单分子技术原理与应用 北京大学 生物医学前沿创新中心(BIOPIC) 单分子与活细胞动态过程实验室 孙育杰 sun_yujie@pku.edu.cn 应用单分子超分辨荧光研究细胞动态过程和精细结构 发展单分子荧光技术研究细胞动态过程 Sun & Goldman, 2011 Sun et al,2007 Sun et al., 2009 Sun et al., 2011 In vitro reconstitution of cellular membrane deformation 应用单分子超分辨荧光研究细胞动态过程和精细结构 超分辨显微技术研究细胞超微结构及功能 10大前沿生物技术方法 2014年10月 Nature Methods 创刊10周年特刊 单分子荧光和超高分辨率荧光显微技术 • Biological questions that motivate the development of single molecule techniques. • Brief historical review of single molecule techniques. • Single molecule imaging/spectroscopic techniques. • Single molecule force/manipulation techniques. • Biological applications of single molecule techniques. • Super-resolution microscopy Cell: a chaotic/hectic yet organized world David C. Goodsell, Scripps Institute Cell: a chaotic/hectic yet organized world Lipid droplets trafficking in embryo cells From Internet Mitosis From Internet Melanosome Movement John A. Hammer, III, NIH Cytoplasmic streaming in plant cells From Internet 为什么细胞生物学和分子生物学需要单分子技术 眼见为实 ---- 在细胞生物学领域,单分子研究的终极目标之一就是在体内实时观测 单分子,包括生物大分子的运动并以此在分子水平上理解生理条件下的细胞过程 The wonders of a tiny cell Cytoskeleton and motor protein dynamics 活细胞内过程需要单分子观察 Molecular Motors drive many processes in the cell • Polymerization motors: Actin, Microtubule • Cytoskeletal motors: Myosin, Kinesin, Dynein Kinesin Myosin actin Dynein Green = microtubule Red = actin microtubule Pollard & Earnshaw, Cell Biology, 2nd ed. • Rotary motors: FoF1-ATP synthase The bacterial flagellum Dimroth et al. EMBO, 2006 Credit: Fuller, N.R., NSF • Nucleic acid motors: RNA & DNA polymerase, Helicase, etc. Johnson et al. Cell, 2007 Wang et al. Nucleic Acids Res., 2004 什么是单分子技术 研究对象 技术手段 应用范围 单一小分子、生物大分子和分子复合体/聚 合体 观察 在纳米空间尺度和 毫秒时间尺度上精 确测量单个分子的 距离、位置、指向、 分布、结构以及各 种动态过程 操纵 在皮牛到纳牛力学尺 度上操纵和检测单个 分子的力学行为和动 态过程 单分子技术简要分类 光谱/荧光 力学操纵 荧光 Fluorescence • Optical tweezers • Magnetic tweezers • Scanning probe microscopy • TIRF: Total-internal reflection fluorescence • SMFP: Single molecule fluorescence polarization • FRET: Förster resonance energy transfer • FLIM: Fluorescence lifetime imaging microscopy • Confocal microscopy • Two-photon microscopy 荧光超分辨 Fluorescence Super-resolution • STORM: Stochastic optical reconstruction microscopy • PALM: Photo-activated localization microscopy • STED: Stimulated emission depletion • SIM: Structured-illumination microscopy 光谱 Spectroscopy • SRS: Stimulated Raman Scattering • FCS: Fluorescence correlation spectroscopy …… …… • Electron microscopy • Tethered particle motion (TPM) …… 单分子测量技术的优点除了可以提供与集合系统 测量相近的结果,还体现在下述几点 (1)超高灵敏度和超小的样品用量; (2)得到的是实际几率分布,而不是平均值; (3)测量单一分子及其相互作用的差异性和多样性; (4)实时直接观测单个分子的反应动力学路径; (5)观察分子复合体形成、解离等动态过程; (6)可以捕捉单分子随机过程和分子构象变化的中间态; (7)可以测量稀发但重要的信号和分布,而在集合系统测量时,这些 事件通常都被主要信号掩没; (8)可以测量非平衡态和不同步的体系。很多的细胞分子生物学过程 都是这样的体系,比如分子移动和转动。 