲激光和一个探测器的简易光学成像系统下突破光学衍射极限、实现多色超分辨成像的纳米显微成像技术，降低多色超分辨显微成像系统的复杂性和成本，提升多色超分辨成像稳定性，为低成本的活细胞等活体超分辨成像及亚细胞结构相互作用的动力学过程研究提供技术支撑。
技术解决方案
本发明实施例的技术方案是这样实现的：一种单波长激发的荧光调制多色超分辨显微成像方法，包括以下步骤：
S1、首先，根据激光扫描共聚焦成像和荧光寿命成像原理，搭建一套荧光调制显微成像系统；
S2、用两种光谱相似但荧光寿命不同的染料对样品进行标记，对制备好的样品进行荧光寿命成像，此时由光源分出的两束激光光斑同时照射样品，采集荧光信号并保存荧光寿命数据；
S3、对上述荧光寿命数据的时间维度进行分离，以环形激光脉冲所在时间通道为界，将其分为两个时间序列图栈；
S4、通过正余弦变换分别将两个时间序列图栈转换到相量空间，形成两个图栈各自的相量图；
S5、利用相量图分析方法分离和提取不同标记结构的目标光子，得到两种染料分别在高斯和环形激光激发下产生的四幅荧光图像；
S6、然后对图像每一个像素的强度值进行空间调制，从高斯信号 光子中减去环形图像的荧光信号光子，压缩高斯激发点扩展函数实现超分辨成像；
S7、最后，将两幅超分辨图像施加不同的伪彩色后再叠加，即得到一幅双色超分辨图像；选择多个光谱相似但荧光寿命不同的染料并对样品进行多目标结构标记，利用上述原理和流程即可实现三色及以上的多色超分辨成像。
优选的：所述步骤S1中，该光学成像系统的光源是一个单波长的皮秒脉冲激光。
优选的：所述步骤S3中，所述两个时间序列图栈分别为高斯光和环形光激发的荧光信号。
优选的：所述步骤S2中，所述荧光寿命数据包含x、y和t三个维度。
优选的：所述步骤S5中,所述相量图分析方法为通过将时间域的拟合转化为频域的数学计算来获得荧光寿命信息，荧光寿命信息分布的二维视图，使观察者可以快速地区分一幅荧光寿命图像中不同的寿命种群。
优选的：所述S5中，高斯图像和环形图像之间的时间差为纳秒量级，实时记录高斯光斑和环形光斑的空间位置信息。
优选的：所述S6中，还包括引入权重因子对环形图像进行强度增强，通过空间调制过程调节对高斯光斑周围发光区域的抑制效果，用以去除衍射受限的低频信号和背景噪音。
一种单波长激发的荧光调制多色超分辨显微成像系统，包括
脉冲激光器，由皮秒激光器产生40MHz脉冲型激发光；
半波片，用于调节激光的偏振方向；
格兰激光棱镜，用于分离不同偏振方向的激光；
透镜，会聚平行传输的激光光束；
分束器，用于激光分束和合束；
螺旋相位板，用于将激光的波前由高斯型转换成环形；
反射镜，用于改变激光的传输方向；
角反射器，调节其位置改变环型激发光斑的光程，用于在时间上控制高斯型激光和环形激光之间的脉冲间隔；
四分之一玻片，将线偏振激光转换成右旋圆偏振光，提升环形光斑的质量；
双色镜，用于透射激发光，反射荧光信号；
振镜，用于对激发光进行同步扫描，实现对样品的面阵成像；
扫描透镜，放置于振镜之后，用于收集面阵扫描的激光光束；
管镜，与物镜搭配构成显微镜系统；
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；
载物台，用于放置和固定样品，并对样品进行三维移动；
滤光片，用于去除荧光以外的杂散光，提高图像信噪比；
多模光纤，用于将荧光信号传输至光电倍增管探测器，其纤芯用作共聚焦系统的探测小孔；
光电倍增管，用于收集荧光光子，并对荧光信号进行放大，输出电信号到电脑；
高速光电二极管探测器，用于探测格兰激光棱镜分束后的激光，作为测量荧光寿命的参考信号；
时间相关单光子计数器，用于测量和记录荧光光子的时空信息；
电脑，用于控制软件采集图像，存储数据和图像数据处理等。
优选的：所述角反射器调节的位置延长或缩短改变环型激发光斑的光程。
有益效果
本发明实施例由于采用以上技术方案，其具有以下优点：
一、本发明通过在空间维度上，该方法实现超分辨成像需要高斯激光光斑和环形激光光斑在焦平面精准重合。可以使用金纳米颗粒样品，通过散射成像实时调节两束激光光斑的位置，使其在空间上精准重合。
二、本发明选择多个光谱相似但荧光寿命不同的染料并对样品进行多目标结构标记，利用上述原理和流程即可实现三色及以上的多色超分辨成像，进而揭示活细胞中线粒体、溶酶体和微管等细胞器之间相互作用的动力学过程；
