Marcia Levitus
Research
Overview Nucleosome Dynamics Photophysics FCS-FRET
Photophysical Properties of Single-Molecule Fluorophores
Photophysical processes are a source of artifacts in single-molecule experiments that have not been thoroughly explored in many cases. Photochemical reactions, such as isomerizations, produce results that can be interpreted as conformational changes of the macromolecule to which the fluorophore is attached. A careful characterization of the photophysical properties of these fluorophores is critical for correct interpretation of experimental results.
We are interested in studying the effect of the environment, attachments, and effect of surfaces on the photophysical properties of fluorophores commonly used in single-molecule applications
For example, we investigated the fluorescence properties of Cy3, one of the most common fluorescent dyes used in single-molecule spectroscopy, and found that its fluorescence efficiency depends strongly on its environment
As an example, the fluoerscence quantum yield of the dye covalently attached to the 5' of a single strand of DNA (A) is 0.38, while the value measured after annealing to the complementary strand (B) drops to 0.17. The fluorescence efficiency of the free dye in solution (C) is 0.08.
We have determined that this variability is caused by a photo-isomerization process which has a quantum efficiency that depends on the local rigidity of the Cy3 molecule.