Fluorescent proteins have revolutionized imaging due to their biological compatibility. However, the red fluorescent protein (RFP) variants, whose emission and excitation maxima lie in a window optically favorable for in vivo imaging, remain sub optimal with respect to their quantum yield, photostability, and blinking (dark-state conversion) properties. To optimize these properties, a deeper understanding of the excited state proteinsâ€™ flexibility is required. This project used ultrafast spectroscopy techniques to determine the flexibility of the chromophore and chromophore pocket for a series of RFP mutants. Flexibility was then correlated to specific point mutations in order to determine a given residueâ€™s effect on dark-state formation. Time-resolved fluorescence upconversion spectroscopy and time correlated single photon counting was used to reconstruct the time-dependent emission spectra of each RFP. From these spectra, the time-dependent Stokes shift and spectral density was used to determine pocket flexibility and a given residueâ€™s perturbation of the spectral density. Time-dependent anisotropy decays for a given wavelength were also collected to establish chromophore flexibility. Collectively, this data helps clarify how given mutations to a chromophore pocket correlate to changes in dark-state formation and will be used to design future RFP mutants in an attempt to decrease dark-state conversion and photobleaching. Furthermore, this project had broader impacts that are equally as important as the scientific progress made. Since 1990, approximately 3,700 papers have been published on fluorescent proteins. Of these, Japan and the United States have contributed the vast majority (approximately 2,300). However, only approximately 80 papers (< 4%) have been the result of collaborative attempts between the two countries. This project fostered collaboration between labs in the USA and Japan, which are of synergistic interests but lack collaborative communication. This project also presented the unique opportunity to experience Japanese culture and better understand the scientific climate in Japan and will facilitate future collaborative attempts between the USA and Japan. Projects such as these play an important roll in driving science in the USA forward during this present era where collaborative attempts facilitate scientific success.