单分子技术对生物基础研究非常重要 Diverse views of VDAC structures and functioning In the field of Cell Biology: advancement has been made in molecular motors, protein folding, 305 replication, transcription, and translation as well Emerging Single Molecule as some exciting new Techniques I tools “single molecule genome sequencing” and “single molecule immunoprecipitation”. The occurrence in the scientific literature of papers on single-molecule methods. The number is calculated from a PubMed search displayed as a function of the year from the 1980s until the end of 2006. A Survey of Single-Molecule Techniques in Chemical Biology, Cornish and Ha, ACS Chem. Biol., 2007 概要 • Biological questions that motivate the development of single molecule techniques. • Brief historical review of single molecule techniques. • Single molecule imaging/spectroscopic techniques. • Single molecule force/manipulation techniques. • Biological applications of single molecule techniques. • Super-resolution microscopy Richard Feynman's talk at the 1959 meeting of the American Physical Society at Caltech Detecting Single Molecules Ion channels < ----- > patch clamp Pioneer Work Nobel Prize in Physiology or Medicine in 1991 Erwin Neher Bert Sakmann Detecting Single Molecules Pioneer Work 最早用光学手 段检测单分子 的工作 Detecting Single Molecules Daniel Axelrod 于1981年发明了TIRF – 完全内反射荧光显微镜技术 Pioneer Work Detecting Single Molecules Pioneer Work 其后Moerner和Kador在1989年在低温下实现了用光学手段观测单分子 1990年,Orrit 和 Bernard 也用光学手段实现了单分子观测 Detecting Single Molecules Pioneer Work Eric Betzig在1993年用近场光学显微 镜实现了室温下对单分子的观测 Sunney Xie在1998年在生理环境下用 单分子荧光显微镜研究酶学 Detecting Single Molecules Pioneer Work Taekjip Ha在1996年实现了单分子FRET Toshio Yanagida 于1997-2000年发展了基于物镜的 TIRF技术并首次成功应用于活细胞内单分子成像 Manipulating Single Molecules Pioneer Work 光镊 Optical Trap Arthur Ashkin 于1970年发明了光镊 (Optical Trap/Optical Tweezers) In 1987, Arthur Ashkin and Joseph M. Dziedzic demonstrated the first application of the technology to the biological sciences, using it to trap an individual tobacco mosaic virus and Escherichia coli bacterium Manipulating Single Molecules 光镊 Optical Trap 1997 Nobel Prize in Physics 光镊束缚和控制单个原子 Pioneer Work Manipulating Single Molecules Pioneer Work 光镊 Optical Trap Since 1990, Optical tweezers have been particularly successful in studying a variety of biological systems Carlos Bustamante Dynamics and Forces of molecular motors James Spudich Steven Block Pioneer Work Manipulating Single Molecules 磁镊 Magnetic Tweezers A magnetic tweezer is a scientific instrument for exerting and measuring forces on magnetic particles using a magnetic field gradient. David Bensimon Vincent Croquette Nynke Dekker Neuman & Nagy, Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy, Nat Methods, 2008, 5(6):491 Manipulating Single Molecules Pioneer Work 原子力显微镜 Atomic Force Microscopy The precursor to the AFM, the scanning tunneling microscope, was developed by Gerd Binnig and Heinrich Rohrer in the early 1980s Nobel Prize for Physics in 1986 Binnig, Quate and Gerber invented the first atomic force