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.
Subject(s)
Image Enhancement/instrumentation , Lenses , Microscopy, Fluorescence/instrumentation , Nanotechnology/instrumentation , Quartz , Equipment Design , Equipment Failure Analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The working principle of an optical isolator made of two corrugated dielectric gratings is introduced. One grating acts as a polarizer, and the other acts as a quarter-wave plate used in conical incidence converting linearly polarized light into circularly polarized light. Global maxima of diffraction efficiency for surface-corrugated gratings with binary, sinusoidal, and pyramidal ridge shapes with dependence on the material index are identified. Regarding technological feasibility for use in the visible wavelength range, high-frequency gratings with a binary shape were realized. With these gratings, an extinction ratio of more than 40 dB for the polarizer is theoretically possible, and more than 20 dB was experimentally achieved. A good correlation between theoretically calculated efficiencies and birefringences based on rigorous methods and the experimental results is demonstrated.
