Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy
Research output: Contribution to journal › Research article › Contributed › peer-review
Contributors
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 language | English |
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Pages (from-to) | 3523-3539 |
Number of pages | 17 |
Journal | Optics Express |
Volume | 21 |
Issue number | 3 |
Publication status | Published - 11 Feb 2013 |
Peer-reviewed | Yes |
External IDs
ORCID | /0000-0002-0750-8515/work/142235485 |
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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