Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils.
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.
Borejdo
Julian
J
University of North Texas Health Science Center, Department of Molecular Biology and Immunology, Fort Worth, Texas 76107, USA. jborejdo@hsc.unt.edu
Muthu
Priya
P
Talent
John
J
Gryczynski
Zygmunt
Z
Calander
Nils
N
Akopova
Irina
I
Shtoyko
Tanya
T
Gryczynski
Ignacy
I
D000199:Actins / Q000378:metabolism / Q000528:radiation effects* / Q000648:ultrastructure*; D000818:Animals; D016477:Artifacts; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D018482:Muscle, Skeletal / Q000000981:diagnostic imaging / Q000378:metabolism / Q000528:radiation effects; D009210:Myofibrils / Q000378:metabolism / Q000528:radiation effects* / Q000648:ultrastructure*; D038761:Photobleaching / Q000528:radiation effects; D014463:Ultrasonography
D000199:Actins