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Ultramicroscopy. 2014 Aug;143:3-14. doi: 10.1016/j.ultramic.2014.02.001. Epub 2014 Feb 22.

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells.

Author information

1
Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
2
Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands.
3
Department of Biology, The University of York, Heslington, York, UK.
4
Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
5
Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.

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.

KEYWORDS:

Correlative light and electron microscopy; Diacylglycerol; GFP; In-resin fluorescence; Integrated light and electron microscopy; Mammalian cells

PMID:
24637200
PMCID:
PMC4045205
DOI:
10.1016/j.ultramic.2014.02.001
[Indexed for MEDLINE]
Free PMC Article

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