Super-resolved total internal reflection fluorescence microscopy using random illuminations

Abstract

A benefit of random illumination microscopy (RIM) is that it improves the resolution and linearity of the brightness of structured illumination microscopy using minimally controlled speckled illumination. Here, we implemented RIM in the total internal reflection fluorescence (TIRF) configuration for imaging biological processes close to the coverslip surface. Using standard TIRF objectives, we separated fluorescent lines 60 nm apart and achieved high contrast 86 nm resolution on fixed biological samples. Applied to live macrophages, TIRF-RIM provided two-color dynamic images of paxillin nanoclusters with remarkable spatial (96–120 nm) and temporal (1–8 Hz) resolutions, respectively. The simple experimental setup and imaging protocol together with the robustness of the data processing to leaks and aberrations make TIRF-RIM a method of choice for super-resolution TIRF imaging.