Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Christopher J Peddie, Marie-Charlotte Domart, Xenia Snetkov, Peter O'Toole, Banafshe Larijani, Michael Way, Susan Cox, Lucy M Collinson

Research output: Contribution to journalArticlepeer-review

42 Citations (SciVal)


Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP).

Original languageEnglish
Pages (from-to)120-131
Number of pages12
JournalJournal of Structural Biology
Issue number2
Early online date30 May 2017
Publication statusPublished - Aug 2017

Bibliographical note

Copyright © 2017. Published by Elsevier Inc.


  • Bacterial Proteins/analysis
  • Diglycerides/analysis
  • Equipment Design
  • Green Fluorescent Proteins/analysis
  • Luminescent Proteins/analysis
  • Microscopy, Electron, Scanning/methods
  • Microscopy, Fluorescence/methods
  • Signal-To-Noise Ratio
  • Single Molecule Imaging/methods
  • Vacuum


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