三、本发明提出的方法可以在一个单波长的脉冲激光和一个荧光探测器、以及微瓦级激光功率激发下实现多色超分辨成像，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。
上述概述仅仅是为了说明书的目的，并不意图以任何方式进行限制。除上述描述的示意性的方面、实施方式和特征之外，通过参考附图和以下的详细描述，本发明进一步的方面、实施方式和特征将会是容易明白的。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像系统的光路图；
图2a～2b为本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像方法中激发光在时间和空间维度上的效果示意图；
图3a～3e是本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像方法的流程示意图；
图4a～4g是本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像的荧光珠样品成像结果。
本发明的实施方式
在下文中，仅简单地描述了某些示例性实施例。正如本领域技术人员可认识到的那样，在不脱离本发明的精神或范围的情况下，可通过各种不同方式修改所描述的实施例。因此，附图和描述被认为本质上是示例性的而非限制性的。
下面结合附图对本发明的实施例进行详细说明。
实施例
本发明提供了一种基于单波长激发的荧光调制多色超分辨显微成像方法，是属于在激光扫描共聚焦成像系统的基础上突破光学衍射极限，降低多色超分辨成像系统复杂性和成本、提高成像系统稳定性的新方法。
一种单波长激发的荧光调制多色超分辨显微成像方法，包括以下步骤：
S1、首先，根据激光扫描共聚焦成像和荧光寿命成像原理，搭建一套荧光调制显微成像系统；
S2、用两种光谱相似但荧光寿命不同的染料对样品进行标记，对制备好的样品进行荧光寿命成像，此时由光源分出的两束激光光斑同时照射样品，采集荧光信号并保存荧光寿命数据；
S3、对上述荧光寿命数据的时间维度进行分离，以环形激光脉冲所在时间通道为界，将其分为两个时间序列图栈；
S4、通过正余弦变换分别将两个时间序列图栈转换到相量空间，形成两个图栈各自的相量图；
S5、利用相量图分析方法分离和提取不同标记结构的目标光子，得到两种染料分别在高斯和环形激光激发下产生的四幅荧光图像；
S6、然后对图像每一个像素的强度值进行空间调制，从高斯信号光子中减去环形图像的荧光信号光子，压缩高斯激发点扩展函数实现超分辨成像；
S7、最后，将两幅超分辨图像施加不同的伪彩色后再叠加，即得到一幅双色超分辨图像；选择多个光谱相似但荧光寿命不同的染料并对样品进行多目标结构标记，利用上述原理和流程即可实现三色及以上的多色超分辨成像。
本发明实施例是这样实现的，一种基于激光扫描共聚焦荧光寿命成像系统，包括：
脉冲激光器(Pulsed laser)，由皮秒激光器产生40MHz脉冲型激发光；
半波片(Half-wave plate,HWP)，用于调节激光的偏振方向；
格兰激光棱镜(Glan-laser polarizer,GLP)，用于分离不同偏振方向的激光；
透镜(Lens,L)，会聚平行传输的激光光束；
分束器(Beam splitter,BS)，用于激光分束和合束；
螺旋相位板(Vortex phase plate,VPP)，用于将激光的波前由高斯型转换成环形；
反射镜(Mirror,M)，用于改变激光的传输方向；
角反射器(Retro reflector,RR)，调节其位置改变(延长或缩短)环型激发光斑的光程，用于在时间上控制高斯型激光和环形激光之间的脉冲间隔；
四分之一玻片(Quarter-wave plate,QWP)，将线偏振激光转换成右旋圆偏振光，提升环形光斑的质量；
双色镜(Dichroic mirror,DM)，用于透射激发光，反射荧光信号；
振镜(GS)，用于对激发光进行同步扫描，实现对样品的面阵成像；
扫描透镜(Scan lens,SL)，放置于振镜之后，用于收集面阵扫描的激光光束；
管镜(Tube lens,TL)，与物镜搭配构成显微镜系统；
物镜(Objective,Obj)，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；
载物台(3D Stage)，用于放置和固定样品，并对样品进行三维移动；
滤光片(Filter)，用于去除荧光以外的杂散光，提高图像信噪比；
