Abstract
Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Sali A, Glaeser R, Earnest T et al (2003) From words to literature in structural proteomics. Nature 422:216–225
Stahlberg H, Walz T (2008) Molecular electron microscopy: state of the art and current challenges. ACS Chem Biol 3:268–281
Pierson J, Sani M, Tomova C et al (2009) Toward visualization of nanomachines in their native cellular environment. Histochem Cell Biol 132:253–262
Kourkoutis LF, Plitzko JM, Baumeister W (2012) Electron microscopy of biological materials at the nanometer scale. Annu Rev Mater Res 42:33–58
Hell SW (2007) Far-field optical nanoscopy. Science 316:1153–1158
Lippincott-Schwartz J, Manley S (2009) Putting super-resolution fluorescence microscopy to work. Nat Methods 6:21–23
de Jonge N, Peckys DB, Kremers GJ et al (2009) Electron microscopy of whole cells in liquid with nanometer resolution. Proc Natl Acad Sci U S A 106:2159–2164
Ring EA, de Jonge N (2010) Microfluidic system for transmission electron microscopy. Microsc Microanal 16:622–629
Peckys DB, Mazur P, Gould KL et al (2011) Fully hydrated yeast cells imaged with electron microscopy. Biophys J 100:2522–2529
Dukes MJ, Peckys DB, de Jonge N (2010) Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid. ACS Nano 4:4110–4116
Ring EA, Peckys DB, Dukes MJ et al (2011) Silicon nitride windows for electron microscopy of whole cells. J Microsc (Oxford) 243:273–283
Lidke DS, Nagy P, Heintzmann R et al (2004) Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat Biotechnol 22:198–203
Acknowledgements
We are grateful to Protochips Inc. (NC, USA) for providing the silicon microchips and specimen holder. We thank Eduard Arzt for his continuous support through INM. Research in part supported by NIH grant 2R44-EB008589 (to J. Damiano).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Peckys, D.B., Dukes, M.J., de Jonge, N. (2014). Correlative Fluorescence and Electron Microscopy of Quantum Dot Labeled Proteins on Whole Cells in Liquid. 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_23
Download citation
DOI: https://doi.org/10.1007/978-1-62703-776-1_23
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-775-4
Online ISBN: 978-1-62703-776-1
eBook Packages: Springer Protocols