The development of near-infrared fluorescent proteins has unlocked new potential for deep-tissue imaging due to the low scattering, cellular autofluorescence, and lower absorption by cellular constituents in this portion of the spectrum. These proteins are characterized by low quantum yields, which must be improved to fully utilize these proteins. The dark state photophysics of near-infrared fluorescent proteins have yet to be characterized, and understanding this behavior is needed to select the proper protein for an experiment and to inform generation of mutants with improved quantum yields. This work discusses my study of the dark state conversion of two near-infrared fluorescent proteins, smURFP and miRFP67,0 which both use biliverdin as a chromophore. To study this problem, I designed and optimized the fabrication of microwell devices, a technique which sped up collection time and decreased measurement variability. The microwell devices were used to perform time-resolved fluorescence measurements of fluorescent proteins, the results of which can be used to inform generation of new mutants as well as experimental applications of these proteins.
Samantha Allen / JILA (Advisor: Ralph Jimenez)
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