Nanosizing by Spatially Modulated Illumination (SMI) Microscopy and Applications to the Nucleus

In this chapter we present the method of spatially modulated illumination (SMI) microscopy, a (far-field) fluorescence microscopy technique featuring structured illumination obtained via a standing wave field laser excitation pattern. While this method does not provide higher optical resolution, it has been proven a highly valuable tool to access structural parameters of fluorescently labeled macromolecular structures in cells. SMI microscopy has been used to measure relative positions with a reproducibility of <2 nm between fluorescing objects. Among others, we have measured size distributions of protein clusters with an accuracy much better than the resolution achievable e.g. in confocal microscopy. The advantages of the SMI microscope over other (ultra-)high resolution light microscopes are its easy sample preparation and microscope handling as well as the comparably fast acquisition times and large fields of view.