Dresden 2017 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 2: Bioimaging and Spectroscopy I

BP 2.7: Vortrag

Montag, 20. März 2017, 11:45–12:00, HÜL 386

Investigating DNA binding kinetics by camera-based total internal reflection fluorescence correlation spectroscopy (TIR-FCS)Jonas Mücksch1,3, •Philipp Blumhardt1,3, Maximilian Strauss1,2, Ralf Jungmann1,2, and Petra Schwille11Max Planck Institute of Biochemistry, Martinsried, Germany — 2Ludwig Maximilian University, Munich, Germany — 3equal contribution

Fluorescence correlation spectroscopy (FCS) has been extensively used to study the kinetics of various in vitro and in vivo systems on a molecular level. The vast majority of FCS studies is performed using confocal setups, which feature well-defined detection volumes but suffer from low surface selectivity. Combining FCS with total internal reflection fluorescence (TIRF) illumination drastically enhances the spatial selectivity and enables the investigation of reversible binding of fluorescently labeled ligands to surface-confined receptors. So far, this potential to observe and quantify surface binding using TIR-FCS has been used only to minor extent. Here, we present a versatile optical setup for exploring surface-binding kinetics with TIRF illumination and point- (APD) or camera-based (EMCCD) fluorescence detection. In a first application, our camera-based assay facilitated the investigation of the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of a surface-immobilized DNA origami scaffold. We varied the nucleotide overlap, yielding different binding times in the range of milliseconds to seconds. Using this highly tunable system, we systematically explored the parameter space accessible to EMCCD-based TIR-FCS.

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