Methodical approaches

Methodical approaches and developments

In our lab we operate two fluorescence microscopes for single-molecule detection. In addition to a confocal microscope (MicroTime 200, PicoQuant) with pulsed excitation and simultaneous two color detection, we are running a widefield/TIRF microscope, which also allows us to detect two colors simultaneously. 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 brightness-gated two-color coincidence detection (BTCCD). 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. (2022) Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution. Biomolecules 18;12(3):468
  • Katranidis A. and Fitter J. (2019) Single-Molecule Techniques and Cell-Free Protein Synthesis: A Perfect Marriage. Anal. Chem., 91 (4), 2570–2576
  • Kempe D., Cerminara M., Poblete S., Schöne A., Gabba M., Fitter J. (2017) Single-molecule FRET measurements in additive-enriched aqueous solutions. Anal. Chem., 89, 694-702
  • Kempe D., Schöne A., Fitter J., Gabba M. (2015) Accurate Fluorescence Quantum Yield Determination by Fluorescence Correlation Spectroscopy. J. Phys. Chem. B, 119, 4668-4672
  • Höfig H., Gabba M., Poblete S., Kempe D., Fitter J. (2014) Inter-dye Distance Distributions Studied by a Combination of Single-Molecule FRET-Filtered Lifetime Measurements and a Weighted Accessible Volume (wAV) Algorithm. Molecules, 11, 19269-19291
Last Modified: 04.06.2022