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

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)3-14
Number of pages12
JournalUltramicroscopy
Volume143
DOIs
Publication statusPublished - 1 Aug 2014

Keywords

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

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Instrumentation

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