title: Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy.
abstract: Illumination based on objective-type total internal reflection (TIR) is nowadays widely used in high-performance fluorescence microscopy. However, the desirable application of such setups for dark-field imaging of scattering entities is cumbersome due to the spatial overlap of illumination and detection light, which cannot be separated spectrally. Here, we report a novel TIR approach based on a parabolically shaped quartz prism that allows for the detection of single-molecule fluorescence as well as single-particle scattering with high signal-to-noise ratios. We demonstrate homogeneous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, we quantitatively compare the fluorescence performance of our setup to objective-type TIR and demonstrate sub-nanometer localization accuracies for the scattering of 40 nm gold nanoparticles (AuNPs). When bound to individual kinesin-1 motors, the AuNPs reliably report on the characteristic 8 nm stepping along microtubules.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: René
authors.initials: R
authors.lastname: Schneider
authors.forename: Tilman
authors.initials: T
authors.lastname: Glaser
authors.forename: Michael
authors.initials: M
authors.lastname: Berndt
authors.forename: Stefan
authors.initials: S
authors.lastname: Diez
mesh_terms: D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D007909:Lenses; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D036103:Nanotechnology / Q000295:instrumentation*; D011791:Quartz; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011791:Quartz