Nanoscale structure and deformation in soft materials revealed by single-molecule localization and orientation

Fluorescent dyes have enabled dramatic advances in the life sciences, where protein-specific labeling enables structure/function correlations to be made. In addition, the ability to engineer fluorescent dye lifetimes to respond to e.g., viscosity, pH, and oxygen, or Ca²⁺ concentration, makes them powerful probes of dynamic phenomena. The advent of single-molecule super-resolution methods has served to extend this powerful technique to the nanoscale. However, the use of single-molecule fluorescence imaging methods in materials science has been slower to develop. One principal reason for this is that, in materials, fluorophore orientation is often fixed. Single-molecule images therefore have a complex, orientation-dependent structure, that, if not correctly accounted for, can lead to large errors in localization. More sophisticated approaches to fitting single-molecule images can provide not only accurate localization data, but also information on the orientation of individual fluorophores. I will discuss our progress in making accurate and precise measurements of fluorophore position and orientation in materials to enable high-resolution imaging, our development of a straightforward approach to determine how localization uncertainty and fluorophore labeling density together limit our ability to resolve nanoscale structures, how lithographic patterning enables us to partially overcome that limit, and how single-molecule orientation measurements can provide information on deformation in polymers at the 10 nm length scale. Finally, I will speculate on how measurement of single-molecule fluorescence lifetimes might provide information on local polymer dynamics in complex systems.