Biophysikalische Bildgebung

Interferometric Scattering Microscopy (iSCAT) enables label free, prolonged and high frame rate image acquisition. Typically, iScat setups employ scanning illumination schemes to achieve uniform sample illumination. However, this implementation limits the field of view (FoV) and maximum sampling rate, while increasing hardware requirements and setup size. We demonstrate that it is possible to achieve a large (80μm x 80μm) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. Thus, we developed a scanning-free Interferometric Scattering Microscopy setup in combination with an objective based Total Internal Reflection Fluorescence Microscopy (TIRF) channel. This setup has the additional advantage of a compact footprint and a freely available software implementation. We show that we can acquire images with a uniform contrast and a ≤10 nm localisation precision throughout the FoV. The instrument is capable of performing quantitative single molecule measurements as Single Particle Tracking and imaging Fluorescence Correlation Spectroscopy. We demonstrate these measurements on model membrane systems. This illumination-detection scheme for iSCAT has the potential to drive further innovation and applications of iSCAT microscopy in the life sciences.