microscope (also abbreviated as AFM) in 1986 概要 • Biological questions that motivate the development of single molecule techniques. • Brief historical review of single molecule techniques. • Single molecule imaging/spectroscopic techniques. • Single molecule force/manipulation techniques. • Biological applications of single molecule techniques. • Super-resolution microscopy Single cell gene expression (mRNA) analysis using single molecular resolution fluorescence imaging in fixed and living cells 1959 40 years • • • • Zenklusen D, et al. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 2008 Raj A, et al. Stochastic mRNA Synthesis in Mammalian Cells. PLoS Biol 2006. Golding I, et al. Real-Time Kinetics of Gene Activity in Individual Bacteria. Cell 2005 Chubb JR, et al. Transcriptional Pulsing of a Developmental Gene. 2006 Study of Single Molecules used to be very hard Small signal Large noise Light Current Force … Background noise Thermal noise Circuit noise … S/ N 单分子检测的技术难点 • 一个荧光分子在光漂白前大约可发射 300,000个光子。 • 检测效率: Objective (30%)´ Dichromatic mirror (85%)´ Barrier filter (80%)´ Detector (50%) = 10% Room light = 5x107 photons/μm2/s • 系统噪音:dark current, readout noise, shot noise etc. • 样品噪音: (1) Substrate和光学器件的荧光 (2) 其它荧光分子的荧光 S/N 与单分子定位精度 光学衍射 Diffraction-limited Spot Point Spread Function (PSF) 体外量子点 体内荧光蛋白 Improvement in all aspects of contemporary microscopy makes Single Molecules eventually VISIBLE Microscope Light sources (illumination/excitation) Magnification/Objectives Detectors (Camera etc.) Stages/Displays … Stage: less vibration and drift 单分子荧光显微镜基本配置 显微镜 物镜 激光 研究级倒置 荧光显微镜 Olympus 100x 1.4NA Oil UPlanSApo 405 nm (~50 mW) for photoactivation of most fluorophores Nikon 100x 1.49NA Oil Optical modulation AOTF TIRF-level flatness 405/488/561/647 polychroic mirrors 相机 EMCCD Quad-band emission filters. 561 nm laser (~100 mW) for EosFP or Cy3B 647 nm (~200 mW) for Cy5/Alxea647. Filter/ Splitter Mechanical shutter sCMOS Nikon Ti-E Dual-view or Quad-view ND filters Polarizor/Wave plate TTL modulation Laser Conventional Light Sources Laser Magnification/Objectives • Larger collection of light • Higher transmission • Less aberration Detectors: Electron Multiplied Charge Coupled Device (EMCCD) Cameras S/N vs. EM gain S/N Single Photon Sensitivity: • Higher Quantum efficiency • Lower Dark Current • Higher amplification EM gain Fluorescence Probes: Organic dyes Fluorescence Probes: Fluorescent Proteins “A protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite [a handheld ultraviolet lamp], has also been isolated from squeezates” ---- Shimomura et al. 1962 Fluorescence Probes: Quantum Dots Highly fluorescent nanometer-sized single crystals semiconductor materials 量子点 如何评价一个荧光基团 Extinction coefficient/Absorption cross-section Quantum yield Photostability Brightness 比较三种荧光探针: 有机染料、荧光蛋白和量子点 • Size: if it matters for target proper functions. [Organic dyes] • Toxicity: if it poisons cells or animals. [Fluorescent proteins] • Brightness: extinction, quantum yield. [Quantum dots] • Photostability: photobleach, quench, blinking. [Quantum dots] • Labeling specificity: approaches to targeted labeling. [Fluorescent proteins] • Environment sensitivity: pH, ionic, temperature etc. [?] 