@phdthesis{12816,
  author = {Srijit Mukherjee},
  title = {Spectroscopic evaluation of excited state depopulation in red fluorescent proteins developed using fluorescence lifetime selections},
  abstract = {Fluorescent Proteins are intriguing molecules that are ubiquitous tools for biological research. This thesis investigates the relationship between engineering improved fluorescent proteins and fundamental physical chemistry principles that drive the molecular design of brighter and more photostable variants of existing fluorescent proteins. This work primarily focuses on designing brighter variants of the red-fluorescent proteins, FusionRed and mCherry, and the consequent physical investigations that lead to increments in brightness. To achieve this, droplet-based and single stream microfluidic sorters that discriminate members of a cell-based mutagenesis library of variants on their excited state lifetime and fluorescence intensity were developed and utilized. These lifetime-based selections led to variants with greater than 3-fold higher values of fluorescence quantum yield in comparison to their progenitors. Finally, the mechanisms and pathways that lead to excited state depopulation after photoexcitation, which govern observables like fluorescence quantum yield, fluorescence brightness, reversible and permanent photobleaching, and blinking at the single molecule level were investigated. This work highlights the progress and the many knowledge gaps in understanding the interwoven relationships of observed photophysics in fluorescent proteins, and suggests ways in which one might further examine the physical basis of brightness and photostability in these molecules.},
  year = {2022},
  journal = {Department of Physics},
  volume = {Ph.D},
  pages = {334},
  month = {2022-07},
  publisher = {University of Colorado Boulder},
  address = {Boulder},
}