DOI: 10.1083/jcb.201009037. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

10.1083/jcb.201009037

Crossref journal-article

Rockefeller University Press

Journal of Cell Biology (291)

Abstract

Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale.

Bibliography

Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., & Briggs, J. A. G. (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. Journal of Cell Biology, 192(1), 111â119.

Authors 6

Wanda Kukulski (first)
Martin Schorb (additional)
Sonja Welsch (additional)
Andrea Picco (additional)
Marko Kaksonen (additional)
John A.G. Briggs (additional)

References 25 Referenced 419

10.1016/j.ceb.2004.11.001 / Curr. Opin. Cell Biol. / Microtubule plus-end-tracking proteins: mechanisms and functions by Akhmanova (2005)
10.1038/nrm2369 / Nat. Rev. Mol. Cell Biol. / Tracking the ends: a dynamic protein network controls the fate of microtubule tips by Akhmanova (2008)
10.1016/j.cub.2004.03.029 / Curr. Biol. / The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules by Busch (2004)
10.1016/j.chom.2009.05.021 / Cell Host Microbe. / Membrane rupture generates single open membrane sheets during vaccinia virus assembly by Chlanda (2009)
10.1083/jcb.129.5.1311 / J. Cell Biol. / Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates by Chrétien (1995)
10.1016/j.devcel.2007.01.020 / Dev. Cell. / Organization of interphase microtubules in fission yeast analyzed by electron tomography by Höög (2007)
10.1083/jcb.200708060 / J. Cell Biol. / Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane by Idrissi (2008)
10.1038/ncb1414 / Nat. Cell Biol. / Molecular architecture of a kinetochore-microtubule attachment site by Joglekar (2006)
10.1016/S0092-8674(03)00883-3 / Cell. / A pathway for association of receptors, adaptors, and actin during endocytic internalization by Kaksonen (2003)
10.1016/j.cell.2005.09.024 / Cell. / A modular design for the clathrin- and actin-mediated endocytosis machinery by Kaksonen (2005)
10.1042/BC20090096 / Biol. Cell. / A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms by Kolotuev (2009)
10.1006/jsbi.1996.0013 / J. Struct. Biol. / Computer visualization of three-dimensional image data using IMOD by Kremer (1996)
10.1016/0304-3991(88)90322-1 / Ultramicroscopy. / A method for monitoring the collapse of plastic sections as a function of electron dose by Luther (1988)
10.1128/JVI.78.19.10803-10813.2004 / J. Virol. / Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative by Müller (2004)
10.1016/S0091-679X(06)79004-5 / Methods Cell Biol. / Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis by Müller-Reichert (2007)
10.1111/j.1600-0854.2008.00859.x / Traffic. / A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos by Nixon (2009)
10.1091/mbc.10.6.2017 / Mol. Biol. Cell. / High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae by O’Toole (1999)
10.1083/jcb.200304035 / J. Cell Biol. / Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans by O’Toole (2003)
10.1016/j.copbio.2009.03.008 / Curr. Opin. Biotechnol. / Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes by Plitzko (2009)
10.1016/j.jsb.2007.07.011 / J. Struct. Biol. / Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography by Sartori (2007)
10.1111/j.1365-2818.2007.01794.x / J. Microsc. / Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching by Schwartz (2007)
10.1016/j.ejcb.2009.07.002 / Eur. J. Cell Biol. / Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells by van Driel (2009)
10.1038/nmeth.1263 / Nat. Methods. / Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections by van Rijnsoever (2008)
10.1111/j.1365-2818.2008.01989.x / J. Microsc. / Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing by Verkade (2008)
10.1046/j.1365-2818.2002.01064.x / J. Microsc. / Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water by Walther (2002)

Dates

Type	When
Created	14 years, 7 months ago (Jan. 3, 2011, 11:19 p.m.)
Deposited	2 years, 1 month ago (July 29, 2023, 12:35 a.m.)
Indexed	4 days, 12 hours ago (Aug. 24, 2025, 7:01 p.m.)
Issued	14 years, 7 months ago (Jan. 3, 2011)
Published	14 years, 7 months ago (Jan. 3, 2011)
Published Online	14 years, 7 months ago (Jan. 3, 2011)
Published Print	14 years, 7 months ago (Jan. 10, 2011)

Funders 0

None

BibTeX

@article{Kukulski_2011, title={Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision}, volume={192}, ISSN={0021-9525}, url={http://dx.doi.org/10.1083/jcb.201009037}, DOI={10.1083/jcb.201009037}, number={1}, journal={Journal of Cell Biology}, publisher={Rockefeller University Press}, author={Kukulski, Wanda and Schorb, Martin and Welsch, Sonja and Picco, Andrea and Kaksonen, Marko and Briggs, John A.G.}, year={2011}, month=jan, pages={111–119} }

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