Single-molecule imaging in live cell using gold nanoparticles.
Methods Cell Biol
; 125: 13-27, 2015.
Article
em En
| MEDLINE
| ID: mdl-25640421
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Nanopartículas Metálicas
/
Imagem Molecular
/
Ouro
Limite:
Animals
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article