Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes
- 2 April 2012
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 109 (16), 6136-6141
- https://doi.org/10.1073/pnas.1121558109
Abstract
Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.Keywords
This publication has 40 references indexed in Scilit:
- Facile and General Synthesis of Photoactivatable Xanthene DyesAngewandte Chemie-International Edition, 2011
- Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precisionThe Journal of cell biology, 2011
- Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructureProceedings of the National Academy of Sciences, 2009
- A bright and photostable photoconvertible fluorescent proteinNature Methods, 2009
- Assembly of the oxidative phosphorylation system in humans: What we have learned by studying its defectsBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009
- Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samplesNature Methods, 2008
- Serial Section Scanning Electron Microscopy of Adult Brain Tissue Using Focused Ion Beam MillingJournal of Neuroscience, 2008
- Organization and dynamics of human mitochondrial DNAJournal of Cell Science, 2004
- Evidence for a two membrane–spanning autonomous mitochondrial DNA replisomeThe Journal of cell biology, 2003
- Low Levels of Mitochondrial Transcription Factor A in Mitochondrial DNA DepletionBiochemical and Biophysical Research Communications, 1994