Correlative Light- and Electron Microscopy in Peroxisome Research

Correlative light and electron microscopy (CLEM) combines the advantages of protein localization by fluorescence microscopy with the high resolution of electron microscopy. Here, we describe a protocol that we developed for yeast peroxisome research. First, cells are fixed, using conditions that preserve the properties of fluorescent proteins and avoid the introduction of autofluorescence. Next, cryosections are prepared and imaged by fluorescence microscopy. The same sections are used for electron microscopy. Both images are aligned and merged, allowing to localize fluorescent proteins in electron microscopy images. This method was successfully used for peroxisomal membrane contact site research and allows to precisely localize contact site resident proteins at regions where membranes are closely associated at distances far below the resolution of conventional fluorescence microscopy.