Correlative single-molecule and structured illumination microscopy of fast dynamics at the plasma membrane

Abstract

AbstractTotal internal reflection fluorescence (TIRF) microscopy offers powerful means to uncover the functional organization of proteins in the plasma membrane with very high spatial and temporal resolution. Traditional TIRF illumination, however, shows a Gaussian intensity profile, which is typically deteriorated by overlaying interference fringes hampering precise quantification of intensities – an important requisite for quantitative analyses in single-molecule localization microscopy (SMLM). Here, we combined flat-field illumination by using a standard πShaper with multi-angular TIR illumination by incorporating a spatial light modulator compatible with fast super-resolution structured illumination microscopy (SIM). This unique combination enabled quantitative multi-color SMLM with a highly homogenous illumination. By using a dual camera setup with optimized image splitting optics, we achieved versatile combination of SMLM and SIM with up to three channels. We deployed this setup for establishing robust detection of receptor stoichiometries based on single-molecule intensity analysis and single-molecule Förster resonance energy transfer (smFRET). Homogeneous illumination furthermore enabled long-term tracking and localization microscopy (TALM) of cell surface receptors identifying spatial heterogeneity of mobility and accessibility in the plasma membrane. By combination of TALM and SIM, spatially and molecularly heterogenous diffusion properties could be correlated with nanoscale cytoskeletal organization and dynamics.