Optimized sample preparation for single-molecule localization-based superresolution microscopy in yeast

Single-molecule localization-based superresolution microscopy methods allow the resolution of cellular structures in the range of tens of nanometers. However, these techniques are of limited use in current yeast labeling protocols, owing to problems with structural preservation. Here we describe an optimized sample preparation protocol that enables single-molecule localization microscopy at high resolution combined with improved structural preservation in Saccharomyces cerevisiae. The protocol uses small binders called nanobodies and an enzymatic labeling strategy to deliver organic dyes to the target protein. These small binders readily penetrate through the yeast cell wall and thus eliminate the requirement for its prior degradation, and they allow structural preservation. In addition, the small size of the binders reduces the distance of the dye to the target protein, and thus it reduces the localization error. The preparation of S. cerevisiae cells for superresolution imaging takes 2-4 h to perform. Researchers should have skills in yeast molecular biology, immunolabeling techniques and access to a microscope equipped for single-molecule imaging.