Syllabus
1) Introduction into fluorescence: Jablonski diagram; absorption and emission spectra; fluorescentprobes; kinetics of fluorescence deexcitation; fluorescence decay; average fluorescence lifetime;quantum yield of fluorescence; fluorescence quenching;2) Detection of individual molecules: principal differences between a classical fluorescencemeasurements from a large ensemble of molecules and a single molecule fluorescence measurement;photophysics of individual molecules; methods of fluorescence detection; confocal versus wide-filedmicroscope; total internal reflection fluorescence (TIRF); detection of different protein configurationsby sm-FRET; rotations and reorientations of individual molecules;
3) Single particle tracking (SPT): diffusion in two dimensions; random walk; MSD diagrams; differentmodes of diffusion: free, hindered and hop-diffusion. Methods of SPT measurement; DerivedTechniques: brightness analysis and TOCCSL (thinning out clusters while conserving stoichiometryof labeling); colocalization analysis4) Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) I: theory of FCS:autocorrelation and cross-correlation functions; translational diffusion in FCS; inter-system crossingin FCS;5) FCS and FCCS II: FCS in two dimensions: applications to lipid membranes; FLCS technique;practical applications of FCS: reaction kinetics, protein binding to the membrane, clustering ofproteins on the membrane;6) PCH - photon counting histogram: principles and applications in biophysics; shot noise; Number andbrightness method (N&B); fluorescence anti-bunching;7) Fluorescence depolarization: definition of anisotropy; measurement of anisotropy; excitation andemission anisotropic spectra; causes of fluorescence depolarization; kinetics of fluorescencedepolarization; rotational diffusion and its impact on fluorescent anisotropy;8) Förster resonance energy transfer (hetero-FRET): FRET within one donor-acceptor pair; migration ofenergy between two donors (homo-FRET); kinetics of fluorescence deexcitation and depolarization;single molecule FRET9) Förster resonance energy transfer and migration in the field of many donors and acceptors:fluorescence deexcitation kinetics in the field of many acceptors, FRET on lipid bilayer,determination of the thickness of a lipid bilayer; MC-FRET; detection of lipid nanodomains;oligomerization of proteins on the membrane - quantification using homo- and hetero-FRET10) Raster Image Correlation Spectroscopy (RICS) and Imaging-FCS: Principles and practicalapplications
Annotation
Single molecule fluorescence spectroscopy has recently experienced unprecedented rapid development and has become one of the indispensable methods in biophysics. The aim of this course is to make students familiar with this field.
Emphasis is placed on understanding the physico-chemical principles on which these methods are based. The usefulness of these fluorescence techniques is demonstrated on many practical examples from the field of biophysics.
