徕卡SP8 SMD技术原理及应用.pdf 
徕卡SP8 SMD技术原理及应用.pdf
方策 9/1/2016 徕卡显微系统（上海）贸易有限公司 内容： 一、常规共聚焦的原理、应用及参数设置 二、FCS荧光相关光谱及FLIM荧光寿命成像 1、技术原理 2、生物医学应用案例 3、操作注意事项 显微镜的不同观察方式 明场 DIC 相差 荧光 共聚焦最主要的观察对象：荧光信号 荧光蛋白或荧光染料 荧光蛋白与荧光染料 细胞核、黏着斑蛋白和微丝 9 肾脏组织学切片 蓝色：细胞核，绿色胞浆， 红色：平滑肌细胞 信号分子标记 Ca2+ Fluo-3, Fluo-4, Calcium Green, Fura2等 pH BCECF AM ROS H2DCFDA (DCF) Singlet Oxygen Sensor Green NO DAF 膜电势 JC-1 激发光谱和发射光谱 激发光谱 发射光谱 激发光波段控制 发射光波段控制 荧光显微镜 荧光滤块转轮 荧光显微镜光路图 宽场荧光显微镜的局限 Camera 相机 分光镜 激发光源 焦点外信号 焦点信号 激光共聚焦显微镜 激光共聚焦显微镜原理 P M T 探测器 针孔 分光镜 激发光源 光学扫描器 焦点外信号 焦点信号 点光源 共聚焦 vs 宽场显微镜 Leica TCS SP8 扫描头光路图 激光器 固体激光器 1、405nm 激光器： DAPI，Hoechest 33342蓝色荧光 2、488nm激光器： FITC，GFP，Alexa 488等绿色荧光 3、 5552nm 激光器： RFP，mCherry，Cy3，Alexa 555等红色荧光 4、633nm 激光器： Cy5，Alexa 633， Alexa 647等近红外荧光 AOTF：精确控制激光能量 Desired lines in (to scan device) Undesired lines out (to photon dumpster) Acoustic Scout (activation by sound) 1、精准调节激光使用的能量 All lines in (from laser crowd) 2、感兴趣区域（ROI）扫描或光激活 激光控制: AOTF - ROI 扫描 ROI1: 488nm for FITC only. (Green colour) ROI2: 405nm for DAPI, 488nm for FITC, and 561nm for TRITC ROI3: 561nm for TRITC only (Red colour) ROI4: 405nm for DAPI only (Blue colour) Leica TCS SP8 扫描头 扫描振镜 激光点扫描成像光学放大 A B C D E F 上方为63×物镜下不放大扫描图像，下方为zoom放大图像 Nyquist采样定律 采样频率必须要达到信号频率的2-3倍。 为了获得最佳分辨率，需要达到 “像素点尺寸(pixel size)”=“当前所使用物镜 的横向光学分辨率大小”的1/2 – 1/3。 Page 30 Magnification 63 40 10 Numerical Aperture 1.4 1.25 0.4 Optical Resolution [µm] 0.14 0.16 0.5 Field (Edge) [µm] 238 375 1500 # Resel (Field / Resolution) 1700 2344 3000 2 x Oversampling 3400 4688 6000 3 x Oversampling 5100 7031 9000 扫描图像参数设置 pixel size < 0.5 * xy侧向分辨率 20x/0.7 NA Objective + 1024x1024 pixels = loss of detail 8192x8192 detail 1024x1024 detail Courtesy: Dr. Michael E. Calhoun, Department of Cellular Neurology, Hertie Institut for Clinical Brain Research, Tübingen, Germany Leica TCS SP8 扫描头 视场旋转器 实时视场旋转 Cardiac muscle cell Calcium waves & sparks, 1st ch Fluo 3 2nd ch TLD Courtesy Prof. Neumann, University Halle, Germany 34 Leica TCS SP8 扫描头 二向色镜 二向色镜 荧光 激光 激光 + 荧光 Leica TCS SP8 扫描头 针孔 共聚焦针孔（pinhole） 检测器 针孔 物镜 焦平面 三维成像 光学层切 y z x 共聚焦针孔参数设置 针孔越大，信号越强，但分辨率越低， 针孔越小，信号越弱，但分辨率越高， 各种性能最平衡的针孔大小为1 AU； 当荧光很弱时，可适当调大针孔直径 当使用内置检测器进行多光子成像时， 应把针孔开到最大。 Leica TCS SP8 扫描头 分光部件 滤色片分光与棱镜分光 Leica SP2 – SP8 棱镜分光，光谱检测 荧光接收范围设置 双击滑条可手动更改波长范围 光谱检测器的参数调节 Emission 4 3 2 1 Wavelength 1 2 3 4 序列扫描 DAPI与其他染料之间的窜色 DAPI GFP Alexa Fluor 488 Rhodamine 123 Alexa Fluor 532 由于染料之间的荧光发射光谱的交叉（窜色）， 所以多色标记时经常需要通过序列扫描进行成像。 序列扫描 一个序列中只开一根激光谱线和一个检测器 成像模式 XYZ扫描 神经细胞 MIP最大强度投影 毛细胞 50 三维重构模块 斑马鱼头部原始数据 GFP标记肿瘤细胞 mCherry标记血管 54 斑马鱼头部 GFP标记肿瘤细胞 mCherry标记血管 55 FITC标记的小鼠大脑血管 57 FITC标记的小鼠大脑血管 58 3D Visualization 显微CT 61 层切厚度设置 层切厚度设置 层切厚度设置 层切厚度设置 层切厚度设置 xyt 扫描 有丝分裂 神经细胞轴突延伸 活细胞成像 小鼠胚胎异染色质的形成需要关键的组蛋白变体H3.3. 显微注射oregon green后细胞的生存实验 Courtesy of ME Torres-Padilla (Team L. Tora) & Marc Koch (Imaging Centre IGBMC). Courtesy of Adrien Eberlin (Team L Tora) & Marc Koch (Imaging Centre IGBMC). 68 共振扫描头 OFF：高分辨率扫描头，8192 x 8192，7 fps@512 x 512， 常规成像及慢速动态成像 ON： 高速扫描头，1024 x 1024，28 fps@512 x 512， 快速动态成像 xyt 扫描 Calcium waves & sparks, Cardiac muscle cell 1st ch Fluo 3, 2nd ch TLD Courtesy Prof. Neumann, University Halle, Germany 70 xyzt 扫描 Arabidopsis thaliana protoplast Monitoring mitochondrial (GFP-green) and chloroplast (autofluorescence-red) movement. 