Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron mi
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ORIGINAL ARTICLE
Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope Elisabeth Brama 1 & Christopher J. Peddie 1 & Martin L. Jones 1 & Marie-Charlotte Domart 1 & Xenia Snetkov 2 & Michael Way 2 & Banafshe Larijani 3 & Lucy M. Collinson 1
Received: 16 April 2015 / Accepted: 23 June 2015 / Published online: 8 July 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dualmodality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging
Elisabeth Brama and Christopher J. Peddie are joint first authors. * Lucy M. Collinson [email protected] 1
Electron Microscopy, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, London WC2A 3LY, UK
2
Cellular Signalling and Cytoskeletal Function, The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, London WC2A 3LY, UK
3
Cell Biophysics Laboratory, Ikerbasque, Basque Foundation for Science and Unidad de Biofisica (CSIC-UPV/EHU), Barrio de Sarriena, 48940 Leioa, Spain
strategies for standard FPs acting as dual-modality probes in a single ‘multi-colour’ integrated microscope system. Keywords GFP . YFP . Fluorescent protein . In-resin fluorescence . Vacuum . Integrated light and electron microscopy
Introduction From the discovery of Aequorea victoria green fluorescent protein (GFP) [1], fluorescent proteins (FPs) have developed into ubiquitous tools for localising macromolecules in biological samples [2]. The GFP molecule consists of 238 amino acids in a single chain, folded into a -barrel, at the centre of which sits the fluorescent chromophore parahydroxybenzylidene imidazolone (PHBI). Four water molecules sit within the cavity that surrounds the chromophore, contributing to a hydrogen-bonding network that is important for the photoactive properties of GFP [3]. On excitation of GFP by light in the near-UV range, a p
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