降低背景噪音的单分子荧光技术 降低背景噪音的单分子成像手段 Ø Light-sheet荧光 显微镜:适用于细胞内 部,如细胞核内的单分 子过程. Ø 双光子或共聚焦荧光 显微镜:常用于较深较大 的组织样品,可用于细胞 内部,如细胞核内的单分 子过程. Ø TIRFM –– 全内反射 荧光显微镜:限于较薄 的细胞和细胞区域. 𝑆 𝑁 TIRFM –– Total Internal Reflection Fluorescence Microscopy Snell’s law n1 /n2 = sin(θ2)/sin(θ1) Beads Fluorescence Intensity vs. Piezo Mirror Votage 4.0E+05 water n2 = 1.330 Intensity (A.U.) Critical Angle 3.5E+05 3.0E+05 V Increase V Decrease 2.5E+05 2.0E+05 1.5E+05 1.0E+05 5.0E+04 glass n1 = 1.513 Total Internal Reflection Nikon Microscope Website 0.0E+00 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 Votage (V) Cytoskeletal actin polymerization and branching Epi Blanchoin et al., (2000) Nature, 404:1007-1011 TIRF Sun Y. et al., Curr. Biol. 2013 Light sources (illumination/excitation) Magnification/Objectives Detectors (Camera etc.) Stages/Displays … Fluorescent labeling Imaging mode Single Molecules Imaging 概要 • Biological questions that motivate the development of single molecule techniques. • Brief historical review of single molecule techniques. • Single molecule imaging/spectroscopic techniques. • Single molecule force/manipulation techniques. • Biological applications of single molecule techniques. • Super-resolution microscopy 单分子技术在体外的应用 单分子技术在体内的应用 Nucleosome Membrane Helicase Gyrase Focal adhesion Lac repressor RNA polymerase Molecular motors Splicesome NPC Transcription Ribosome mRNA Cytoskeletal motors Virus tracking Flagellar motor F-type ATP synthase 复制 转录 RNA转运 剪切 翻译后处理/修饰 翻译 蛋白转运/分布 Ion channels Calcium pump 蛋白-蛋白相互作用 信号响应—信号传导 EGF receptor 离子通道 Ras Peters, Annu. Rev. Biophys. Biomol. Struct. 2007 转录调控 能量过程(线粒体) ……. 单分子技术研究DNA別构效应 Direct observation of Binding and Unbinding 別构效应 Allostery = “allos” (other)+ “stereos” (object) 当效应因子结合到远离活性中心的变构位点处能够通过蛋白质的长程构象变化 来影响蛋白质活性中心功能(酶活性)的现象 koff koff 可能的功能:转录调控? http://202.204.115.67/jpkch/jpkch/2008/wswx/ koff of LacR is modulated by binding of T7 RNAp as a function of their separation Science, 2013 体外 实验 单分子荧光实验研究 DNA別构效应 LacR L T7RNAp t N (t ) = A exp(- ) t 10 µm Average binding time = t Standard error = t /sqrt(N) koff = 1/ t Science, 2013 koff(saturating T7 RNAp) / koff(no T7 RNAp) STABILITY OF LACR AS A FUNCTION OF SEPARATION FROM T7 1.6 If DNA is nicked If DNA has mismatched bases 1.4 LacR dissociates faster 1.2 1.0 0.8 LacR binds longer 0.6 0.4 0 5 10 15 20 25 30 35 Separation, L (bp) 40 45 50 Science, 2013 核小体影响其附近转录因子DNA 结合强度的实验结果 Science, 2013 单分子技术研究马达蛋白 Direct observation of Motion FoF1-ATPase (ATP合成酶) 位于线粒体内膜基质一边,由F0和F1构成的复合体。是一种ATP驱动的质 子运输体,当质子顺电化学梯度流动时催化ATP的合成;当没有氢离子梯 度通过质子通道Fo时,F1的作用是催化ATP的水解。 