Multichannel imaging is used as a readout of relative localization of two or more components and is often the first step in investigating functional ensembles in cells. However, the localization volume of diffraction-limited light microscopy (approx. 200 nm by 500 nm) can accommodate hundred of proteins, calling for increased resolution for these types of analyses. Here, we present a protocol for 4-channel imaging using structured illumination microscopy (SIM), which increases resolution by a factor of two. We use adherent, fixed cells to identify the localization of adhesion proteins using immunofluorescence and fluorescent proteins. We discuss how labeling with the necessary brightness is achieved and how data has to be processed for colocalization analysis.
Keywords: Actin; Chromatic aberration; Colocalization; Cytoskeleton; Fluorescent protein; Focal adhesion; Structured-illumination microscopy; Super-resolution.