Berlin 2012 – scientific programme

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BP: Fachverband Biologische Physik

BP 31: Imaging

BP 31.2: Talk

Friday, March 30, 2012, 10:00–10:15, H 1058

Revival of prism-based TIR microscopy - Versatile tracking of fluorescent and scattering probes with nm-precision — •René Schneider and Stefan Diez — B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden

Current single-molecule microscopy experiments mostly rely on the fluorescence signal of fluorescent proteins, organic dyes or quantum dots attached to proteins of interest. However, these probes suffer from limitations, namely limited number of photons before photobleaching, photon blinking, and fluorescence saturation. Consequently, the temporal and spatial resolution by which single molecules can be tracked is limited. Promising candidates for replacing fluorescent probes in-vitro are gold nanoparticles (GNPs). GNPs exhibit a large scattering cross section in the optical spectrum due to plasmon resonance, provide long-term stability and allow for versatile surface chemistry. Furthermore, their emission rate does not saturate.

Here, we present a camera-based wide-field imaging technique for GNP-labeled proteins using a novel parabolic prism-type total-internal reflection (TIR) microscope. We demonstrate the advantages of GNPs over commonly used fluorescent probes and discuss the pros and cons of prism-type versus objective-type TIR microscopy. We demonstrate that prism-based TIR microscopy allows imaging of fluorescent and scattering probes with high signal-to-noise and excellent control over a wide range of incidence angles.

Our method allows for precise localization of biomolecules within short acquisition times over long time scales.