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

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


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).This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001999), the UK Medical Research Council (FC001999), and the Wellcome Trust (FC001999); and by the MRC, BBSRC and EPSRC under grant award MR/K01580X/1 to LMC and POT, and awards MR/K015664/1 and BB/K01563X/1 to SC. SC acknowledges support from a Royal Society University Research Fellowship. We would like to thank Sander den Hoedt (DELMIC B.V.) for useful discussions, and Bernd Rieger (T.U. Delft) for advice on using the FRC code

Topics: CLEM, ILSEM, correlative, volume, electron microscopy, super-resolution, integrated, YFP, GFP, fluorescence, protein localisation, 3-dimensional, blinking, In-resin fluorescence, vaccinia virus, localization microscopy, cryoelectron tomography, structural basis, dynamics, photoactivation, cells, light, resolution, nanoscopy
Publisher: 'Elsevier BV'
Year: 2017
DOI identifier: 10.1016/j.jsb.2017.05.013
OAI identifier:
Provided by: Communities in ADDI
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