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Sci Rep. 2015 Mar 31;5:9583. doi: 10.1038/srep09583.

Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.

Author information

1
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
2
Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
3
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
4
1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK [2] Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Abstract

We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

PMID:
25823571
PMCID:
PMC4379466
DOI:
10.1038/srep09583
[Indexed for MEDLINE]
Free PMC Article

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