Abstract
Correlative light and electron microscopy (CLEM) aims at combining data acquired from the same sample through both imaging modalities. Many combinations can be found in the literature where almost any kind of light microscopy (LM) has been associated to different processing in electron microscopy (EM) and applied to a wide variety of specimen, from cultured cells to multicellular organisms. In this chapter, we focus on a technique that intends to combine LM acquisition on living cells with transmission EM (TEM) analysis. A specific attention is given to the description of a method to bring precise coordinates to the object of interest, to allow a straightforward correlation between LM and EM. Moreover, we describe how, by using high-pressure freezing as a fixation technique, dynamic events observed at the LM are captured and studied at the ultrastructural level.
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References
Robinson JM, Takizawa T, Pombo A et al (2001) Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap. J Histochem Cytochem 49:803–808
Schwarz H, Humbel BM (2007) Correlative light and electron microscopy using immunolabeled resin sections. Methods Mol Biol 369:229–256
Kukulski W, Schorb M, Welsch S et al (2011) Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol 192:111–119
Watanabe S, Punge A, Hollopeter G et al (2011) Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8:80–84
Price ZH (1967) The micromorphology of zeiotic blebs in cultured human epithelial (HEp) cells. Exp Cell Res 48:82–92
Price ZH (1968) A study of membrane ruffing in cultured cells by time-lapse cinephotomicrography and electron microscopy. J Biol Photogr Assoc 36:93–99
Hayat MA (1974) Principles and techniques of electron microscopy. Biological applications. Van Nostrand Reinhold Company, New York
Colombelli J, Tangemo C, Haselman U et al (2008) A correlative light and electron microscopy method based on laser micropatterning and etching. Methods Mol Biol 457:203–213
Small JV (1985) Simple procedures for the transfer of grid images onto glass coverslips for the rapid relocation of cultured cells. J Microsc (Oxford) 137:171–175
Hanson HH, Reilly JE, Lee R et al (2010) Streamlined embedding of cell monolayers on gridded glass-bottom imaging dishes for correlative light and electron microscopy. Microsc Microanal 16:747–754
van Rijnsoever C, Oorschot V, Klumperman J (2008) Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nat Methods 5:973–980
Jimenez N, van Donselaar EG, de Winter DA et al (2010) Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB-SEM. J Microsc (Oxford) 237:208–220
McDonald KL (2009) A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. J Microsc (Oxford) 235:273–281
Auinger S, Small JV (2008) Correlated light and electron microscopy of the cytoskeleton. Methods Cell Biol 88:257–272
Rinnerthaler G, Herzog M, Klappacher M et al (1991) Leading edge movement and ultrastructure in mouse macrophages. J Struct Biol 106:1–16
Sartori A, Gatz R, Beck F et al (2007) Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol 160:135–145
Verkade P (2008) Moving EM: the rapid transfer system as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing. J Microsc (Oxford) 230:317–328
Spiegelhalter C, Tosch V, Hentsch D et al (2010) From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy. PLoS One 5:e9014
Daguenet E, Baguet A, Degot S et al (2012) Perispeckles are major assembly sites for the exon junction core complex. Mol Biol Cell 23:1765–1782
Gibbings D, Leblanc P, Jay F et al (2012) Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes. Nat Struct Mol Biol 19(517–524):S511
Kolotuev I, Bumbarger DJ, Labouesse M et al (2012) Targeted ultramicrotomy: a valuable tool for correlated light and electron microscopy of small model organisms. Methods Cell Biol 111:203–222
Brown E, Van Weering J, Sharp T et al (2012) Capturing endocytic segregation events with HPF-CLEM. Methods Cell Biol 111:175–201
Kolotuev I, Schwab Y, Labouesse M (2010) A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms. Biol Cell 102:121–132
Karreman MA, Agronskaia AV, van Donselaar EG et al (2012) Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen. J Struct Biol 180:382–386
Biel SS, Kawaschinski K, Wittern KP et al (2003) From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging. J Microsc (Oxford) 212:91–99
Nixon SJ, Webb RI, Floetenmeyer M et al (2009) A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic 10:131–136
Lucic V, Kossel AH, Yang T et al (2007) Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography. J Struct Biol 160:146–156
Acknowledgements
We would like to thank Heinz Schwarz for fruitful discussions and great inspiration in performing CLEM experiments and Matthia Winter-Karreman for discussions and careful reading of this manuscript.
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Spiegelhalter, C., Laporte, J.F., Schwab, Y. (2014). Correlative Light and Electron Microscopy: From Live Cell Dynamic to 3D Ultrastructure. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_21
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DOI: https://doi.org/10.1007/978-1-62703-776-1_21
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