Single molecule microscopy / spectroscopy

  Picture shows optic table Copyright: Fitter Typical view on the optical excitation light path of a fluorescence microscope.

In our lab we operate two fluorescence microscopes for single-molecule detection. In addition to a confocal microscope (MicroTime 200, PicoQuant) with pulsed excitation at 440, 485, 510 and 638 nm and simultaneous two color detection, we are running a widefield/TIRF microscope, which also allows us to detect two colors simultaneously at 488, 532, and 640 nm excitation. By employing single-photon counting (TCSPC) with sub nanosecond time resolution we make use of extensive multi-parameter fluorescence analysis. Major methods applied in our group are single molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), time resolved anisotropy decay (TRA), photo-induced electron transfer (PET), and two-color coincidence detection (TCCD). In this respect many of our activities are related to methodical developments. For example, a strong linkage between cell-free protein synthesis and applications of single-molecule fluorescence spectroscopy was established in our lab. We make use of this approach to produce tailor-made samples for single-molecule fluorescence spectroscopy and, on the other hand, employ specific in vitro assays to investigate aspects of the protein biosynthesis.

 

Publications

Katranidis A and Fitter J
Single-Molecule Techniques and Cell-Free Protein Synthesis: A Perfect Marriage
Anal. Chem., 91 (4), 2570–2576 (2019)

D. Kempe, M. Cerminara, S. Poblete, A. Schöne, M. Gabba, and J. Fitter
Single-molecule FRET measurements in additive-enriched aqueous solutions
Anal. Chem., 89, 694-702, (2017)

D. Kempe, A. Schöne, J. Fitter, and M. Gabba
Accurate Fluorescence Quantum Yield Determination by Fluorescence Correlation Spectroscopy
J. Phys. Chem. B, 119, 4668-4672, (2015)

J. Fitter, A. Katranidis, T. Rosenkranz, D. Atta, R. Schlesinger, G. Büldt
Single molecule fluorescence spectroscopy: A tool for protein studies approaching cellular environmental conditions .
Soft Matter, 7, 1254-1259, (2011)