Biophysikalische Bildgebung

Interferometric scattering microscopy (iSCAT) enables prolonged and high frame rate single particle tracking (SPT) for studying molecular dynamics. Typical iSCAT setups employ conventional widefield or scanning illumination schemes. However, these implementations limit the field-of-view (FoV), the uniformity of the illumination and thus comparable accuracy over the whole FoV, and/or the maximum sampling rate, while in parts increasing hardware requirements and setup size. We demonstrate the realization of a large (60 µm x 60 µm) uniformly illuminated FoV through a passive refractive optical element in the iSCAT illumination path. This scanning-free iSCAT microscope 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 it 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 constant ≤10 nm localization precision throughout the whole FoV. The performance is further demonstrated through dynamic iSCAT SPT and imaging fluorescence correlation spectroscopy (imaging FCS) of lipid diffusion in a model membrane system, highlighting the ability to track a large number of molecules with the same accuracy over a large FoV. Our iSCAT setup thus depicts an accurate and improved way of recording fast molecular dynamics in life sciences.