细胞骨架分子马达 • Polymerization motors: Actin, Microtubule • Cytoskeletal motors: Myosin, Kinesin, Dynein Property Fact Hydrolyze ATP ~ 20 kBT cycle time diffusion coeff. ~ 20 ms ~ 10 nm (head) ~ 100 nm (overall) ~ 10 µm2/s stiffness ~ 1 pN/nm force ~ 5 pN speed from 0.5 to 30 µm/s size Molecular Motor Toolbox ELC Myosin V Calmodulins 37 nm 5.5 nm Actin Filament Motor Domain Coiled Coil Cargo Binding Domain Molecular Motor Toolbox ELC Myosin V Calmodulins 37 nm Coiled Coil Cargo Binding Domain Motor Domain Microtubule 8 nm 5.5 nm Kinesin-1 Actin Filament Coiled Coil Cargo Binding Domain Motor Domain Molecular Motor Toolbox ELC Myosin V Calmodulins 37 nm Coiled Coil Cargo Binding Domain Motor Domain Microtubule Cytoplasmic Dynein IC Dynactin Binding Domain 5.5 nm Microtubule Binding Stalk LIC 8 nm IC Actin Filament Kinesin & Dynein in Neuron MOLECULAR MOTORS AND MECHANISMS OF DIRECTIONAL TRANSPORT IN NEURONS Hirokawa and Takemura NATURE REVIEWS NEUROSCIENCE 2005, 6:201 The Myosin Superfamily The Myosin Superfamily The Myosin Superfamily Cytoplasmic streaming in plant cells Myosin X Myosin V Lipid droplets trafficking in embryo cells Myosin VI Myosin X Helical Pitch = 72 nm Adapted and modified from Rock et al., PNAS (2001) and Krendel & Mooseker, Physiology (2005 ) In vitro motility assays are used to measure motor activity Toyoshima et al. (1987) Nature 328:536-539. Myosin V Strolling Single Molecule Study of Molecular Motors Myosin Stepping Mechanisms and Dynamics by Single Molecule Position and Orientation Measurements Sun et al., 2007 Sun et al., 2010 Ø Width of l/2 » 250 nm Diffraction limited spot Prism-type TIR 0.2 sec integration center 280 240 Photons 200 160 120 width 80 40 0 5 10 15 Y ax is 20 Z-Data from Columns 1-21 photon pixelation noise background noise With enough photons (signal to noise) … Center can be determined to » 1 nm. Yildiz et al., 2003, Science 20 25 25 15 X Da 10 ta 5 0 measurements of myosin motor stepping Streptavidin-QD 120 NEMMyosin Biotin Tag Cover glass Y pixels 118 116 114 404 406 408 410 412 414 X pixels 800 120 600 500 y position (nm) 118 Y pixels 400 300 200 116 100 0 100 200 0 2 4 6 8 10 12 114 404 14 Time (s) Time (s) 406 408 410 x position (nm) X pixels 412 414 800 700 600 500 t (nm) Displacement (nm) Displacement (nm) 700 400 Sun, 2010, Nat Struct Mol Biol measurements of myosin motor stepping QD labeled labeled Myosin Myosin XX motors motors QD Label Position Position 11 Label Position 2 Label short P(t) = ke−kt long <Dwell time> = 0.89 s P(t) = tk2e−kt <Dwell time> = 1.62 s 0 Super long Sun, 2010, Nat Struct Mol Biol Ø Precise localization proves single myosin motors step hand-over-hand short long Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity Warshaw, D.M. et al. Biophys. J. 88, L30–L32 (2005). Correlation between the movement of MyoV-HMM and the binding /dissociation of deacaminonucleotide. Images of