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NanoBiotechnology Protocols pp 71–84Cite as

Quantum Dot-Based Single-Molecule Microscopy for the Study of Protein Dynamics

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1026))

Abstract

Real-time microscopic visualization of single molecules in living cells provides a molecular perspective of cellular dynamics, which is difficult to be observed by conventional ensemble techniques. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. This chapter provides an overview of single quantum dot tracking for protein dynamic studies. First, we review the fundamental diffraction limit of conventional optical systems and recent developments in single-molecule detection beyond the diffraction barrier. Second, we describe methods to prepare water-soluble quantum dots for biological labeling and single-molecule microscopy experimental design. Third, we provide detailed methods to perform quantum dot-based single-molecule microscopy. This technical section covers three protocols including (1) imaging system calibration using spin-coated single quantum dots, (2) single quantum dot labeling in living cells, and (3) tracking algorithms for single-molecule analysis.

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Acknowledgments

The authors thank Drs. David Piston and Sam Wells for helpful advice with single-quantum-dot tracking experimental setup. We thank colleagues in the group, especially to Dr. James McBride and Oleg Kovtun, for helpful discussions and suggestions. This work was supported by grants from National Institutes of Health (R01EB003778).

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Authors and Affiliations

  1. Department of Chemistry, Vanderbilt University, Nashville, TN, USA

    Jerry C. Chang & Sandra J. Rosenthal

Authors
  1. Jerry C. Chang
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  2. Sandra J. Rosenthal
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Editor information

Editors and Affiliations

  1. Dept. Chemistry, Vanderbilt University, Stevenson Center 7330, Nashville, 37235, Tennessee, USA

    Sandra J. Rosenthal

  2. Dept. Chemistry, Vanderbilt University, Stevenson Center 7330, Nashville, 37235, Tennessee, USA

    David W Wright

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Chang, J.C., Rosenthal, S.J. (2013). Quantum Dot-Based Single-Molecule Microscopy for the Study of Protein Dynamics. In: Rosenthal, S., Wright, D. (eds) NanoBiotechnology Protocols. Methods in Molecular Biology, vol 1026. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-468-5_6

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  • DOI: https://doi.org/10.1007/978-1-62703-468-5_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-467-8

  • Online ISBN: 978-1-62703-468-5

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