Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • René Schneider - , Chair of BioNano-Tools, Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Tilman Glaser - , Carl Zeiss Jena GmbH (Author)
  • Michael Berndt - , Max Planck Institute of Molecular Cell Biology and Genetics (Author)
  • Stefan Diez - , Chair of BioNano-Tools, Max Planck Institute of Molecular Cell Biology and Genetics (Author)

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.

Details

Original languageEnglish
Pages (from-to)3523-3539
Number of pages17
JournalOptics Express
Volume21
Issue number3
Publication statusPublished - 11 Feb 2013
Peer-reviewedYes

External IDs

ORCID /0000-0002-0750-8515/work/142235485
PubMed 23481810
Scopus 84874026760

Keywords

Research priority areas of TU Dresden

Keywords

  • Equipment Design, Equipment Failure Analysis, Image Enhancement/instrumentation, Lenses, Microscopy, Fluorescence/instrumentation, Nanotechnology/instrumentation, Quartz, Reproducibility of Results, Sensitivity and Specificity

Library keywords