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.


Related publications

Yukhnovets O., Höfig H., Bustorff N., Katranidis A. ,Fitter, J.
Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution
Biomolecules 12, 468 (2022)

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)