现代分子荧光检测技术及应用-稳态及荧光寿命测试的解决方案

现代分子荧光检测技术及应用稳态及荧光寿命测试的解决方案,邱海林 博士天美（中国）科学仪器有限公司,Who is who?,邱海林工学博士，高级工程师天美(中国）科学仪器有限公司市场部分子荧光产品专家联系方式,天美（中国）科学仪器有限公司是国内最大的分析仪器,生命科学设备和实验室仪器的制造和销售商.公司的总部设在香港,工厂在上海和天津,在中国共有14家办事处和新加坡、印度、澳大利亚分公司.Hitachi 分子光谱产品亚洲地区总经销Jobin Yvon稳态及瞬态荧光产品大陆地区总经销,分子荧光产品“超市”,需要您关注的内容,功能,稳态：激发谱、发射谱、三维谱、同步谱；波长范围（紫外可见近红外200nm - 850，1550，2600nm and longer）变温测量；气氛测量快速反应动力学；自动滴定光致发光量子产率及发光色度分析,性能,瞬态荧光：荧光寿命、磷光寿命样品形态：固体粉末，液体，散光样品，薄膜及特殊样品在线、微区测量（光纤附件）灵敏度及配置，分辨率样品稳定性,选择荧光光谱仪的时候，会考虑什么？激发波长范围发射波长范围是否要兼顾稳态和瞬态？寿命衰减范围灵敏度数据采集时间样品特性满足功能附件选择维护要求预算及执行时间,1819: Creation of the Company, in Paris, by Jean-Baptiste Soleil1923: The company becomes A. Jobin & G. Yvon1997: HORIBA acquired Jobin Yvon2004: “ One Company Project ” , Jobin Yvon became,R&D and Manufacturing Center,Chilly-Mazarin,Villeneuve dAscq,Longjumeau : Headquarters,Edison, New Jersey,Glasgow, Scotland,Other Facilities,Stanmore, England,Munich, Germany,Milano, Italy,Bensheim, Germany,Facilities,China : Beijing and ShanghaiJapan : Kyoto and TokyoKorea : Seoul,Shared office and operations with HORIBA,Core Technology : Diffraction Gratings,Raman,Fluorescence,Ellipsometry,Gratings,I.C.P,Spectrometers,Detectors,6 Main products lineDistribution in 70 Countries,HORIBA Jobin Yvon凝聚 SPEX, IBH, SLM-Aminco顶级品牌在荧光研究领域的精华，提供卓越性能的荧光分光光度计，用于稳态和瞬态荧光测量。唯一同时拥有时间分辨测定和相分辨测定瞬态荧光分光光度计产品供应商。产品包括：紧凑型的FluoroMax系列；模块化的Fluorolog系列光谱仪，或者，你需要搭建自己的荧光系统，无论常量试样或者显微成像技术，都能得到特殊的满足。,The Various types of luminescence,Fluorescence,First observed from quinine by Sir John Frederick William Herschel in 1845, blue fluorescence 450nm.,Blue glass Filter 400 nm,G.G. Stoke,1853 (G.G. Stokes) Introduction of the term of “fluorescence”1858 (E. Becquerel) First phosphoroscope1867 (F. Goppelsrder) First fluorometric analysis (determination of Al(III) by the fluorescence of its morin chelate)1888 (E. Wiedemann) Introduction of the term “luminescence”1926 (F. Perrin) Theory of fluorescence polarization (sphere). Perrins equation. Indirect determination of lifetimes in solution1926 (Gaviola) First instrument for the determination of lifetime (phase fluorometer operating at a single frequency)1935 (A. Jablonski) Jablonskis diagram1960 First Jobin Yvon Spectrofluorometer 2003 IBH joined Jobin Yvon,Brief History,Phosphoroscope from Becquerel,Many materials emits fluorescenceLiquids, solids, gasOrganic compounds:aromatic hydrocarbons(anthracene, perylene,naphthalene), fluorescein,rhodamines, aminoacids(tryptophan, tyrosine), etc.Inorganic compounds: lanthanide ions (Eu3+, Tb3+), doped glasses (e.g. with Nd, Mn, Ce , Sn, Cu, Ag), crystals (ZnS, CdS, ZnSe, GaS), etc.Organometallic compounds: ruthenium complexes, complexes with lanthanide ions, complexeswith fluorogenic chelating agents, etc.,Fluorophore Samples,Ground StateElectrons,S1 excited state,S2 excited state,Absorbance energy,Fluorescence,Absorption,Nonradiative dissipation,Blue Excitation,Internal Conversion,Ground StateElectrons,S1,S2,Absorbance energy,Phosphorescence,Absorption,Phosphorescence,T1,IntersystemCrossing,Triplet state (T,1,),Singlet state (S,1,),Singlet state (S,2,),Absorption of light,Fluorescence emission,Phosphorescence,Intersystem crossing =,Ground state (S,0,),CHARACTERISTIC TIMESabsorption10-15 svibrational relaxation10-12-10-10 slifetime of the excited state S110-10-10-7 sintersystem crossing10-10-10-8 sinternal conversion10-11-10-9 