Increasing Resolution in Live Cell Microscopy by Structured Illumination (SIM)

Abstract

In the context of various approaches to super-resolution microscopy, structured illumination microscopy (SIM) offers several advantages: it needs rather low light doses (with a low risk of phototoxicity or photobleaching), is comparably fast and flexible concerning the use of microscopes, objective lenses and cameras, and has potential for 3D imaging. This paper describes an experimental setup for SIM with first diffraction orders of a spectral light modulator (SLM) creating an interference pattern in two dimensions. We kept this system rather compact with a comparably large illuminated object field, validated it with nano-beads and applied it further to living cells for imaging the cytoskeleton, mitochondria or cell nuclei with a resolution slightly above 100 nm. Its advantages, challenges and limitations—concerning cameras, acquisition time, depth of imaging, light exposure, and combining it with further super-resolving methods—are discussed.