Recent work has demonstrated that imaging platforms based on the integration of total internal reflection fluorescence microscopy (TIRFM)/spectroscopy with scanning probe microscopy are well-suited for investigations of biological phenomena, ranging from lipid bilayer dynamics and membrane protein assembly to cellular dynamics and structures. We have shown that this approach is particularly compelling for studies of protein-membrane interactions. While this functional imaging strategy has proved to be very useful for studies of cell dynamics, and most recently, in the study of single-molecule dynamics by TIRF-AFM-based force spectroscopy, a number of key challenges remain to be resolved.