slifetime of the excited state T110-6-1 s,Fluorescence:from the ps to the s scalePhosphorescence:from the s to the s scale,Jablonski Diagram Fluorescence & Phosphorescence Pathways,Fluorescence Spectra of Perylene,Typical aromatic molecules,Typical aromatic molecules,Different excitation wavelengthDifferent emission wavelengthIn the UV and visibleLong or narrow stokes shift,Emission and excitation spectraQuantum yield: FLifetime: tAnisotropy and polarization: r and PQuenching of fluorescenceFRET,Basic Definitions,Main Applications,Molecular Distances: Fluorescence Resonance Energy Transfer (FRET)Intrinsic Tryptophan: Quenching, Anisotropy, pHLigand Binding: Anisotropy, FRET, QuenchingGene regulation/Cell Imaging: Luminescent Molecular Markers, GFPs, LuciferaseBiosensing: Conjugation with Dyes, Nanoparticles,Efluo labs,lphos lfluoEnergy of the lowest vibrationallevel of the triplet state T1 is lower than that of the singlet state S1,Perrin-Jablonski diagram,Effect of Optical Density on Fluorescence Intensity,Inner Filter Effect, Fluorophores OD 0.1 to avoid the inner filter effect,Quantum Yield: , = = kr : rate constant for radiative desactivationknr : rate constant for non radiative desactivation,Fluorescence Lifetimes: ,Fluorescence lifetime is the average time that a molecule remains in the excited state : ps-ns.emission is a statistical processPhosphorescence involves intersystem crossing: singlet-tripletQuantum mechanically forbiddenPhos lifetimes ms to seconds,Photoselection: under polarized excitation, fluorophores whose transition moment are oriented in a direction close to that of the electric vector of the incident beam are preferentially excitedEmitted fluorescence is anisotropicAny change in direction of the transition moment during the lifetime of the excited state will cause the anisotropy to decrease,Anisotropy and Polarization,Polarized emission with polarized excited lightP =r =P = ; r =,I - I ,I + I ,I - I ,I + 2I ,3r,2 + r,2P,3- P,x,z,y,Photoselection,Anisotropy and Polarization,Anisotropy - measure polarized emissionUses polarizers in excitation and emission pathsMeasure vertical (V) and horizontal (H) intensitiesCalculate from these intensity measurements,Anisotropy Measurements in Steady State,Anisotropy: steady state,V,H,V,H,IVV,IVH,IHH,IHV,Anisotropyr=Grating FactorG=,IVV,IVH,-,+2,G,G,x,x,Information obtained with the fluorescencedepolarization: On molecular mobility, size and shape Flexibility of the molecule Fluidity of the medium Fluorescence lifetime,Anisotropy and Polarization,Anisotropy observation versus sample temperature,Monomers: SmallRapid rotationUnhinderedLow anisotropyDepolarized,Dimers: LargerSlow rotationHindered by ViscosityHigh anisotropyPolarized,Quenching: Any interaction decreases the intensity of the fluorescence emission Collisional quenching dynamic quencher reacts with molecules in excited state shorter lifetimes; involves O2, NO, Cl- Static quenching Quencher reacts with molecules in the ground state no changes in lifetimes; N2, heavy metals,Quenching of Fluorescence,Fluorescence Resonance Energy Transfer (FRET)Occurs when the emission of a donor (D) overlaps with the absorption of an acceptor (A).Dipole Dipole interactionEnergy transfer depends on distance between D and Aand the spectral overlapE = ; D ARo Forster distance = 15-70 , 