30 µm xt扫描 250 ms Consecutive line-scan images from HAMs showing two calcium sparks and a calcium wave. Anna Llach et al., European Heart Journal (2011) Quantify Life! – The Challenge Concentration? Molecule motility? Molecule interaction? Diffusion behavior? Molecule identification? Reaction kinetics? Signal transduction? Translocation? Explain phenomena! Predict models! Verify assumptions! Optimize agents! Improve procedures! Validate data! Understand life! Possibilities: FCS - FCCS - FLIM – FRET - FLCS - gated FCS 84 Quantify Life! – Possibilities TCS SMD Series: System block diagram SMD: Single Molecule Detection Covers 3 basic technologies (FLIM, FCS, FLCS) LAS X integrated workflow New possibilities of data generation and analysis PicoQuant Leica SMD analysis SP8 confocal ext. excitation int. excitation fiber cables int. detection ext. detection communication The TCS SMD series • A platform that integrates the confocal system TCS SP8 with SMD specific components from Picoquant • Used for FLIM, FLIM-FRET, FCS, FCCS, gated FCS, FLCS, and FLCCS SMD specific components TCS SP8 87 Integration of SMD Software SPT to LASAF LAS X与SPT 整合 Global control by full system integration Comprehensive network connection between confocal and SMD components 共焦跟SMD 部件全面连接 TCS SP8 User controls LAS X PC only 只须对应 LAS X 电脑，单一界面 Easy and reliable data handling 简单操控 User support by dedicated application wizards 专用界面 FLIM (FLUORESCENCE LIFETIME IMAGING MICROSCOPY) 荧光寿命成像显微技术 荧光寿命: 激发-发射 循环 Vibrational Fluorescen Relaxiation ce life time ps (10-12 s) ns(10-9 s) 影响荧光寿命的因素: • 染料自身特性 异构 质子化 蛋白质折叠 其他的亚状态 • 低于光学分辨率的微环境: 分子结合 离子浓度 pH 亲脂环境 • 胞膜电位 Excitation fs (10-15 s) 什么是FLIM? Fluorescence Lifetime Imaging Microscopy – FLIM - 普通共聚焦亮度成像 建立在荧光基础上的显微技术 分析荧光分子在激发态的寿命 通过和成像信息的结合分析 荧光寿命的空间分布信息 获得附加信息 （分子识别，微环境，分子结合 ） 荧光寿命成像 /ps Sample: Prionium, stained with Safranin and Fast green FLIM – 工作流程 Time/ns • 在每个像素重复测量激光脉冲 发出和探头收到荧光光子之间 的时间 • 获得光子数量对到达时间的分 布图 • 对每个分布图进行指数衰变拟 和. t 即为所得荧光寿命 • 使用伪色显示寿命图像 典型 FLIM 应用 无需染色样本（自发荧光）: 测定代谢状态 癌症研究：识别转化的细胞 植物生理：病原宿主反应 形态学：识别甲壳素和木质素结构 FLIM image of scale insect antenna (non-stained): Image was reconstructed from a 3dimensional FLIM-z-stack, using Amira SW Courtesy of Kees Jalink 典型 FLIM 应用 无需染色样本（自发荧光）: 测定代谢状态 癌症研究：识别转化的细胞 植物生理：病原宿主反应 形态学：识别假壳素和木质素结构 Image 1 Image 7 Image 13 Image 19 Image 22 Image 25 Image 31 Image 37 Image 43 Image 47 百合花粉的 3-D FLIM 整个扫描深度 z: 37 µm 典型 FLIM 应用 染色样本: 为图像信息增加一个新的维度 能量转移(FRET)用以测量分子结合和分子间距离 局部离子浓度 (Ca2+, Na+, pH), 配体, 氧含量 微环境研究 (粘滞度， 折射系数，胞膜电位） 染料分离 染料的光物理特性 Conventional confocal intensity image Fluorescence lifetime image of cells expressing CFP-YFP Courtesy: G. Hams, University of Würzburg Advantages of FLIM Immune to effects like: Concentration fluctuations caused by diffusion or photo-bleaching (without photo-conversion) Shading in thick samples Fluctuations of excitation intensity Light source noise Internally calibrated FRET Unperturbed conditions in unstained samples Lifetime dependency on local ion concentration or pH Probe: exc/em ta (ns) tb (ns) BCECF 490/520 3.0 (acid) 3.8 (base) Fluo-3 Lucifer Yellow Sodium Green 490/520 2.44 (no Ca2+) 3.3 1.1 (low Na+) 0.79 (Ca2+) 2.2 (no acc., 7-AAD) 1.4 (acceptor,7-AAD) Hoechst 2.4 (high Na+) FITC TRITC 490/520 543/ 4.0 (pH > 7) 2.0 3.0 (pH < 3) Rhodamine 700 Rhodamine 700 Cy3 Cy5 659/669 1.6 (pH 9) 1.55 (H2O) 0.27 1.0 1.55 (pH 6) 2.99 (Ethanol) 0.5 (antibody conjug.) GFP free (S65T) CFP YFP 488/507 550/570 