按一下以編輯母片標題樣式,按一下以編輯母片,第二層,第三層,第四層,第五層,*,此处编辑母版单击标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Fluorescence Spectroscopy,Part I.Background,Fluorescence SpectroscopyPart,1,Perrin-Jablonski diagram,Perrin-Jablonski diagram,2,S is singlet and T is triplet.,The S,0,state is the ground state and the subscript numbers identify individual states.,S is singlet and T is triplet.,3,n,p,*,p,p,*,n,s,*,s,p,*,s,s,*,Energy level of MO,n p*p p*n s*s,4,D,S,0,Singlet&Triplet,DS0Singlet&Triplet,5,Characteristics of Excited States,Energy,Lifetime,Quantum Yield,Polarization,Characteristics of Excited Sta,6,Stokes shift,The,Stokes shift,is the gap between the maximum of the first absorption band and the maximum of the fluorescence spectrum,loss of vibrational energy in the excited state as heat by collision with solvent,heat,Stokes shiftThe Stokes shift i,7,Example,:7-amino-4-methylcoumarin(AMC),Example:7-amino-4-methylcouma,8,Example,Example,9,Example,fluorophores,fluorescein,ethidium bromide,bound to DNA.,Example fluorophoresfluoresce,10,Fluorescence-Specroscopy-朝阳科技大学荧光光谱朝阳科技大学-资料课件,11,Lifetime,Lifetime,12,Lifetime,Excited states decay exponentially with time,I=I,0,e,-t/,t,I,0,is the initial intensity at time zero,I,is the intensity at some later time t,t,is the lifetime of the excited state.,k,F,=1/,t,where k,F,is the rate constant for fluorescence.,LifetimeExcited states decay e,13,Quantum Yield,Quantum Yield=,F,F,F,F,=number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state,F,F,=ratio of photons emitted to photons absorbed,Quantum yield is the ratio of photons emitted to photons absorbed by the system:,Quantum YieldQuantum Yield=F,14,Quantum Yield,Quantum Yield,15,Quantum Yield&Structure rigidity,Quantum Yield&Structure rigi,16,Polarization,Molecule of interest is randomly oriented in a rigid matrix(organic solvent at low temperature or room temperature polymer).And plane polarized light is used as the excitation source.,Degree of polarization is defined as,P,I,|,and,I,are the intensities of the observed parallel and perpendicular components,a,is the angle between thee mission and absorption transition moments.,If,a,is 0 than P=+1/2,and if,a,is 90 than P=-1/3,.,PolarizationMolecule of intere,17,Steady-state measurements:,F,I,Time-Resolved measurements:,t,Experimental Measurements,Steady-state measurements:F,18,Instruments,Instruments,19,Inner Filter Effect,At low concentration the emission of light is uniform from the front to the back of sample cuvette.,At high concentration more light is emitted from the front than theback.,Since emitted light only from the middle of the cuvette is detected the concentration must be low to assure accurate,F,F,measurements.,Inner Filter Effect At low con,20,Inner Filter Effect,Inner Filter Effect,21,I,f,(em),=I,Abs(,ex),.,f,.,f,(em),.,K,I,0,(,ex,),em,em,em,em,measured intensity of,fluorescence at,em,absorbed intensity at,ex,fluorescence quantum yield,fraction of intensity emitted at that,particular wavelength,fraction of total fluorescence,that is detected,If A,0,If we measure the sample and a standard under the,same experimental conditions,keeping,ex constant:,Important:the index of refraction of the two solvents,(sample and standard)must be the same,Standards:,Quinine sulfate in H,2,SO,4,1N:,f,=0.55,Fluorescein in NaOH,0.1N:,f,=0.93,Measurement of fluorescence quantum yields,If(em)=IAbs(ex).f.f(,22,The TCSPC measurement relies on the concept that the probability distribution for emission of a single photon after an excitation yields the actual intensity against time distribution of all the photons emitted as a result of the excitation.By sampling the single photon emission after a large number of excitation flashes,the experiment constructs this probability distribution.,Time correlated single photon counting:,#events,.,.,.,.,t(nsec),different excitation flashes,Start PMT,Stop PMT,sample,exc.monochromator,emission monochromator,pulsed source,t,Measurement of fluorescence lifetimes,The TCSPC measurement relies o,23,Lifetime,ns,Absorption,Fluorescence,Wavelength,nm,Absorptivity,Wavelength,nm,Quantum,Tryptophan,2.6,280,5,600,348,0.20,Tyrosine,3.6,274,1,400,303,0.14,Phenylalanine,6.4,257,200,282,0.04,Intrinsic Fluorescence of Proteins and Peptides,LifetimeAbsorptionFluorescence,24,Fluorescence-Specroscopy-朝阳科技大学荧光光谱朝阳科技大学-资料课件,25,Fluorescence-Specroscopy-朝阳科技大学荧光光谱朝阳科技大学-资料课件,26,Tryptophan,the dominant intrinsic fluorophore,is generally present at about 1mol%in proteins.A protein may possess just one or a few Trp residues,which facilitates interpretation of the spectral data.,Tryptophan,is very sensitive to its local environment.It is possible to see changes in emission spectra in response to conformational changes,subunit association,substrate binding,denaturation,and anything that affects the local environment surronding the indole ring.Also,Trp appears to be uniquely sensitive to collisional quenching,either by externally added quenchers,or by nearby gro