50% transfer efficiency,Ro6,Ro6 + r6,r,FRET,r = 23.6,Mellitin is a rigid a-helix with an intrinsic tryptophan,tryptophan,dansyl,Dansyl label with fixed distance from tryptophan,Trp=DAbs,Trp=DFlu,Dan=AAbs,Dan=AFlu,dipole-dipoleinduction,radiationlesstransfer,Frster Overlap Integral D(Flu) and A(Abs),Frster Resonance Transfer Mechanism,kT(r)=,QD,2,tD,r6,FD(),A(),4,d,QD :Quantum yield of donor in absence of acceptortD :Lifetime of donor in absence of acceptor2 :Dipole orientation factor = 2/3 with random orientationsr6 :Distance dependence of transferN:Avogadros numbern:Refractive index of solventOverlap integral:FD() :Corrected fluorescence intensity of donorA() :Extinction coefficient of acceptor,Rate constant of transfer as a function of r,Frster Resonance Transfer Equation: Quantum Mechanical Version,N,n4,What is a Fluorescence Lifetime?,Population of Molecules Excited With Instantaneous Flash,Random Decay Back to Ground State:Each Molecule Emits 1 Photon,Why measure lifetimes?,Absolute quantities- not merely ratios or time-averaged intensitiesLargely independent of sample concentration and absorbance cross-section-in contrast to- steady stateDynamic information-rotation-correlation times, collisional quenching rates and energy transfer processesAdditional dimensions for fluorescence data increased specificity, sensitivity and selectivityLifetime senses local molecular environment (e.g. polarity, pH, temperature, electrostatics etc),Time-resolved FluorometryMain Instrumentations,Systems with streak cameraProvides the best time resolution: a few picoseconds scale Very sophisticated and expensive Phase-modulation fluorometersProvide good time resolution : a few tens of picoseconds Reasonable price Single-photon timing fluorometersProvide good time resolution : a few tens of picoseconds Reasonable price Stroboscopic instrumentsLower time resolution : a few hundred of picoseconds Unexpensive devices ,- Time resolution +,The Pulse Fluorometry: “Time Domain” techniqueUses short exciting pulse of lightGives the d-pulse response of the sample convoluated by the instrumental responseThe Phase-Modulation Fluorometry:”Frequency Domain” techniqueUses modulated light at variable frequencyGives the harmonic response of the sample that is the Fourier transform of the d-pulse response,Time-resolved FluorometryTwo Time-resolved techniques,Theoretically equivalent techniques but principles and instrumentation are different,Excitation by a sinusoidally modulated light at high frequencySinusoidally modulated fluorescence emission at the same frequencyBut delayed in phase: FAnd partially demodulated: M=m/mo,mo=B/A,Excitation,Response,A+B.cos(wt),a+b.cos(wt-F),Convolution product of the d-pulse response,intensity,phase shift F,A,B,b,a,m=b/a,Tan F = w.tM =1/(1+ w2.t2)1/2,Phase Fluorometry Principle,f,Frequency Domain Transform Principle,Variation of F and M as a function of frequencyI(t) = f( N, ai, ti ),No deconvolution,Curve fitting(variable: freq.),Optimum frequency: such as wt=1, i.e. f=1/2Pt10ps16GHz, 1ns160MHz, 100ns1,6MHz,Phase Fluorometry Principle,FluoroLog-Tau3: Multifrequency Cross-Correlation Fluorometer,sample turret,R928PPMT,FFT: Fast Fourier Transform,MHz,f,M,sample,reference,filter,Spectracq,450Wcw xenon,Pockels Cell,amp,MASTER Rf,SLAVE Rf + Df,amp,Rf+Df,Rf,Df=cross correlation frequency,X,GFP: the Green