633/ 2.68 1.3 3.7 Measurement of local pH in live cells via FLIM Protein activity is often affected by the local environment, in particular by pH, ion concentration and polarity. FLIM measures such changes both spatially resolved and over time. Example: GFP fusion protein expressed in Hela cells. Sub-cellular localization of the GFP labeled protein gives rise to two populations displaying a different average lifetime. The protein responds to pH changes in the organelle by a shortened lifetime. Courtesy: M. Weiss, J. Szymanski, DKFZ, Heidelberg Lifetime analysis of local pH As revealed by the lifetime histogram GFP possesses two fluorescence lifetimes . Their relative contribution to the average lifetime changes with localization to the cytoplasm (yellow line) or the intracellular organelle (red line). Thus, FLIM proves a differential nnanoenvironment. Courtesy: M. Weiss, J. Szymanski, DKFZ, Heidelberg Unperturbed investigation of host-pathogen interaction by spectrally resolved FLIM 413 – 766 nm FLIM lamda stack Intensity image: Low contrast FLIM image: Complex behavior Sample (non-stained): Hyphae of pathogenic fungus invading tomato fruit Analysis of fluorescence intensity and lifetime λ-stack 415-465 nm 465-515 nm 515-565 nm 565-615 nm 615-665 nm 665-715 nm 715-765 nm What is FRET? Fluorescence Resonance Energy Transfer – FRET Fluorescence based method Describes the non-radiative transfer of energy stored in an excited fluorescent molecule (the donor) to a non-excited different fluorescent molecule (the acceptor) in its vicinity Thus probes the proximity of fluorescently labelled molecules Energy transitions in FRET pair: Light energy matching a transition in the donor molecule is absorbed (blue arrow). The excited donor can relax either by fluorescence (left gey dotted arrow) or by resonance energy transfer to the acceptor molecule (black arrow). Conditions for FRET Three conditions must be fulfilled for FRET to take place: 1. Overlap of donor emission spectrum with acceptor excitation spectrum 2. Molecules must be in close proximity on an Angstrom (10-10 m) scale. 3. Molecules must have the appropriate relative orientation. Example: FRET pair CFP-YFP Emission spectrum of donor (here ECFP, blue line) must overlap with excitation spectrum of acceptor (here EYFP, yellow line). Distance dependency of enery transfer 1. Donor and acceptor are separated by a distance r >> R0. 2. At this distance no FRET can occur. Only the donor emission is detected. 3. At a small distance r ~ R0 FRET gives rise to acceptor fluorescence. FLIM-FRET in live cells: typical FRET pairs CFP-YFP GFP-YFP Cerulean-Venus mTurquoise-YFP GFP-HcRed Biological heterogeneity of FRET cells (CFP-YFP fusion), Courtesy: G. Hams, University of Würzburg GFP-mCherry GFP-mKate FLIM-FRET (CFP-YFP) in live cells: donor lifetime images Donor lifetime images of FRET and control cells: Donor only (CFP) Sample: RBKB78 cells transfected with a CFP donor only or CFP-YFP fusion. Data Acquisition: The detection band was set between 445 – 495 nm using spectral FLIM detectors. Data Analysis: The colored region has been used for analysis. Colors represent intensity modulated fluorescence lifetimes. Result: In the presence of acceptor the donor lifetime is decreased. Courtesy: G. Hams, University of Würzburg FRET pair (CFP-YFP tandem ) FLIM-FRET (CFP-YFP) in live cells: Quantitative data analysis Fluorescence lifetime distribution histogram of donor only (yellow) and FRET (green) samples using average lifetimes. There is a clear shift of 0.7 ns towards shorter lifetimes in the FRET sample. Computation of FRET Efficiency: From lifetime distribution histograms one obtains: Average lifetime t of the donor is: 2.1 ns. Donor lifetime of the FRET construct is: 1.4 ns. FRET efficiency is: E = 33 %. FLIM-FRET (GFP-mCherry) in live cells: donor lifetime image Donor lifetime image of a mixture of FRET and control cells: Sample: HeLa cells expressing either GFP (donor) only or GFP-mCherry tandem (FRET pair) Data Analysis: Colours represent intensity modulated fluorescence lifetimes. Result: As indicated by the colour shift from yellow to green the average donor lifetime decreases in the presence of acceptor. Courtesy: M. Weiss, J. Szymanski, DKFZ, Heidelberg FLIM-FRET (GFP-mCherry) in live cells: Quantitative data analysis Fluorescence lifetime distribution histogram of donor only (yellow) and FRET (green) samples using average lifetimes. There is a clear shift of 0.2 ns towards shorter lifetimes in the FRET sample. Computation of FRET Efficiency: From lifetime distribution histograms one obtains: average lifetime t of the donor is: 2.15 ns. donor lifetime of the FRET construct is: 1.95 ns. FRET efficiency is: E = 10 %. Integration of SMD specific components Leica SP8 Single photon counting detector: HyD-SMD “stop” Synchronization of scanner (pulse interface, trigger unit) PC Leica “LAS AF” Synchronization of data acquisition Pulsed laser system “Stop watch” Time-Correlated Single Photon Counting (TCSPC) unit PC Pico Quant “SPT” “start” Towards FLIM Measurements within 4 Steps Step 1: Adjust correction ring of the objective. Step 2: Find your sample using “normal” imaging mode Step 3: Optimize FLIM measurement conditions Step 4: Setup and start the FLIM measurement series or stacks The SMD FLIM Wizard: special properties Easy handling by intuitive user interface and user guidance through the experiment. Global control over the experiment within the SMD FLIM wizard. Intelligent presets allowing fast results: automated configuration of filters wheels, coupling ports, detectors etc. Data handling made easy: User-definable experiment names are used in both, confocal software LASAF and FLIM software SPT. All relevant instrument parameters and user comments are automatically transferred from LASAF to SPT and saved with the experiment file. The SMD FLIM Wizard: new applications FLIM time series FLIM xy z volume stacks FLIM xz y volume stacks FLIM lambda emission stacks (SP FLIM) FLIM lambda excitation stacks (WLL FLIM) Combination of FLIM stacks with time series ABC option (automated brightness control) Definition of FLIM xz y- stack in the FLIM wizard FCS （FLUORESCENCE CORRELATION SPECTROSCOPY) 荧光相关光谱检测 什么是 FCS? FCS: 建立在荧光现象基础上的测量方法。它能够测量和分析单个分子进入和离开一个 特定的很小的观察体积动态过程。这个观测体积一般为共聚焦成像的焦点(约 0.15fl). 对分子的运动引起的荧光强度变化的分析提供个研究者附加的信息。 