多模光纤(Multimode fiber,MMF)，用于将荧光信号传输至光电倍增管探测器，其纤芯用作共聚焦系统的探测小孔；
光电倍增管(Photomultiplier,PMT)，用于收集荧光光子，并对荧光信号进行放大，输出电信号到电脑；
高速光电二极管探测器(High speed photodiode detector,HSPD)，用于探测格兰激光棱镜分束后的激光，作为测量荧光寿命的参考信号；
时间相关单光子计数器(Time-correlated single photon counting,TSCPC)，用于测量和记录荧光光子的时空信息；
电脑(Personal computer,PC)，用于控制软件采集图像，存储数据和图像数据处理等；
图1中红色实线代表高斯型激光，激光出射后透过半波片，经格兰激光棱镜一分为二，其中一束反射后经透镜聚焦于高速光电二极管探测器，作为荧光寿命成像时的参考信号，另一束透射后再次经分光镜(BS)分束。分光镜透射的光仍然是高斯激光(红色实线)，反射的光束经0～2π螺旋相位板(VPP)调制后形成环形激光(红色空心线)，上述两束激光在第二个分光镜处重合。绿色实线代表样品反射回来的荧光信号。将光电倍增管采集的信号传输至TCSPC并将数据保存至电脑。工作时，打开激光器，将脉冲频率调至40MHz，通过移动角 反射器(RR)来调节高斯激光和环形激光之间的脉冲间隔。
为了使本发明的目的、技术方案及优点更加清晰明白，以下结合附图及实施例，对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不限定于本发明。
以下结合实施例对本发明的实现进行详细描述。
图1为本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像系统光路图。从图中可以看到，本系统仅使用一个脉冲型皮秒激光作为光源，而探测端收集荧光也仅使用一个光电倍增管探测器。激光出射后透过一个半波片，经格兰激光棱镜和分光镜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，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，其中一路光的波前不调制，仍为高斯型；另一路光经螺旋相位板调制后波前呈中空的环形分布。通过调节角反射器改变环形激光的光程，使其相对高斯激光脉冲产生一定的时间延迟，且延迟时间大于所测染料的荧光寿命，小于激光光源脉冲周期的一半。上述两路激发光经第二个分光镜后合束，依次经过λ/4玻片、双色镜、振镜、扫描透镜和管镜后，通过物镜聚焦在样品上。被染料标记的样品被激光激发产生荧光，荧光被同一物镜收集后原路返回，双色镜反射后经过透镜和滤光片聚焦于多模光纤的纤芯，荧光在多模光纤中传输后最终到达光电倍增管探测器。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，并将数据传输至电脑进行存储和处理。
图2为本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像方法中激发光在时间和空间维度上的效果示意图。图2a中第一行是高速光电二极管探测器采集到的激光脉冲序列，作为荧光寿命成像的参考信号(起始点或终点)；图2a中第二行是高斯激光脉冲序列，以及在高斯激光激发下产生的荧光衰减曲线(黑色实线)； 图2a中第三行是环形激光脉冲序列，以及在环形激光激发下产生的荧光衰减曲线(黑色虚线)；两个激光脉冲序列之间具有一定的时间间隔，该时间间隔的具体数值介于所测染料的荧光寿命(τ)和激光光源脉冲周期(T)之间，其中τ<T；图2a中最后一行为两束激光同时照射样品时，样品发射荧光的衰减曲线。在本发明中，相对于参考信号脉冲，可以是高斯激光脉冲在前、环形激光脉冲在后；也可以是环形激光脉冲在前、高斯激光脉冲在后。在空间维度上，该方法实现超分辨成像需要高斯激光光斑和环形激光光斑在焦平面精准重合。可以使用金纳米颗粒样品，通过散射成像实时调节两束激光光斑的位置，使其在空间上精准重合，如图2b。
