Detailed Understanding of Dark State Dynamics of Far-Red Fluorescent Proteins

Author
Abstract
The development of fluorescent proteins in the far-red and near-infrared has unlocked new potential for deep-tissue imaging, significantly reducing scatter, cellular autofluorescence, and absorption by cellular constituents compared to their blue-shifted counterparts. These proteins are characterized by low quantum yields, which must be improved to fully utilize these proteins for imaging. The dark state photophysics of near-infrared fluorescent proteins have yet to be characterized, which is necessary to render them useful for imaging and to inform generation of mutants with improved quantum yields. This work discusses my study of the dark state conversion of two far-red fluorescent proteins, smURFP and miRFP670, which both use biliverdin as a chromophore. I designed and optimized the fabrication of microwell devices for the isolation of single proteins. Use of these devices speeds data collection and decreases measurement variability compared to the preceding technique. The microwell devices were used to perform time-resolved fluorescence measurements of smURFP and miRFP670, the results of which indicate the proteins’ effectiveness and guide future generations of far-red mutants.
Year of Publication
2020
Academic Department
Department of Physics
Degree
Ph.D.
Number of Pages
151
Date Published
2020-06
University
University of Colorado Boulder
City
Boulder
JILA PI Advisors
Advisors - Other
Amy E. Palmer
PDF
Publication Status