Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.
J Biomed Opt
; 13(3): 034021, 2008.
Article
em En
| MEDLINE
| ID: mdl-18601566
ABSTRACT
Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Técnicas de Sonda Molecular
/
Actinas
/
Microscopia de Força Atômica
/
Microscopia de Fluorescência
/
Miofibrilas
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Biomed Opt
Assunto da revista:
ENGENHARIA BIOMEDICA
/
OFTALMOLOGIA
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos