Bimodal endocytic probe for three-dimensional correlative light and electron microscopy
Fermie, Job; de Jager, Leanne; Foster, Helen E; Veenendaal, Tineke; de Heus, Cecilia; van Dijk, Suzanne; Ten Brink, Corlinda; Oorschot, Viola; Yang, Lin; Li, Wei; Müller, Wally H; Howes, Stuart; Carter, Andrew P; Förster, Friedrich; Posthuma, George; Gerritsen, Hans C; Klumperman, Judith; Liv, Nalan
(2022) Cell Reports Methods, volume 2, issue 5
(Article)
Abstract
We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and
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has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications.
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Keywords: correlative light and electron microscopy, cryoelectron microscopy, electron tomography, endolysosomal system, nanogold fiducials, volume-electron microscopy, Biotechnology, Biochemistry, Biochemistry, Genetics and Molecular Biology (miscellaneous), Genetics, Radiology Nuclear Medicine and imaging, Computer Science Applications
ISSN: 2667-2375
Publisher: Cell Press
Note: Funding Information: We acknowledge funding from the Netherlands Organisation for Scientific Research (NWO), Stichting voor de Technische Wetenschappen (Technology Foundation STW), grant 12715 (to H.G. and J.K.) and the Netherlands Organisation for Health Research and Development (ZonMW) grant 40-00812-98-16006 (to J.K.). The microscopy infrastructure in this work is partially subsidized by the Roadmap for Large-Scale Research Infrastructure (NEMI) of Netherlands Organisation for Scientific Research (grant number 184.034.014 ) (to J.K.). The Cell Microscopy Core of UMC Utrecht is part of the Dutch Correlative Light Electron Microscopy node of EuroBioImaging. Publisher Copyright: © 2022 The Author(s)
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