| Chapter title |
Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells
|
|---|---|
| Chapter number | 14 |
| Book title |
Super-Resolution Microscopy
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-7265-4_14 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7264-7, 978-1-4939-7265-4
|
| Authors |
Errin Johnson, Rainer Kaufmann, Johnson, Errin, Kaufmann, Rainer |
| 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. |
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