Superresolution Optical Microscopy of Isolated Cardiac Mitochondrial Proteins

Abstract

To study the structural organization of mitochondrial proteins, we applied Stimulated Emission Depletion (STED) microscopy in isolated mitochondria. In STED microscopy, two laser beams are used: one for excitation of fluorophores and the other, with doughnut shape, to deplete them in order to allow fluorescence emission only from the excited volume located at the doughnut's center. With STED a lateral resolution of ∼45 nm was achieved in images of isolated mitochondria. We investigated the localization pattern and distribution of MaxiKα, COX4 and VDAC1. After a combined analysis of classical confocal and STED images, we found distinct distributions for VDAC1, MaxiKα and COX4. COX4 distribution was consistent with localization in the cristae. We established that there are 7-15 clusters of MaxiKα, 10-15 clusters of VDAC1, and 15-20 clusters of COX4 per mitochondria. We have demonstrated that protein clusters in the mitochondria can be resolved with a separation power of ∼45 nm, and that it is possible to retrieve quantitative information about the number of clusters and density of proteins in mitochondria. This approach can be extended to eins in mitochondria and subcellular organelles. Supported by NIH.View Large Image | View Hi-Res Image | Download PowerPoint Slide