INTRODUCTION TO FLUORESCENCE(two-photon, three steps, phase incoherent process)

•Einstein’s coefficients, dipole transitions.• Experimental setup•T-configuration•Jablonsky diagram•Franck-Condon principle, reflection rule and Stokes shift•Lifetime and quantum yield•Fluorescence quenching (collisional) •London’s theory of molecular forces

•RET resonant Energy Transfer•FRAP (Fluorescence Recovery after Photo Bleaching

Experimental setup

Gregorio Weber’s T-configuration

Stokes shift

Protein Intrinsic Fluorophores

stati di tripletto e di singoletto

Kasha’s rule: emission spectra are independent of the excitation wavelength.

Quantum yield Q and lifetime τ

Γ=spectral emissivity, knr rate of non radiative transitions

teoricamente

Fluorescence Quenching

Stern-Volmer equation

Collisional quenching

Fluorescence Anisotropy

anisotropy

polarization

What is lost in steady state measurements?

Green-fluorescent proteins GFP

Non radiative energy transfer (RET)

Center for fluorescence spectroscopy (J. Lakowicz) http://cfs.umbi.umd.edu/ISS http://www.iss.com/Fluorescence foundation http://www.fluorescence-foundation.org/Enrico Gratton’s homepage http://www.lfd.uci.edu/~gratton/Laboratory of fluorescence dynamicsGregorio Weber http://www.lfd.uci.edu/weber/

CORSO DI MICROSCOPIA OTTICA DI CLEGGhttp://online.physics.uiuc.edu/courses/phys598om/spring12/