Alexa-Fluor-568–MyoV-HMM and deac-aminoATP fluorescence were acquired simultaneously with a DualView system. Ø Kinetics features of myosin motors Trailing head Leading head Vale, J. Cell Biol., 2003 Coordination between the two legs of Myosin V An Optical Trap study Deterministic Working Stroke Thermal Motions This image cannot currently be displayed. Completion of Step 单分子体内实验应用 3D movement of GLUT4-containing vesicles in living adipocytes Sun et al., Nano Lett, 2009 Next Level: Ex vivo Single Molecule 体外单分子荧光实验研究分子马达驱动的膜形变 lysosome Kinesin 1 Autolysosome Microtubule Network Mitochondria 500 nm In collaboration with Li Yu, Tsinghua University Membrane deformation in the cell -- Two model systems Autophagic Lysosome Reformation (ALR) 1. Mechanism for budding or fission of tubules and proto-lysosome? 2. Curvature formation and maintenance? 3. Sorting mechanism for cargo segregation and membrane separation? Yu et al, Nature, 2010 Mitochondrial Network Formation 1. Fission and fusion? 2. Morphology and functional states? In vitro and In vivo Filter paper Slides DoubleSide Tape Pipette tips Flow Coverslip Controllable ingredients Controllable concentration Low background Physiological In vitro Reconstitution lysosome Kinesin 1 P PE Biotin P PIP2 PE Rhod Autolysosome Microtubule Network AP2 K560 Biotin Clathrin Full Length Kif5b Mitochondria Fluorescent Microtubule 500 nm Tubulin Antibody In vitro Reconstitution 体外重构 (In vitro reconstitution) Autolysosome Mitochondria Working model Coordination? Deforming Concentrating Recruiting Budding Kenisin Clathrin AP2 PIP2 Remaining questions 1. Fission of tubules and proto-lysosome --- Dynamin ? 2. Sorting mechanism for cargo segregation and membrane separation --- PI4P, PI5K1, PI(4,5)P2 , Clathrin, and Curvature? 概要 • Biological questions that motivate the development of single molecule techniques. • Brief historical review of single molecule techniques. • Single molecule imaging/spectroscopic techniques. • Single molecule force/manipulation techniques. • Biological applications of single molecule techniques. • Super-resolution microscopy 生命科学对高分辨率 显微成像技术的需求 细胞的尺度 ~0.02mm 人眼的分辨率 ~0.1mm 光学衍射极限与生物超高分辨率成像的需求 细胞骨架 荧光显微镜图像 细胞骨架 细胞骨架 电子显微镜图像 Green = microtubules Red = actin Medalia et al. Science, 2002. Abbe衍射极限 分辨极限 无法分辨 可分辨 爱里斑 爱里斑 ~ 300 nm 1873年,德国科学家Ernst Abbe 提出“光学衍射极限理论”。 电磁波波长范围和光学显微镜空间分辨率 波长 ≈ λ/2 ≈ 300 nm 数值孔径和光学显微镜空间分辨率 数值孔径 NA = n*sin α 折射率 n 折射率 孔径角 光学衍射极限与生物超高分辨率成像的需求 荧光显微镜 电子显微镜 哺乳动物细胞 蛋白质 核糖体 病毒 细胞骨架 线粒体 光学显 微镜 分辨率 300nm • 尺度:在细胞内,很多亚细胞结构、细胞器和生物大分 子的尺寸都在微米和几十纳米的级别。 • 浓度:细胞内非常拥挤,1 μM 的浓度相当于 1 μm3中 有 ~600 个分子,也相当于衍射极限体积内有 5个分子。 细菌 细胞骨架超高分辨率 荧光显微镜图像 2014年诺贝尔化学奖 “突破”衍射极限 超高分辨率显微成像技术 突破衍射极限的超高分辨率光学成像 突破衍射极限的超高分辨率光学成像 Nature:2008年度方法 超高分辨率荧光成像 Marta Fernández-Suárez and Alice Y. Ting, Nature Review: Mol. Cell Biol. 2008 突破衍射极限的超高分辨率光学成像 活细胞超高分辨率 RESOLFT 显微镜 S. W. Hell: "Diffraction-unlimited all-optical imaging and writing with a photochromic GFP" Nature 478, 204 – 208 (2011) S. W. Hell: "Nanoscopy of Living Brain Slices with Low Light Levels" Neuron 75, 992 – 1000 (2012) S.W. Hell. "rsEGFP2 enables fast RESOLFT nanoscopy of living cells“ eLIFE (2012) S.W. Hell. "Nanoscopy with more than 100,000 ‘doughnuts’ ” Nature Methods (2013) 突破衍射极限的超高分辨率光学成像 2014, 2015 活细胞超高分辨率显微镜 lattice light sheet based NL-SIM Bi-chang Chen … Eric Betzig, Science 2014 Dong Li … Eric Betzig, Science 2015 “突破”衍射极限和实现超高分辨率成像的基本思想 任何光源聚焦成的光斑的大 所有分子同时发光,其“爱 小也是受衍射极限限制的! 里斑”重合,导致无法区相 邻分子。 基本思想一 用小于衍射极限的 光斑来扫描样品 缩小照明光斑点 扩散函数 + 扫描 控制每次“可见”的分 子数目,使得相邻分子 不同时发光 单分子定位 +重构 基本思想二 Single molecule localization microscopy 基于随机重构的突破衍射极限的超高分辨率光学成像 STochastic Optical Reconstruction Microscopy (STORM) Photoactivated Localization Microscopy (PALM) From FIONA to Super-Resolution Imaging 利用光控的荧光基团进行随机重构 Imaging Laser 光控荧光基团 Photoactivable Photoswitchable Photoconvertable 利用光控的荧光基团进行随机重构 Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 精确的单分子定位 荧光强度 X 轴 (像素) 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 Weak Activation Laser 利用可激活的荧光基团进行 随机重构 Strong Imaging Laser 利用可激活的荧光基团进行 随机重构 利用可激活的荧光基团进行 随机重构 “突破”衍射极限和实现超高分辨率成像的基本思想 STED和STORM/PALM技术的对比 超高分辨率光学成像技术简要比对 技术名称 空间 分辨率 (nm) STED STORM PALM SIM NSIM RESOLFT SOFI# µ n 3B 40-60 10-20 20-30 100 70 40-60 (up to 4) ~80 nm ~50 (扫描区域 100x100μm2) ~10 s ~1 s ~30 s ~10 s ~1 s ~1 s* 1-4 s ~4 s 探针 主要为有机 染料 时间分辨率 其它 使用较强激 光,可能伤 害生物样品 饱和原理 有机染料, 荧光蛋白, 都可以 不易做活 适于活细 细胞 胞 • • • 最高z轴分辨率 可定量Couting 单分子追踪 最相似于 传统显微 镜,简单 易用 单分子定位原理 干涉 荧光蛋白, 荧光蛋白, 适合用量 子点 适于活细 适于活细 胞 胞 对荧光蛋 白光稳定 性要求高 对荧光蛋 白光稳定 性要求高 饱和原理 对SNR要 求低,基 于算法, 高阶可能 有假象 需抗漂白且 闪烁多 对SNR要求 低,基于算 法,计算量 巨大 荧光涨落原理 * 基于平行化的扫描方式 #: Super-resolution optical fluctuation imaging (SOFI) achieves resolution enhancement based on the statistical analysis of temporal fluorescence fluctuations. Nucleoli Nucleus Endoplasmic Reticulum Mitochondia Many biological structures and processes in cell biology, microbiology and neurobiology Plasma Membrane Golgi Microtubule Lipid Rafts Peroxisomes Cytosol Actin Lysosomes RESOLFT 实验数据 Live-cell imaging with parallelized RESOLFT nanoscopy S.W. Hell. "Nanoscopy with more than 100,000 ‘doughnuts’ ” Nature Methods (2013) STORM超分辨显微镜解析神经细胞突触区分蛋白分子分布 Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, and Xiaowei Zhuang Neuron, 2010 双物镜STORM解析神经细胞轴突中Actin周期结构 超高分辨率显微技术的发展趋势 从显“微”镜时代带入到显“纳”镜时代 • 更高: 更高的空间分辨率 • 更快: 更快的时间分辨率 • 更深: 更深的成像深度 活体研究 超高分辨率显微技术的发展趋势 • 原理创新: 发展新型的超分辨技术和标记技术 高速超分辨:超快扫描元件,高灵敏检测元件等 深层超分辨:自适应光学,组织透明化(CLARITY)等 • 发展和优化荧光探针: 优化现有探针:亮度、光稳定性、转换速率以及发射波长等 开发新型探针:上转换纳米材料,荧光钻石,石墨烯量子点等 • 模态融合: 没有哪种技术能兼顾时间分辨率、空间分辨率、空间尺度。对于超分辨技术而 言,至少有三种值得融合的技术。 双光子荧光显微镜:成像深度深 电子显微镜:即光电融合显微技术CLEM (Correlative Light&Electron Microscope) 片层光 light-sheet: 2014年Nature Methods年度方法 • 高效的图像处理软件和算法:超高分辨率动态成像以及大样品成像 Seeing what’s going on in the cell is very fun! 谢谢! 单分子技术和应用的书 推荐 Phil Nelson Rob Philips UPenn Caltech