Peddie, Christopher J, Blight, Ken, Wilson, Emma et al. (7 more authors) (2014) Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells. Ultramicroscopy. pp. 3-14. ISSN 0304-3991
Abstract
Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | Copyright © 2014 The Authors. Published by Elsevier B.V. Open access under CC BY-NC-ND license. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) |
Funding Information: | Funder Grant number MEDICAL RESEARCH COUNCIL (MRC) MR/K01580X/1 BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/F004729/1 |
Depositing User: | Pure (York) |
Date Deposited: | 16 Mar 2015 14:16 |
Last Modified: | 26 Nov 2024 00:29 |
Published Version: | https://doi.org/10.1016/j.ultramic.2014.02.001 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1016/j.ultramic.2014.02.001 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:84254 |
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Description: Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells