title: Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
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.
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Kopek
authors.forename: Gleb
authors.initials: G
authors.lastname: Shtengel
authors.forename: C Shan
authors.initials: CS
authors.lastname: Xu
authors.forename: David A
authors.initials: DA
authors.lastname: Clayton
authors.forename: Harald F
authors.initials: HF
authors.lastname: Hess
mesh_terms: D016475:3T3 Cells; D000818:Animals; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008857:Microscopy, Interference / Q000379:methods*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000378:metabolism / Q000648:ultrastructure*; D024101:Mitochondrial Proteins / Q000378:metabolism / Q000648:ultrastructure*; D015203:Reproducibility of Results
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; C081930:Tfam protein, mouse