This study has been carried out to explore the potential cell culture applications for molecular rotors, a family of fluorescent molecules with the ability to undergo intramolecular rotation upon photoexcitation. The relationship between intramolecular rotation and fluorescence quantum yield is proportional to environmental free volume, which can be related to viscosity. New insights into the biomechanical properties of cells, which includes plasma membrane viscosity, are revealing the importance of these properties and how they relate to cell state and disease. Membrane viscosity describes the ease of movement within the phospholipid bilayer, and directly influences physiological properties such as membrane-bound enzyme activity, carrier-mediated transport, and membrane-bound receptor binding. Many methods exist that directly or indirectly report membrane physical properties, but these methods are time-consuming and of limited spatial resolution. Molecular rotors are ideally suited for characterizing membrane viscosity because of their excellent spatial and temporal response. We have synthesized and characterized a ratiometric family of molecular rotors for this purpose. Human fibroblast cells are grown on polyacrylamide gels to induce changes in cell mechanics, which is verified by atomic force microscopy. Molecular rotors are applied to demonstrate the relationship between cell stiffness and membrane viscosity. Another objective was to establish molecular rotors as a tool for measuring membrane viscosity in cells grown as 3D cultures. We design and fabricate a custom 3D culture platform for this purpose. To optimize these efforts, we have customized a Nikon PCM2000 confocal microscope system for the purpose of imaging the ratiometric molecular rotor emission.