图3为本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像方法的流程示意图。如图3a，首先，根据激光扫描共聚焦成像和荧光寿命成像原理，搭建一套荧光调制显微成像系统。该光学成像系统的光源是一个单波长的皮秒脉冲激光，而探测端仅使用一个光电倍增管探测器来收集荧光信号。以双色成像为例，用两种光谱相似但荧光寿命不同的染料对样品进行标记，对制备好的样品进行荧光寿命成像，此时由光源分出的两束激光光斑(分别为高斯型和环形波前)同时照射样品，采集荧光信号并保存数据，该数据包含x、y和t三个维度，如图3b。对荧光寿命数据的时间维度(t)进行分离，以环形激光脉冲所在时间通道为界，将其分为两个时间序列图栈，它们分别包含高斯光和环形光激发的荧光信号，如图3c。通过正余弦变换分别将两个时间序列图栈转换到相量空间，形成两个图栈各自的相量图。相量图分析(Phasor plot analysis)是一种常用的荧光寿命数据分析方法，通过将时间域的拟合转化为频域的数学计算来获得荧光寿命信息，因此无需任何拟合过程。基于相量图分析的荧光寿命数据处理方法是简单直接的，它提供了寿命分布的二维视图，使观察者可以 快速地区分一幅荧光寿命图像中不同的寿命种群。根据两种染料各自的荧光寿命，利用相量图分析方法分离和提取不同标记结构的目标光子，得到两种染料分别在高斯和环形激光激发下产生的四幅荧光图像(Ig1、Ig2、Id1和Id2)，如图3d。由于激光脉冲周期为40MHz，因此高斯图像和环形图像(Ig和Id)之间的时间差为纳秒量级(～10ns)，相当于实时记录了高斯光斑和环形光斑的空间位置信息。然后对图像每一个像素的强度值进行空间调制，从高斯信号光子中减去环形图像的荧光信号光子(Ig-Id)，压缩高斯激发点扩展函数实现超分辨成像。引入权重因子(β)对环形图像进行强度增强，通过空间调制过程调节对高斯光斑周围发光区域的抑制效果，去除衍射受限的低频信号和背景噪音，进一步提升分辨率，即Is＝Ig-β×Id。最后，将两幅超分辨图像(Is1和Is2)施加不同的伪彩色后再叠加，即得到一幅双色超分辨图像(IS)，如图3e。选择多个光谱相似但荧光寿命不同的染料并对样品进行多目标结构标记，利用上述原理和流程即可实现三色及以上的多色超分辨成像，进而揭示活细胞中线粒体、溶酶体和微管等细胞器之间相互作用的动力学过程。
图4是本发明实施例提供的基于单波长激发的荧光调制多色超分辨显微成像的荧光珠样品成像结果。图4a为分别标记染料STAR 635P和Alexa 647的两种荧光珠颗粒(直径均为23nm)的共聚焦强度图像，从图像中无法对两种荧光颗粒进行区分和辨别。在两束激光光斑(功率均为微瓦量级)同时照射下进行荧光寿命成像，得到荧光衰减曲线，如图4b。其中，第一个完整的荧光衰减过程为高斯光激发下产生的自发荧光信号，第二个完整的荧光衰减过程为环形光激发下产生的自发荧光信号。将该数据进行时间通道分离后得到两个时间序列图栈，将其正余弦变换后得到两个相量图，如图4c。分别在两个相量图中进行两次光子提取(STAR 635P为蓝色实线和虚线区域， Alexa 647为红色实线和虚线区域)形成四幅图像，如图4d。对图像每一个像素的强度值进行空间调制，当β＝1时，得到两幅超分辨图像(Is1＝Ig1-Id1和Is2＝Ig2-Id2)，如图4e。对两种染料标记荧光珠的图像施加不同的伪彩色后叠加得到一幅双色超分辨图像，如图4f。对双色超分辨图像中两个荧光的强度分布进行高斯拟合，分别得到87nm和92nm的超分辨点扩展函数，如图4g。对比图4a和图4f可以看出，图4f不仅分辨率更高，而且能区分出不同染料标记的两种荧光珠，实现了双色超分辨成像。所以，本发明提出的方法可以在一个单波长的脉冲激光和一个荧光探测器、以及微瓦级激光功率激发下实现多色超分辨成像，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。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到其各种变化或替换，这些都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

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  1. 一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于，包括以下步骤：
    S1、首先，根据激光扫描共聚焦成像和荧光寿命成像原理，搭建一套荧光调制显微成像系统；
