(490h) Single-Molecule Localization and Orientation for Super-Resolution Microscopy

Structural characterization by super-resolution microscopy has become increasingly widespread, particularly in the biological community. The technique is powerful because it can produce real-space images with resolutions of tens of nanometers, while sample preparation is relatively non-invasive. Previous studies have applied these techniques to important scientific problems in the life sciences, but relatively little work has explored the attainable limit of resolution using samples of known structure. In this work, we apply photo-activated localization microscopy (PALM) to polymer films that have been nanopatterned using electron-beam lithography. Trace amounts of a rhodamine spiroamide dye are dispersed into nanostructured poly(methyl methacrylate), and UV-induced switching of the fluorophores enables nanoscale localization of single molecules to generate a final composite super-resolution image. Features as small as 25 nm half-pitch are clearly resolvable.

We also explore the effect of molecular orientation of fluorophores, which can be used to report nanoscale chemical environment and dynamics in materials. Fluorophore orientation alters the single-molecule point-spread function, which can introduce systematic mislocalizations in imaging. Here, we avoid these phenomena by simultaneously determining single-molecule position and orientation. Distributions of orientations are then collected in response to illumination polarization, as well as mechanical stress and deformation.