Introduction to Fluorescence Jablonski Diagram
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1 ntroduction to Fluorescence Jablonski Diagram Excited Singlet Manifold S1 internal conversion S2 k -isc k isc Excited riplet Manifold 1 S0 k nr k k' f nr fluorescence k p phosphorescence Singlet round State ntersystem crossing a method for populating the triplet state nternal conversion Kasha rule riplet state phosphorescence; significantly longer lifetimes than fluorescence What is Fluorescence? defined as the decay from an excited singlet state of a fluorophore the result of bsorption(1) of a photon Jablonski Diagram leading to an excited singlet state, S 1 followed by a decay (2) from S 1 (timescale of nanoseconds; other processes can occur in this time) yielding emitted light of lower energy, i.e. redshifted (3) in wavelength (Stokes shift) the Stokes shift allows efficient discrimination of the excitation, making fluorescence a very sensitive technique lternative pathways for relaxation of excited molecule
2 Lifetimes of different processes
3 ritical Fluorescence Parameters bsorption spectroscopy o characterize the photoexcited emission from molecules in a system of unknown complexity, we should determine the spectral distribution, photon yield, lifetime of the excited state, and polarization of the fluorescence emission, as a function of the wavelength of emission. regorio Weber, Meth. Enzym. 278, p. 1 (1997) = 010 ε ( λ ) cl Spectral distribution Emission spectra: Fix the excitation wavelength and scan through emission wavlengths; usually independent of excitation wavelength Excitation spectra: Fix emission wavelength and scan through excitation spectra; usually same as absorption spectrum = 0 ransmittance pathlength l = log bsorbance Lambert-Beer = log 0 = log = ε ( λ )cl Spectral distribution Determination of spectral distribution Normalized Fluo. ntensity ryptophan EFP chromophore 500 nm det. 434 nm exc. Source Monochromator Sample Detector Spectrophotometer Fluorescence Excitation Fluorescence Emission Wavelength [nm] Wavelength, nm
4 Measuring Fluorescence Fluorescence Lifetime(s) Spectrofluorometer Source Monochromator Sample all competing processes affect the fluorescence lifetime Measured lifetime dn dt = N ( k f + k ic + k isc ) Fluorescence Excitation Fluorescence Emission Detector τ int N= Ne = 1 ( kf + kic+ kisc) t 0 k + k + k f ic isc Wavelength, nm Q = k f τ ritical Fluorescence Parameters Photon Yield/Quantum Efficiency a measure of the emission efficiency of the fluorophore Q = # of photons emitted # of photons absorbed Brightness Q = k proportional to ability to absorb light (extinction coefficient, ε) ND Quantum Yield, Q otal Fluorescence F = 0 ε[]lq c where 0 is incident light intensity, l is pathlength [c] is fluorophore concentration f k + k f ic + k isc Fluorescence Lifetime(s) Fluorescence lifetime (τ) is the characteristic time that the fluorophore spends in the excited state. τ = 1/( k + k + k During this time in the excited state, the fluorophore undergoes multiple interactions with the environment collisional quenching fluorescence energy transfer intersystem crossing rotational motion int f ic homogeneous system (fluorophore+uniform solvent) should, in principle, exhibit a single lifetime Heterogeneous systems (most real systems) such as cells typically show multiple lifetimes isc )
5 Fluorescence spectra of different amino acids fluorescence intensity (arb. unit) wavelength (nm) What is a fluorophore? Fluorophores any molecule that fluoresces is called a fluorophore typically polyaromatic hydrocarbons some amino-acids, in particular rp, yr and Phe Fluorophores ommon fluorophores exogenous fluorophores - dyes such as Fluorescein, Rhodamine, cridine Orange, Ethidium Bromide, y dyes endogenous fluorophores - NDH autofluorescence, e.g. FLUORESEN Molecular Formula: 20 H 12 O 5 Molecular Weight:
6 ommon Fluorophores Measuring Fluorescence Spectrofluorometer excitation and emission spectra usually based on diffraction gratings usually for bulk solutions (cuvette experiments) Fluorescence microscope spatially resolved fluorescence cellular samples, e.g. typically filter-based Some applications of fluorophores Measuring Fluorescence mmunofluorescence ion sensitive dyes -K +, Na +, a 2+ specific markers -ph indicators membrane potential increased intracellular fluorescence DN dyes determination of protein fluorescence
7 Measuring Fluorescence Filters emitted light Filters Selecting Filters Wild ype FP ypical Filter ube in a Microscope Fluorescence Excitation Fluorescence Emission Excitation light Wavelength, nm sample hroma echnology WFP Bandpass Filter Set
8 Light sources Fluorescence imaging Fluorescence in situ hibridization Denaturation hemically modified DNS-probes arget DNS chromosomes he sample is stained with F (fluorescein isothiocyanate) and Rhodamine-phalloidin to selectively highlight microtubules and actin filaments.
9 Photophysical onsequences of FRE D* * FRE introduces an additional deactivation pathway for the excited donor molecule Upon energy transfer Donor excited state (D*) lifetime decreases Donor fluorescence intensity decreases Donor photobleaching rate decreases cceptor fluorescence intensity (if the acceptor is fluorescent) increases Fluorescence Resonance Energy ransfer (FRE) D* Donor and acceptor far apart - No FRE Donor and acceptor close together - FRE non-radiative (electromagnetic) transfer from excited chromophore (donor) to receptor molecule (acceptor) by dipole-dipole coupling dynamic Förster transfer process strongly distance dependent, rate constant 1/R 6 powerful method for looking at molecule association, protein-protein interactions, receptor-ligand interactions D* * EFP/EYFP Normalized Fluo. ntensity FRE Pairs (FP) EFP, ex EFP, em EYFP, ex. EYFP, em Wavelength, nm
10 Photoselection of fluorophores Detecting FRE Spectral increase of acceptor fluorescence decrease of donor fluorescence Lifetime decrease of donor fluorescence lifetime Donor Photobleaching decrease in donor photobleaching rate in the presence of acceptor (FRE) cceptor Photobleaching create an area free of acceptor by photobleaching increase in donor fluorescence vertically polarized light selected population Distance dependence of FRE efficiency values Sample energy transfer efficiency p = VV VV + VH VH Source Monochromator r = VV VV + 2 VH VH Polarizer E = 6 R0 R + R Detector = HV HH donor-acceptor distance (*R 0 )
11 fluorophore macromolecule motion of fluorophore group motion of a whole molecule 1 r 1 k = 1 + τ r0 Vη ime distribution of first photon upon excitation photon events from detector number of photons time
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