Fluorescence-Specroscopy-朝阳科技大学荧光光谱朝阳科技大学-PPT资料41页课件.ppt

1、Part I. BackgroundS is singlet and T is triplet. The S0 state is the ground state and the subscript numbers identify individual states.n p* p p* n s* s p* s s*DS0 Energy Lifetime Quantum Yield PolarizationThe Stokes shift is the gap between the maximum of the first absorption band and the maximum of

2、 the fluorescence spectrumloss of vibrational energy in the excited state as heat by collision with solventheat: 7-amino-4-methylcoumarin (AMC)fluorophoresfluoresceinethidium bromide bound to DNA.Excited states decay exponentially with time I = I0e-t/t I0 is the initial intensity at time zero, I is

3、the intensity at some later time t t t is the lifetime of the excited state. kF = 1/ t t, where kF is the rate constant for fluorescence.Quantum YieldQuantum Yield = F FF FF = number of fluorescence quanta emitted divided by number of quanta absorbed to a singlet excited state FF = ratio of photons

4、emitted to photons absorbedQuantum yield is the ratio of photons emitted to photons absorbed by the system: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 so

5、urce. Degree of polarization is defined as PI| and I are the intensities of the observed parallel and perpendicular components, a 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. Steady-state measurements: F, I Time-Resolved

6、 measurements: t 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 ac

7、curate FF measurements.Inner Filter EffectIf ( em) = IAbs ( ex). f . f( em). KI0(ex)ememememmeasured intensity of fluorescence at emabsorbed intensity at exfluorescence quantum yieldfraction of intensity emitted at thatparticular wavelengthfraction of total fluorescencethat is detected)(0101)()(exAe

8、xexabsIIIf A0exexexabsIAI0.303. 2KIAKIIfexexemfexabsf.).().(.303. 2.).(0standardsamplestandardsamplestandardsample.ffexexemfemfAAIIIf we measure the sample and a standard under thesame experimental conditions, keeping ex constant:Important : the index of refraction of the two solvents(sample and sta

9、ndard) must be the sameStandards:Quinine sulfate in H2SO4 1N: f =0.55 Fluorescein in NaOH 0.1N: f =0.93The 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 ph

10、otons 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 flashesStart PMTStop PMTsampleexc. monoc

11、hromatoremission monochromatorpulsed sourceLifetimens AbsorptionFluorescenceWavelength nmAbsorptivity Wavelength nmQuantum Tryptophan2.6280 5,6003480.20Tyrosine3.62741,4003030.14Phenylalanine6.42572002820.04, the dominant intrinsic fluorophore, is generally present at about 1mol% in proteins. A prot

12、ein may possess just one or a few Trp residues, which facilitates interpretation of the spectral data. 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 anythi

13、ng 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 groups in the protein.fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr)IIIIIIIVV

14、: Tyrosine and its derivativesIIIIIIIIIIIVIVIIVVEmission spectra of Pseudomonas fluorescens azurin Pfl.For 275-nm excitation, a peak is observed due to the tyrosine residue(s) The position and structure of the fluorescence suggests that the indole residue is located in a completely nonpolar region o

15、f the protein. These results agree with X-ray studies, which show that the indole group is located in the hydrophobic core of the protein. In the presence of a denaturing agent, the TrpP emission loses its structure and shifts to 351nm, characteristic of a fully exposed Trp residue. Changes in emiss

16、ion spectra can be used to follow protein unfolding00.20.40.60.811.205101520Resolution of the contributions of individual tryptophan residues in multi-tryptophan proteins.I(,t)=ai()exp(-t/ti)it1=2ns, t2= 5ns020406080100120500550600650700750t1=2nst2=5nst (ns)Fluorescence intensity (A.U.)Fluorescence

17、intensity (A.U.)wavelength (nm) emIsolated from the Pacific jellyfish Aequorea victoria and now plays central roles in biochemistry and cell biology due to its widespread use as an in vivo reporter of gene expression, cell lineage, protein - protein interactions and protein trafficking One of the mo

18、st important attributes of GFP which makes it so useful in the life sciences is that the luminescent chromophore is formed in vivo, and can thus generate a labeled cellular macromolecule without the difficulties of labeling with exogenous agents.The structure of GFP : eleven-strand beta-barrel wrapp

19、ed around a central alpha-helix core. This central core contains the chromophore which is spontaneously formed from a chemical reaction involving residues Ser 65, Tyr 66, and Gly 67 (SYG)There is cyclization of the polypeptide backbone between Ser 65 and Gly 67 to form a 5-membered ring, followed by

20、 oxidation of Tyr 66.The high quantum yield of GFP fluorescence probably arises from the nearly complete protection of the fluorophore from quenching water or oxygen molecules by burial within the beta-barrel. Ribbon diagram of the Green Fluorescent Protein (GFP) drawn from the wild-type crystal str

21、ucture. The buried chromophore, which is responsible for GFPs luminescence, is shown in full atomic detail. Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively. Its emission peak

22、 is at 509 nm in the lower green portion of the visible spectrum. For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect is prominent with irradiation of GFP by UV light. In a wide range of pH, increasing pH leads to a reduction in fluorescence by 395 nm excitation and an increased sensitivity to 475 nm excitation. Melittin GIGAVLKVLT TGLPALISWI KRKRQQX Example Biochemical Education 28 (2000) 171173Example Example Thank you