BPCPPDYSOE21 – scientific programme

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BP: Fachverband Biologische Physik

BP 11: Poster A: Single Molecule, Multicellular, Bioimaging, Focus Sessions, etc.

BP 11.33: Poster

Monday, March 22, 2021, 16:30–19:00, BPp

Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection — •Valentin Dunsing, Annett Petrich, and Salvatore Chiantia — Universität Potsdam, Potsdam, Deutschland

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy is a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about inter-molecular interactions, but is conventionally limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on standard confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane of cells. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore extend raster spectral image correlation spectroscopy (RSICS) to four species analysis and apply it to determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus. Based on triple correlation analysis of RSICS data, i.e. detection of coincident fluctuations of fluorescence signals emitted by three fluorophore species, we provide direct evidence for the assembly of ternary protein complexes.