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Imaging Intracellular Quantum Dots: Fluorescence Microscopy and Transmission Electron Microscopy

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

Abstract

Quantum dots (QDs) and other nanoparticles require delivery and targeting for most intracellular applications. Despite many advances, intracellular delivery and targeting remains inefficient with many QDs remaining bound to the plasma membrane rather than internalized into the cell. The fluorescence resulting from these extracellular QDs results in a background signal that competes with intracellular QDs of interest. We present two methods for the reduction and discrimination of signal resulting from plasma membrane-bound QDs. The first method, a photophysical approach, uses an extracellular quencher to greatly reduce the fluorescence signal from extracellular QDs. This method is compatible with fast, widefield, fluorescence imaging in live cells. Results are presented for two extracellular quenchers, QSY-21 and trypan blue, used in combination with 655 nm emitting QDs. The use of an extracellular quencher can be extended to a wide variety of fluorophores. The second method uses transmission electron microscopy (TEM) to image thin (60–70 nm) slices of resin-embedded cells. The use of sectioned cells and high-resolution TEM makes it possible to discriminate between plasma membrane-bound and intracellular QDs. To overcome the difficulties associated with using TEM to image individual QDs in cells, we have utilized a silver enhancement method that significantly improves the contrast of QDs in TEM images.

Key words

Fluorescence microscopy Transmission electron microscopy Quantum dot Quencher Silver enhancement 
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Notes

Acknowledgments

C.K.P. gratefully acknowledges financial support from NIH R01-GM086195.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Georgia Institute of TechnologyAtlantaUSA
  2. 2.Emory UniversityAtlantaUSA

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