MFD: Multiparameter fluorescence detection

Fluorescence Spectroscopic Toolbox

To determine structure and dynamics of biomolecules we use a set of Fluorescence Spectroscopic tools such as Multiparameter Fluorescence Detection (MFD), Time-Correlated Single-Photon Counting (TCSPC) at ensemble and single molecule conditions, fluctuation analysis (FRET-FCS, fFCS) and FRET-restrained positioning and screening (FPS).

MFD takes advantage on the fact that fluorescence information has at least eight dimensions: excitation spectrum, fluorescence spectrum, anisotropy, fluorescence lifetime, fluorescence quantum yield, macroscopic time and the fluorescence intensities influenced by the stoichiometry and distance between fluorophores.

Single molecule MFD

FRET-restrained positioning and screening (FPS)

FPS is used to generate structural 3D models of biomolecules based on FRET restraints. FPS follows a flow diagram and comprises three steps: (i) design of the experiment (shaded in red), (ii) measurement and analysis of the samples set (shaded in yellow) and (iii) generation and validation of structural models (shaded in green).

MFD uses pulsed excitation and Time-Correlated Single-Photon Counting (TCSPC) to simultaneously monitor the evolution of fluorescence in real time and for selection of specific single-molecule events for further analysis. MFD experiments of freely diffusing single molecules are analyzed by the FRET indicator FD/FA (ratio of donor over acceptor fluorescence) accumulated during single burst events. Unimodal distributions occur (Orange island) when dynamic conformational mixing is faster than the burst duration and follow the dashed green line.

 

Experimental Molecular Dynamics

We use a set of fluorescence tools to capture and monitor the time evolution of fluorescence indicators over time with picosecond resolution. We cover most of the biologically relevant dynamics.