title: Single-molecule imaging in live cell using gold nanoparticles.
abstract: 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.
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Cécile
authors: C
authors: Leduc
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Satyabrata
authors: S
authors: Si
authors: CNRS, Laboratoire d'Enzymologie et Biochimie Structurales, Gif sur Yvette, France.
authors: Jérémie J
authors: JJ
authors: Gautier
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Zhenghong
authors: Z
authors: Gao
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Edakkattuparambil S
authors: ES
authors: Shibu
authors: CNRS, Laboratoire d'Enzymologie et Biochimie Structurales, Gif sur Yvette, France.
authors: Alexis
authors: A
authors: Gautreau
authors: Univ Bordeaux, Interdisciplinary Institute for Neuroscience UMR 5297, CNRS, Bordeaux, France.
authors: Grégory
authors: G
authors: Giannone
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Laurent
authors: L
authors: Cognet
authors: Univ Bordeaux, Laboratoire Photonique Numérique et Nanosciences, Institut d'Optique & CNRS, Talence, France.
authors: Brahim
authors: B
authors: Lounis
mesh_terms: D000818:Animals; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D004705:Endocytosis; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D057054:Molecular Imaging / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D006046:Gold
keywords: Biofunctionalization; Gold nanoparticles; Nanobodies; Photothermal imaging; Single-molecule tracking