Simonet, Rowan2025-01-282025-01-282022-08https://hdl.handle.net/11299/269547University of Minnesota M.S. thesis. August 2022. Major: Chemistry. Advisors: Ahmed Heikal, Erin Sheets. 1 computer file (PDF); xii, 132 pages.Living cells are complex and encompass heterogeneous and dynamic environments. Here, we investigated a family of protein-based donor-linker-acceptor constructs in controlled environments of varying macromolecular crowding and ionic strength. Once the donor undergoes two-photon (2P) excitation with 850-nm laser pulses, Förster resonance energy transfer (FRET) can occur from the donor to the acceptor. The sensitivity of these sensors was examined using newly developed time-resolved 2P fluorescence depolarization spectroscopy, where the parallel and perpendicular fluorescence polarizations of mostly the acceptor were simultaneously detected. Our results reveal that the sensitivity of the crowding sensors is dependent upon the donor’s mutations. For ionic strength sensing, the sensitivity of a sensor with two oppositely charged α-helices in the linker region was more sensitive in comparison to a single charged α-helix. Taken together, these optimized methodologies represent a step towards our goal of noninvasive, site-specific, quantitative, and sensitive in vivo mapping of environmental conditions.enAnisotropyBiophysicsDepolarizationFluorescenceFRETTime-resolvedThesis or Dissertation