Revival of Prism-Based TIR Microscopy - Versatile Tracking of Fluorescent and Scattering Probes with NM-Precision

Abstract

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 certain 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 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, because generation of photons relies on elastic scattering, 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, excellent control over a wide range of incidence angles, and even illumination intensities within a field of view. When tracking surface-immobilized GNPs (40 nm diameter) we obtained two-dimensional localization precisions as good as 5 angstrom within 30 ms exposure time. We demonstrate localization experiments of kinesin-1 motors labeled with GNPs walking on surface-immobilized microtubules. When GNPs where bound to the tail or motor domains of kinesin-1 we found localization precisions of 3.6 nm and 1.9 nm within 30 ms exposure time, respectively. Furthermore GNP-loaded motors showed 8 nm stepping along microtubules at 3 μM MgATP within image acquisition times of 15 ms. Our method allows for precise localization of biomolecules within short acquisition times over long time scales.