Imperial College London
Professor Michael Way

ProfessorMichaelWay

Faculty of Medicine, Department of Infectious Disease

Professor of Virology
 
 
 
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Contact

 

+44 (0)20 3796 2068michael.way1 Website

 
 
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Location

 

Francis Crick InstituteThe Francis Crick Institute

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Summary

 

Publications

Citation

BibTex format

@article{Brama:2015:10.1007/s12154-015-0143-3,
author = {Brama, E and Peddie, CJ and Jones, ML and Domart, MC and Snetkov, X and Way, M and Larijani, B and Collinson, LM},
doi = {10.1007/s12154-015-0143-3},
journal = {Journal of Chemical Biology},
pages = {179--188},
title = {Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope},
url = {http://dx.doi.org/10.1007/s12154-015-0143-3},
volume = {8},
year = {2015}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - 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 dual-modality probes for ILEM when preserved using the in-resin 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 re-creation 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 strategies for standard FPs acting as dual-modality probes in a single ‘multi-colour’ integrated microscope system.
AU  - Brama,E
AU  - Peddie,CJ
AU  - Jones,ML
AU  - Domart,MC
AU  - Snetkov,X
AU  - Way,M
AU  - Larijani,B
AU  - Collinson,LM
DO  - 10.1007/s12154-015-0143-3
EP  - 188
PY  - 2015///
SN  - 1864-6158
SP  - 179
TI  - Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope
T2  - Journal of Chemical Biology
UR  - http://dx.doi.org/10.1007/s12154-015-0143-3
VL  - 8
ER  -
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