Abstract
Peroxisomes are crucial organelles that occur in almost all eukaryotes. Well known are their roles in various metabolic processes, such as hydrogen peroxide detoxification and lipid metabolism. Recent studies indicated that peroxisomes also have several non-metabolic functions, for instance, in stress response, signaling, and cellular ageing. In mammalian cells, the small size of peroxisomes (~200 nm, near the diffraction limit) hinders unveiling peroxisomal structures by conventional light microscopy. However, in the yeast Hansenula polymorpha, they can reach up to 1.5 μm in diameter, depending on the carbon source. To study the localization of peroxisomal proteins in cells in more detail, super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy can be used. STED enables fast (live-cell) imaging well beyond the diffraction limit of light (30-40 nm in cells), without further data processing. Here, we present optimized protocols for the fluorescent labeling of specific peroxisomal proteins in fixed and living cells. Moreover, detailed measurement protocols for successful STED imaging of human and yeast peroxisomes (using antibodies or genetic tags labeled with dyes) are described, extended with suggestions for individual optimizations.