    S2、用两种光谱相似但荧光寿命不同的染料对样品进行标记，对制备好的样品进行荧光寿命成像，此时由光源分出的两束激光光斑同时照射样品，采集荧光信号并保存荧光寿命数据；
    S3、对上述荧光寿命数据的时间维度进行分离，以环形激光脉冲所在时间通道为界，将其分为两个时间序列图栈；
    S4、通过正余弦变换分别将两个时间序列图栈转换到相量空间，形成两个图栈各自的相量图；
    S5、利用相量图分析方法分离和提取不同标记结构的目标光子，得到两种染料分别在高斯和环形激光激发下产生的四幅荧光图像；
    S6、然后对图像每一个像素的强度值进行空间调制，从高斯信号光子中减去环形图像的荧光信号光子，压缩高斯激发点扩展函数实现超分辨成像；
    S7、最后，将两幅超分辨图像施加不同的伪彩色后再叠加，即得到一幅双色超分辨图像；选择多个光谱相似但荧光寿命不同的染料并对样品进行多目标结构标记，利用上述原理和流程即可实现三色及以上的多色超分辨成像。
  2. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述步骤S1中，该光学成像系统的光源是一个单波长的皮秒脉冲激光。
  3. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述步骤S3中，所述两个时间序列 图栈分别为高斯光和环形光激发的荧光信号。
  4. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述步骤S2中，所述荧光寿命数据包含x、y和t三个维度。
  5. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述步骤S5中,所述相量图分析方法为通过将时间域的拟合转化为频域的数学计算来获得荧光寿命信息，荧光寿命信息分布的二维视图，使观察者可以快速地区分一幅荧光寿命图像中不同的寿命种群。
  6. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述S5中，高斯图像和环形图像之间的时间差为纳秒量级，实时记录高斯光斑和环形光斑的空间位置信息。
  7. 根据权利要求1所述的一种单波长激发的荧光调制多色超分辨显微成像方法，其特征在于：所述S6中，还包括引入权重因子对环形图像进行强度增强，通过空间调制过程调节对高斯光斑周围发光区域的抑制效果，用以去除衍射受限的低频信号和背景噪音。
  8. 一种单波长激发的荧光调制多色超分辨显微成像系统，其特征在于：包括
    脉冲激光器，由皮秒激光器产生40MHz脉冲型激发光；
    半波片，用于调节激光的偏振方向；
    格兰激光棱镜，用于分离不同偏振方向的激光；
    透镜，会聚平行传输的激光光束；
    分束器，用于激光分束和合束；
    螺旋相位板，用于将激光的波前由高斯型转换成环形；
    反射镜，用于改变激光的传输方向；
    角反射器，调节其位置改变环型激发光斑的光程，用于在时间上控制高斯型激光和环形激光之间的脉冲间隔；
    四分之一玻片，将线偏振激光转换成右旋圆偏振光，提升环形光斑的质量；
    双色镜，用于透射激发光，反射荧光信号；
    振镜，用于对激发光进行同步扫描，实现对样品的面阵成像；
    扫描透镜，放置于振镜之后，用于收集面阵扫描的激光光束；
    管镜，与物镜搭配构成显微镜系统；
    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；
    载物台，用于放置和固定样品，并对样品进行三维移动；
    滤光片，用于去除荧光以外的杂散光，提高图像信噪比；
    多模光纤，用于将荧光信号传输至光电倍增管探测器，其纤芯用作共聚焦系统的探测小孔；
    光电倍增管，用于收集荧光光子，并对荧光信号进行放大，输出电信号到电脑；
    高速光电二极管探测器，用于探测格兰激光棱镜分束后的激光，作为测量荧光寿命的参考信号；
    时间相关单光子计数器，用于测量和记录荧光光子的时空信息；
    电脑，用于控制软件采集图像，存储数据和图像数据处理等。
  9. 根据权利要求8所述的一种单波长激发的荧光调制多色超分辨显微成像系统，其特征在于：所述角反射器调节的位置延长或缩短改变环型激发光斑的光程。
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