Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to <30 nm near the antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 +/- 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 +/- 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range.

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We report on the development of micro/nanofabrication processes to create hierarchical surface topographies that expand from 50 nm to microns in size on different materials. Three different approaches (named FIB1, FIB2, and EBL) that combine a variety of techniques such as photolithography, reactive ion etching, focused ion beam lithography, electron beam lithography, and soft lithography were developed, each one providing different advantages and disadvantages. The EBL approach was employed to fabricate substrates comprising channels with features between 200 nm and 10 μm in size on polymethylmethacrylate (PMMA), which were then used to investigate the independent or competitive effects of micro- and nanotopographies on cell adhesion and morphology. Rat mesenchymal stem cells (rMSCs) were cultured on four different substrates including 10 μm wide and 500 nm deep channels separated by 10 μm distances (MICRO), 200 nm wide and 100 nm deep nanochannels separated by 200 nm distances (NANO), their combination in parallel (PARAL), and in a perpendicular direction (PERP). Rat MSCs behaved differently on all tested substrates with a high degree of alignment (as measured by both number of aligned cells and average angle) on both NANO and MICRO. Furthermore, cells exhibited the highest level of alignment on PARAL, suggesting a synergetic effect of the two scales of topographies. On the other hand, cells on PERP exhibited the lowest alignment and a consistent change in morphology over time that seemed to be the result of interactions with both micro- and nanochannels positioned in the perpendicular direction, also suggesting a competitive effect of the topographies.

A technique for imparting micro- and nanostructured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures approximately 60 nm tall and approximately 500 x 500 nm(2) to be produced on freestanding approximately 0.5 mm thick sheets of the polymer when cured at 293 K, and structures approximately 400 nm tall and 5 x 5 microm(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (approximately 200 microm thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

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