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

Optics Express 21 (3), 3523-3539, (2013)

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.

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Reference:

R. Schneider, T. Glaser, M. Berndt, S. Diez: Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy, Optics Express, Vol. 21, Issue 3, pp. 3523-3539 (2013).

doi: 10.1364/OE.21.003523

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