Fluorescence Proteinbiologically interesting naturally fluorescent,Frequency Domain (Phase and Modulation) Advantages: Free from convolution Short, 10ps, lifetime resolved with simple apparatus Use any CW sourceXenon or Hg, Hg(Xe) lampsRelatively low cost laser (HeCd or Ar+ are common)Fast data collection, especially for anisotropy decayEASY TO USE,FL3-TAU: Methodology of Fluorescence Lifetime,Excitation,Response,d(t),I(t),For a Dirac excitation d(t):,d(t): Dirac functionI(t) = exp(-t/t) for a single component,Excitation,Response,P(t),R(t),For any excitation function P(t):,R(t) = P(t) I(t) : convolution function,d-4,d-3,d-2,d-1,d 0,convolveddecay,Pulse Fluorometry Principle,time,measured,R(t)P(t),Deconvolution,P(t),I(t) = f( N, ai, ti ),R(t) = P(t) I(t),Excitation by short pulse of lightpulse duration with respect to time range of the fluorescence lifetimeFluorescence decay is recorded as a function of the time,Curve fitting,Pulse Fluorometry Principle,PRINCIPLE:Based on the fact that the probability of detecting a single photon at time t after an exciting pulse is proportional to the fluorescence intensity at that timeFluorescence decay is reconstructed after timing and recording the single photon following a large number of existing pulse,The time-correlated single-photon counting (TCSPC) method,MCA,S,CFD,SYNC,IBH FluoroCube,TAC rate 1MHzCoaxial Delay 50 NsSync delay 20 ns,TBX-04,nanoled, 2%,statisticalsingle photon events,periodic pulses,Cumulativehistogram,TAC,V,TCSPC Instrument Principle,Continuous xenon lamp for phase fluorometryThe largest emission spectra (250-1000nm)No maintenance Pulsed lamp for TCSPC devices :Spectral range limited to 200-400nmMany peaksRequires gas filling and maintenance (electrodes)Short life timeNon reproducible pulse widthLow intensityA large number of NanoLEDs simulate the continuumNo maintenanceReproducible pulse widthLong life time,Excitation source for time resolved,Spectra of gases,Flash lampIn air or filled with N2, H2 or D2Excitation wavelengths: restricted to200-400 nmNanosecond pulses one to a few hundreds of ps decay timesLow repetition rate (104-105 Hz)Collection period may be quite long (a few tens of minutes to hours)Lamp drift,Conventional TCSPC instrumentExcitation source,Ideal solution is a TCSPC instrument with laser diode or LEDNanoLEDs series :Cover a large wavelength rangeEasy to handleSeveral NanoLEDs can be used on the same instrumentCheap solutionEasy to upgrade,Excitation wavel. range Single wavelength above 280 nm,t = 1.32 ns (chisq = 1.1),Validation on Popop Sample,TCSPC instrumentsThe most economical solution in the nanosecond range (pulsed lamp or LED used)Typical lifetime range : from 100-200ps to a few tens of sFor less than 50psNeeds shorter pulses: mode-locked laserNeeds faster detector (MCP) expensive alternativePhase instrumentsBetter time resolutionTypical lifetime range : from a few tens of ps to a few s,Lifetime range,Pulse fluorometry:TCSPC is characterized by an outstanding sensitivityLow level of light longer acquisition timesPhase fluorometry:Fluorescence intensity must be high enoughTo get an analog signal high enoughTo allow F and M determination,Comparison between pulse and phase fluorometries: Sensitivity,Comparison between pulse and phase fluorometries: Analyses,Pulse fluorometry: direct visualization of the Fluorescence