Modern Approaches to Time-Resolved Single Molecule Microscopy

DIPC Seminars

Dr. Steffen Ruettinger, PicoQuant GmbH, Berlin
Donostia International Physics Center (DIPC).Paseo Manuel de Lardizabal, 4 (nearby the Facultad de Quimica), Donostia
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Modern Approaches to Time-Resolved Single Molecule Microscopy Modern Approaches to Time-Resolved Single Molecule Microscopy PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, Modern time-resolved measurements permit to follow fluorescence dynamics from the sub-nanosecond range up to fluctuations in the second range and even beyond. The underlying data acquisition technique (Time-Tagged Time-Resolved (TTTR) recording) allows to store individually timing as well as spectral and polarization information for every photon, based on a multichannel detection. Having all available information with picosecond time resolution at hand, the recorded data can be analyzed in a manifold manner. Fluorescence lifetime histograms are available from confocal images (FLIM) as well as from diffusing samples (FRET) or from single molecules (SMD). By exploiting the full information content of such a multi-dimensional measurement, classical intensity based analysis schemes like FCS and FRET can be significantly improved by sorting and weighting the detected photons. This approach is for example used in Fluorescence Lifetime Correlation Spectroscopy (FLCS), which allows to study diffusion properties of different species which just differ in their fluorescence lifetime without the need for multicolor labeling. Advanced excitation schemes yield access to absolute diffusion coefficients by Dual Focus FCS (2fFCS) or more accurate FRET measurements utilizing Pulsed Interleaved Excitation (PIE-FRET). The single photon data format enables also the easy incorporation of spatial information, generated by point scanning imaging devices. The whole TTTR data acquisition can be operated in tethered mode and integrated into home-built or commercial imaging devices. Another possibility is the combination of a sample scanning Atomic Force Microscope (AFM) with a single-molecule-sensitive confocal fluorescence microscope in order to record fluorescence and topographic information simultaneously. This enables force studies on a single particle whilst simultaneously monitoring its response using fluorescent probes.