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

Research output: Contribution to journalArticle

Author(s)

  • Christopher J Peddie
  • Ken Blight
  • Emma Wilson
  • Charlotte Melia
  • Jo Marrison
  • Raffaella Carzaniga
  • Marie-Charlotte Domart
  • Peter O'Toole
  • Banafshe Larijani
  • Lucy M Collinson

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Publication details

JournalUltramicroscopy
DateE-pub ahead of print - 22 Feb 2014
DatePublished (current) - Aug 2014
Volume143
Number of pages12
Pages (from-to)3-14
Early online date22/02/14
Original languageEnglish

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.

Bibliographical note

Copyright © 2014 The Authors. Published by Elsevier B.V. Open access under CC BY-NC-ND license.

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