TY - JOUR
T1 - Reduction of photobleaching and photodamage in single molecule detection
T2 - Observing single actin monomer in skeletal myofibrils
AU - Borejdo, Julian
AU - Muthu, Priya
AU - Talent, John
AU - Gryczynski, Zygmunt
AU - Calander, Nils
AU - Akopova, Irina
AU - Shtoyko, Tanya
AU - Gryczynski, Ignacy
N1 - Funding Information:
This work was supported by grant number RO1 AR048622 (to Borejdo) from NIH.
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
KW - actin
KW - nanoparticle monolayers
KW - photobleaching
KW - single molecule detection
UR - http://www.scopus.com/inward/record.url?scp=51649096148&partnerID=8YFLogxK
U2 - 10.1117/1.2938689
DO - 10.1117/1.2938689
M3 - Article
C2 - 18601566
AN - SCOPUS:51649096148
SN - 1083-3668
VL - 13
JO - Journal of Biomedical Optics
JF - Journal of Biomedical Optics
IS - 3
M1 - 034021
ER -