FCS 读出参数和提供的信息： • 分子的平均数量 • 扩散时间 • Fraction of components => 浓度 => 分子大小, 环境黏度 => 结合-自由 比 => 化学反应的动力学参数 => 平衡参数 • 三线态或其他的荧光分子暗态 => 分子自身特性 => 环境参数 (pH, …) 观测体 积 FCS – 工作流程 • 激光束被固定在一个要被测量 的点上（测量体积） 荧光粒子进入和离开测量体积 • 记录荧光信号的变动 I(t) • 计算相关函数 t • 对相关函数进行拟和 => 得到不同的参数(N, τ, α, …) G(t) log t 应用实例：活细胞中的 EYFP 测量 (3) fit 0.02 0.015 0.01 0.005 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 lag time [sec] 0 1.00E-01 1.00E+00 autocorrelation function meas. 0.025 测量结果: HeLa 细胞核中自由移动的 EYFP 的自相关函数: • 测量点内含有 ~45 个分子, 在 大约0.15 fl 的测量体积内，分 子浓度约为 ~60 nM • 扩散时间约为 ~500 sec, i.e., 扩散系数为 ~30 m2/sec, i.e., 黏性比水高三倍 Calculation of autocorrelation (FCS) Photons over time (photon mode data = time between photons) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 Number of photons in time bin (time mode data) Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 1 1 4 4 4 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 (t0) = 20 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 1 2 4 4 2 1 0 0 0 0 0 0 1 1 1 0 0 0 0 0 (t1) = 17 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 2 2 4 2 2 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 (t2) = 14 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 2 2 2 2 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 (t3) = 9 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 2 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (t4) = 5 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (t5) = 2 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (t6) = 1 Calculation of autocorrelation (FCS) 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 (t7) = 0 Calculation of autocorrelation (FCS) correlation function 25 20 15 10 5 0 0 2 4 6 8 tau G(t ) F (t ) F (t t ) F (t ) 2 Normalization of correlation function Logarithmic scale Results from FCS experiments G (t ) 1/N 1/c tcorr 1/D Amplitude of fluctuations concentration Curve shape diffusion model Time of half maximal amplitude Length of fluctuations diffusion coefficient of fluorescently labeled molecules log t Theoretical approach Properties of the optical system Properties of the diffusion process = I (r) = ... G (t ) Analytical autocorrelation function concentration, brightness, diffusion properties of up to 3 species c (r,t) = ... log t Theoretical approach Properties of the optical system I (r) = ... assuming that the product of the illumination PSF and the detection PSF can be approximated as a 3D Gaussian Theoretical approach Properties of the diffusion process c (r,t) = ... solving the diffusion equation for different cases: 1D, 2D, 3D diffusion; anomalous/obstructed diffusion; directed motion; confined diffusion; diffusion and binding; intramolecular fluctuations Model application: Difference in diffusion small molecules generate short fluctuations... I (t ) larger complexes generate longer fluctuations... I (t ) t t G(t) ... and rapidly decaying correlation functions log t ... and slowly decaying correlation functions FCCS: Fluorescence cross correlation Extended concept: • labeling of potential binding partners with spectrally different fluorophores • register intensity fluctuations with two spectrally separated channels • looking for correlations (similarities) between the corresponding signals I(t) No correlation t I(t) Good correlation! t Distinguish bound from unbound state kas + kdis G(t) The higher the cross correlation amplitude in relation to the autocorrelation amplitudes, the higher the degree of binding. log t Calculation of crosscorrelation 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 2 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 2 2 2 1 1 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 