decayPhase fluorometry: analyses of the F and M variations requires more experiences: no direct visualization of the fluorescence decay. But, with experience, lifetime can also be visually estimated,Deconvolution:Pulse fluorometry: Is necessary Requires great care in recording the instrumental response Introduce small error in the lifetime estimationPhase fluorometry: Is not requiredData Analyses:Pulse fluorometry: well defined statistics technique: advantage for data analysesPhase fluorometry: evaluation of the std. dev.of F and M may not be easy,Comparison between pulse and phase fluorometries:,Comparison between pulse and phase fluorometries:,Time-resolved spectra:Evolution of the fluorescence spectra duringthe lifetime of the excited state:Easily recorded in pulse fluorometry (more direct):Lifetime based decomposition of spectra: for multi-components samplesDecomposition of an overall spectrum (overlapping spectra) into its componentsDecomposition is easier with phase fluorometry (faster),= BP = A . (106 cos)/ K x n x LB (Where A is slit width),The bandpass is the width of the spectrum passed by a monochromator when illuminated by a light source with a continuous spectrum. Reducing the width of the slits will decrease the bandpass until a limiting bandpass is reached. The limiting bandpass is called the resolution of the instrument. It is usually obtained around 10 m slit.,Bandpass and resolution,WWW. qiuhailin 2007 年 09年,灵敏度意味着什么,1、可以测量其他仪器无法测试的样品2、更高的准确度3、节约测试时间,Sensitivity: 更小体积的样品得到其他低灵敏度仪器无法获取的低信号；处理更低浓度的样品；用户获得的小体积样品测量；减少昂贵样品的使用； Sensitivity: 更好的光谱分辨率不需要大的带宽；获取更好的信息；更好区分相邻峰。,为什么需要高灵敏度 ?,准确度对应灵敏度（信噪比）的关系,计数光子要求达到1的准确度，至少需要10,000个光子高斯统计，第一标准偏差等于测量数的平方根同样测试条件下，双倍的信噪比会有双倍的准确度！,测量时间对应灵敏度（信噪比）的关系,JY 如何获得高的灵敏度？,光子计数电子；全反射光路，不是透镜为基础的折射光学；平面光栅，不是凹面光栅；全自动操作部件；高质量光学元件；等于： 准确度高，测量速度快，灵敏度高的仪器，值得拥有。,如何表示仪器灵敏度 Sensitivity,1、检测限；(ANSI/ASTM E579-76)The SPEX FLUOROLOGspectrofluorometer is capable of detecting sub-picomolar ( 1012 M) fluorescein.Minimum Detectable Concentration(MDC)ANSI，美国国家标准学会ASTM, 美国材料测试协会计算结果为10 fM荧光素2、水拉曼峰信噪比；Water Raman 水拉曼位移 3380cm1,水的拉曼峰,全波段的高信噪比,Signal-to-noise Sensitivity ratio (S/N):Water Raman emissionEx: 350nm Em:397nm5nm bp Ex & EMInt. time: 1sEstimation:Ip: peak int. (397nm)Ib: base line int. (450nm),HJY荧光光谱仪实际的S/N测量及计算FL3-11 作为检测仪器,测试条件：激发： 350 nm with 5 nm bandpass 发射：360 - 450 nm with 5nm bandpass 采样间隔 1nm, 积分时间 1s，无平滑数据处理 检测器：实际使用的室温红敏R928P,实际测得的数据: 峰信号 (at 397nm) = 501,500 cps 背景信号 (at 450 nm) = 10,500 cps 背景噪声Noise (p-p) (at 450 nm) = 223 (动态扫描测量), 背景信号RMS噪声223/5 = 44.6,采用 HORIBA Jobin Yvon 的 water Raman 信噪比公式，S/N (501500-10500)/ (10500) = 4,791 采用RMS信噪比计算结果water Raman S/N (501500-10500)/ 44.6 = 11,008,对比EDI, PTI, HJY是唯一明确信噪比测试方法和计算方法的仪器公司,HJY 信噪比方法的科学性,第二种方法只是考虑了检测器噪声和电子的起伏噪声；HORIBA Jobin Yvon 采用背景总强度作为噪声测量，虽然得到了较低的数值，但是，这样做更能代表试验的真实情况比如由于系统的散射光、光学部件的质量都是噪声的产生因素，特别在低的信号强度样品测量时候会有很大的影响，不能够被排除。水拉曼信噪比对比：1、同样的测试条件，同样的计算公式；2、全波段比较；HJY FM4 3000:1; FL3-11 4000:1; FL3-21 5000:1; FL3-22 6000:1EDI 6000:1(无论检测器是否更换)PTI 10000:1 或更高，向用户承诺：保证验收 混淆概念确认验收条件！波长要求！,检测器是获得全波段高信噪比的保证,1、R928P PMT：185900nm (HJY 标准配置)2、R1527P PMT : 185-680nm（EDI、 PTI标准配置）,标准配置情况下得到的信噪比数值不能在改变配置后使用！我们采用同样功率的灯源，采用高噪声的光电倍增管得到更高的信噪比：我们有更为高级质量的光学系统！来自JY的高质量光学元件！,检测器范围 1,标配检测器是获取信噪比的依据，EDI 和PTI采用的是185680nm的R1527P 标配PMT获取的信噪比。,UV-Vis & NIR Detectors,HJY FL3 独有NanoLog 型号配置用阵列检测器；有实验室验证及众多科学论文证明合理的配置。快速红外荧光3D M