Search our Database of Scientific Publications and Authors

I’m looking for a
    Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.
    Sci Rep 2015 Mar 31;5:9583. Epub 2015 Mar 31.
    1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK [2] Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
    We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

    Similar Publications

    Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy.
    Methods Cell Biol 2017 5;140:49-67. Epub 2017 May 5.
    University of Oxford, Oxford, United Kingdom.
    There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. Read More
    Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.
    Nano Lett 2014 Jul 4;14(7):4171-5. Epub 2014 Jun 4.
    Division of Structural Biology, Wellcome Trust Centre for Human Genetics and ‡Department of Biochemistry, University of Oxford , Oxford, United Kingdom.
    We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. Read More
    Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.
    J Vis Exp 2012 Dec 3(70):e3995. Epub 2012 Dec 3.
    Department of Biology, Howard Hughes Medical Institute, University of Utah.
    Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Read More
    Fixation-resistant photoactivatable fluorescent proteins for CLEM.
    Nat Methods 2015 Mar 12;12(3):215-8, 4 p following 218. Epub 2015 Jan 12.
    Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA.
    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0. Read More