x 0 0 1 1 2 2 2 1 1 0 0 0 0 0 1 1 1 1 0 0 0 0 Cross correlation - Model application: binding One partner free, one bound: no correlation G (t ) log t Cross correlation - Model application: binding Both partners bound: high correlation G (t ) log t Cross correlation - Model application: binding + kas kdis G (t ) log t Cross correlation - Model application: binding + kas kdis G (t ) log t Cross correlation - Model application: binding + kas kdis G (t ) log t FCCS - Estimation of binding constant (1) Reactants: Atto590-Biotin, Atto488-anti-Biotin-IgG Goals: Estimate bound fraction and Kd from crosscorrelation amplitude Conditions: – Ex: 488 nm, 594 nm – Em1: 500-550 nm – Em2: 607-683 nm – sampling rate: 1 MHz IgG structure by Gareth White 典型 FCS 应用 • • • • • • 浓度测量 扩散和不典型扩散 相位转变 – 磷脂双分子层 低聚反应 – 磷脂或穿膜蛋白的聚合 构象变化 – DNA 解链/成环 结合反应(in vivo 或者 in vitro) – 供体-受体反应 – 蛋白-DNA 反应 – 酶动力学 – 蛋白-蛋白 相互反应 FCS measurement in HeLa cell, Courtesy of Matthias Weiss and Jedrzej Szymanski, DKFZ FCS – Characterization of anomalous diffusion (2) Medium: Dextran polymer matrix of different molecular weight (i.e. molecular length) Probe: Labeled BSA Goal: characterize anomalous diffusion, sample serves as model system for anomalous diffusion observed in cellular compartments Result: Shorter polymers show larger degree of sub-diffusion, hence visco-elastic properties can be simulated FCCS - Estimation of binding constant (2) 活细胞 FCS 测量 优点： • • • • • • • 分子水平的灵敏度 工作在很低的浓度范围: nM 无需蛋白质的超表达 测量在生理条件下进行 无介入检测 量化的分析结果 高空间和时间分辨率 Measurement of EYFP in living cells (1) Sample: HeLa cells expressing pure EYFP which is expected to be freely mobile. Cells courtesy T. A. Knoch, K. Rippe, German Cancer Research Center and KIP, University of Heidelberg) FCS measurement spot Measurement of EYFP in living cells (2) 0.025 fit 0.02 0.015 0.01 0.005 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 lag time [sec] 0 1.00E-01 1.00E+00 autocorrelation function meas. Results: Autocorrelation function of free EYFP in HeLa cell nucleus: ~45 molecules in the focus, concentration of ~60 nM for focal size of 0.15 fl Diffusion correlation time ~500 sec, i.e., diffusion coefficient of ~30 m2/sec, i.e., viscosity ~3fold higher than in water Measurement of EYFP in living cells (3) Diffusion in the nucleus Molecules start at red spot: Covered area after x seconds 0.0 sec 0.1 sec 0.2 sec 0.3 sec 0.4 sec Concentration mapping in living cells Combination of FCS and APD imaging: calibration of the image by FCS APD image: structural information FCS measurements in different structures: Local concentration and diffusion behavior Data courtesy of Dr. Matthias Weiss, Jedrzej Szymanski and Nina Malchus Visualization of concentration on the whole image possible Proof of Caspase6 activity during apoptosis Method: Fluorescence Cross Correlation Spectroscopy (FCCS): FCS in 2 channels, Height of FCCS amplitude corresponds to the bound fraction Sample: Caspase6 dimerization in HeLa cells after induction of apoptosis, Courtesy Prof. Roland Eils, Dr. Joel Beaudouin Choice of dyes for covalent labeling • In binding assays: The smaller reactant must be labeled with an FCS suitable dye. • Criteria for suitable dyes: – High brightness. – High photostability. – Low triplet transition rate. – Amino- and/or thiol-reactive derivatives should be available. – Fluorescence