Microviscosity in the cell membrane and the cell cytoplasm are important parameters in cell signaling and disease. Presently, viscosity on the microscopic scale is determined by fluorescence anisotropy, fluorescence recovery after photobleaching (FRAP), or magnetic nanoparticles. A group of viscosity-sensitive fluorescent molecules, generally termed molecular rotors, allows a new, fast, and convenient approach with minimum requirements of instrumentation and very high temporal and spatial resolution. However, molecular rotors pose one disadvantage - they are intensity based, and local concentration gradients may reduce measurement accuracy. The proposed research program builds on a recently developed ratiometric dye (J.Am.Chem.Soc. 2006;128: 398-399) in which a molecular rotor and a fluorescent reference unit form a covalently linked dye pair for resonance energy transfer (RET). This ratiometric dye has the potential to overcome limitations posed by concentration gradients and optical properties. The overall goal of the proposed research is to develop specific fluorescent ratiometric viscosity sensors to be used in phospholipid bilayers, cell membranes, and the cell cytoplasm. We propose the synthesis, characterization, and optimization of membrane-targeted as well as cytoplasm-targeted ratiometric molecular rotors. The experimental approach includes the testing and application of the new viscosity sensitive dyes in model phospholipid bilayers, red cell ghosts, and living cells. This testing approach allows us to characterize the new probes in environments of increasing complexity. The outcome of the proposed research will be the availability of a series of real-time, microscale viscosity probes for cellular environments with a wide range of applications. Some examples where the new viscosity probes will be useful are: - Analysis of the involvement of the cell membrane in cell signaling under fluid shear stress (vascular endothelial cells) - Analysis of changes in cell membrane biomechanics in atherosclerosis - Studies involving membrane lipid rafts - Analysis of the role of cytoplasmic viscosity in the cryopreservation of cells While it is not the goal of this application to actually apply molecular rotors in the above examples, we will provide the necessary probes as well as their methods of use for investigators involved in any fields listed above or related. Public Health Relevance Statement: Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states. Cell membrane and cytoplasmic viscosity are of high relevance to cell signaling (e.g. blood pressure regulation) and to various disease states (e.g. altered membrane viscosity related to atherosclerosis, cell malignancy, hypercholesterolemia, and diabetes). We propose to develop new, ultrafast and ultra-high resolution methods to determine changes in membrane and cytoplasm viscosity using fluorescent molecular rotors. With these new tools, studies involving viscosity in the cell will be accelerated or made possible in the first place, thus enabling faster study and better understanding of cell signaling processes and the cellular foundations of various disease states.