Homogeneous large field-of-view and compact iSCAT-TIRF setup for dynamic single molecule measurements

Interferometric Scattering Microscopy (iSCAT) enables prolonged and high frame rate Single Particle Tracking (SPT) for single molecule dynamics studies. 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 the realization of a large (60µm x 60µm) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. This scanning-free iSCAT microscopy setup is further combined with an objective based Total Internal Reflection Fluorescence Microscopy (TIRF) channel for a complementary fluorescence readout, a focus-lock system, and a tailored control platform via the open-source ImSwitch software, and has a compact footprint. As a proof-of-principle, we highlight the performance of the setup through the acquisition of iSCAT images with a uniform contrast and a ≤10 nm localization precision throughout the whole FoV. The performance is further demonstrated through dynamic iSCAT SPT and imaging Fluorescence Correlation Spectroscopy of lipid diffusion in a model membrane system. Our iSCAT setup thus depicts an accurate and improved way of recording fast molecular dynamics in life sciences.