Abstract
Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.
Key words
- Correlative light and electron microscopy
- Super-resolution microscopy
- Transmission electron microscopy
- Single-molecule localization microscopy
- SMLM
- GFP
- Tannic acid
- High pressure freezing
- In-resin fluorescence
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Acknowledgments
We are grateful to Elena Seiradake and Marek Drozdz for culture and transfection of cells, and to Ilan Davis, Ian Dobbie, Kay Grünewald, Christoph Hagen, E. Yvonne Jones, Richard Parton, and Jordan Raff for their continuous support and constructive discussions. This work was supported by Wellcome Trust Senior Research Fellowships (090895/Z/09/Z; 096144/Z/11/Z), the Wellcome Trust core award to the Wellcome Trust Centre for Human Genetics (090532/Z/09/Z), a Cancer Research UK programme grant (A10976) and the Micron Strategic Award from the Wellcome Trust (091911).
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Johnson, E., Kaufmann, R. (2017). Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_14
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DOI: https://doi.org/10.1007/978-1-4939-7265-4_14
Publisher Name: Humana Press, New York, NY
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