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Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

Nature Methods volume 2, pages 743–749 (2005)Cite this article

Abstract

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.

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Figure 1: Introduction to QDs.
Figure 2: Application of QDs for multiprotein EM in cultured cells.
Figure 3: Correlated LM-EM of multiple proteins in cultured cells.
Figure 4: Double and triple labeling of proteins for LM and EM imaging in tissue.
Figure 5: Penetration of QD655-conjugated antibodies versus 5-nm gold-conjugated antibodies.

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Acknowledgements

We thank R.Y. Tsien for encouragement and support as well as H. Hakozaki for light microscopy assistance, M.E. Martone for editorial assistance, R.L. Ornberg (Quantum Dot Corporation) for providing QDs and T. Sudhof for providing the IP3R antibody. This work was supported by grants from the US National Institutes of Health to M.H.E. and R.Y. Tsien: NIH-RR04050, NIH-NS27177, NIH-1P20-GM72033.

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  1. Department of Neurosciences, National Center for Microscopy and Imaging Research, and Center for Research in Biological Systems, University of California, San Diego, La Jolla, 92093-0608, California, USA

    Ben N G Giepmans, Thomas J Deerinck, Benjamin L Smarr, Ying Z Jones & Mark H Ellisman

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  1. Ben N G Giepmans
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  2. Thomas J Deerinck
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Correspondence to Mark H Ellisman.

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Quantum Dot Corporation (Hayward, California) supplied QD conjugates and reimbursed a meeting attendance.

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Giepmans, B., Deerinck, T., Smarr, B. et al. Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nat Methods 2, 743–749 (2005). https://doi.org/10.1038/nmeth791

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