Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores.
Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of ~ 20nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F(0)F(1)-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared.
van de Linde
Sebastian
S
Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany.
Sauer
Markus
M
Heilemann
Mike
M
D000818:Animals; D002232:Carbocyanines; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003952:Diagnostic Imaging / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D007150:Immunohistochemistry / Q000379:methods; D007476:Ionophores; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D051336:Mitochondrial Membranes; D024101:Mitochondrial Proteins / Q000032:analysis*; D011506:Proteins
D002232:Carbocyanines; D005456:Fluorescent Dyes; D007476:Ionophores; D024101:Mitochondrial Proteins; D011506:Proteins