lifetime within the lower ns-range (small against diffusion time) • Excitation wavelength criterion: availability of laser line. • Emission wavelength criterion: avoid range of autofluorescence. • Avoid non-specific binding of the dye to buffer components (as BSA, detergents, ...), and the interaction partners, especially the unlabelled partner. Hydrophobic dyes (as rhodamine) tend to bind to chamber surfaces, membranes and proteins. • Regard dependence of photochemical properties of some dyes on measurement conditions as pH, light intensity, …(like GFP, FITC depend on pH and light intensity). Prepare experiment: Choice of dyes List of FCS suitable dyes: Alexa dyes Cy dyes Rhodamin Green, 6G, B, Lissamin EvoBlue DY dyes TAMRA ROX TMR Resorufin Texas Red Atto dyes Molecular Probes Amersham Pharmazia Sigma, … Evotec Dyomics Choice of fluorescent proteins List of FCS suitable fluorescent proteins. - GFP - YFP - mRFP, mCherry - Cross correlation pair: GFP-mCherry Difficult: - CFP - DsRed, HsRed Hardly suitable: - BFP Choice of the right objective Necessary properties: 1. High numerical aperture 2. Water immersion (for some applications: glycerol objective) 3. Correction ring 1. Why is a high numerical aperture necessary ? • FCS is a single molecule detection technique. To capture as many photons as possible a high numerical aperture is essential. The is essential for a good signal to noise ratio. • High NA decreases confocal volume, therefore measurements at higher concentrations are possible. Ideal molecule detection function Molecule detection function (1/e2 isosurface) NA = 1.2 wd = 3 mm tubelens = 180 mm n0 = 1.33 ex = 635 nm = 4.9 mm focus pos. = 10 m em = 670 nm magn. = 60 pinhole radius = 50 m Kindly provided by Jörg Enderlein, Tübingen Towards FCS Measurements within 4 Steps Step 1: Adjust correction ring of the objective. Step 2: Find your sample using “normal” imaging mode. Acquire an image or image stack (xy z or xz y). Step 3: Optimize FCS measurement conditions. Step 4: Define FCS measurement points image or image stacks. Setup and start the FCS measurement time series. FCS向导 嵌套的FCS时间系列 (多点测量，每点时间系列，各点之间循环) xy z 系列中的 FCS 多点测量 xz y 系列中的 FCS 多点测量 在较弱染色的胞膜上进行精确定位: 在 xz 图像上定位FCS测量点 定义FCS 测量点 “on the fly” 进行长时间FCS数据采集时，可定义频率自动拍摄图片—保证对采集过程最大控 制 支持其他的应用方法: FCCS, FLCS, gated FCS, APD 成像 The SMD FCS Wizard: special properties Easy handling by intuitive user interface and user guidance through the experiment. Global control over the experiment within the SMD FCS wizard. Intelligent presets allowing fast results: automated configuration of filters wheels, coupling ports, detectors etc. Data handling made easy: User-definable experiment names are used in both, confocal software LASAF and FCS software SPT. All relevant instrument parameters and user comments are automatically transferred from LASAF to SPT and saved with the experiment file. The SMD FLIM Wizard: tight on-line control of data quality On-line count rate display in LASAF to prevent statistical artifacts (like APD saturation) On-line display of counts per molecule allowing permanent evaluation of optical performance On-line correlation curve display in SPT revealing data quality and correlation analysis already during experiment Count rate monitor in LASAF Welcome to Leica Science Lab http://www.leica-microsystems.com/science-lab/