Single-molecule imaging in live cell using gold nanoparticles.

Cécile Leduc, Satyabrata Si, Jérémie J. Gautier, Zhenghong Gao, Edakkattuparambil S. Shibu, Alexis Gautreau, Grégory Giannone, Laurent Cognet, Brahim Lounis
Methods in Cell Biology. 2015-01-01; : 13-27
DOI: 10.1016/bs.mcb.2014.10.002

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1. Methods Cell Biol. 2015;125:13-27. doi: 10.1016/bs.mcb.2014.10.002. Epub 2015 Jan

Single-molecule imaging in live cell using gold nanoparticles.

Leduc C(1), Si S(1), Gautier JJ(2), Gao Z(1), Shibu ES(1), Gautreau A(2),
Giannone G(3), Cognet L(1), Lounis B(1).

Author information:
(1)Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut
d’Optique & CNRS, Talence, France.
(2)CNRS, Laboratoire d’Enzymologie et Biochimie Structurales, Gif sur Yvette,
(3)Univ Bordeaux, Interdisciplinary Institute for Neuroscience UMR 5297, CNRS,
Bordeaux, France.

Optimal single particle tracking experiments in live cells requires small and
photostable probes, which do not modify the behavior of the molecule of interest.
Current fluorescence-based microscopy of single molecules and nanoparticles is
often limited by bleaching and blinking or by the probe size. As an alternative,
we present in this chapter the synthesis of a small and highly specific gold
nanoprobe whose detection is based on its absorption properties. We first present
a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them
with a nanobody, a single-domain antibody from camelid, targeting the widespread
green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we
describe how to detect and track these individual gold nanoparticles in live cell
using photothermal imaging microscopy. The combination of a probe with small
size, perfect photostability, high specificity, and versatility through the vast
existing library of GFP-proteins, with a highly sensitive detection technique
enables long-term tracking of proteins with minimal hindrance in confined and
crowded environments such as intracellular space.

Copyright © 2015 Elsevier Inc. All rights reserved.

DOI: 10.1016/bs.mcb.2014.10.002
PMID: 25640421 [Indexed for MEDLINE]

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