fastSIM: a practical implementation of fast structured illumination microscopy

Abstract

A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5 × 16.5 µ m 2 , free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.