| To test hypotheses regarding putative roles of water structuring in cell function, a near-field scanning optical microscope system was implemented and adapted for nano-scale fluorescence anisotropy measurement. Technical challenges to be overcome included delivery of polarized light to the sample via a tapered optical fiber, implementation of a suitable shear-force system, choice and design of optimized molecular probes for microviscometry, quantitatively accurate distinction of fluorescence polarization components, and measurement of a small region within a bath of fluorophore. The performance of the system as a near-field microscope and as an accurate monitor of fluorescence anisotropy was verified. Lateral shear force and optical resolutions were found to be 200 nm and 250 nm, respectively, and measured anisotropies in solutions of known viscosity agreed well with theoretical values. When employed in a vertical scanning mode, Z-axis resolution of anisotropy variations was <100μm. Microviscometric results in gels in which water had previously been found to be bound showed a correlation between microviscosity and measured bound fraction (∼1 cp/0.3% bound water; R2 = 0.7435). An untreated glass surface and a hydrophobic surface were assayed for induction of microviscometric deviation in the water above them; none was found (detection limit ∼μm and 10 cp in the case of untreated glass, and ∼30 nm and ∼0.1 cp in the case of the hydrophobic surface). Water above a cleaved mica surface showed an increased microviscosity over a range of several microns. In its first biological application, the system was used to investigate the triple-banding pattern seen in previous shear-force images of myofibril bundles, showing them to be optical in origin. |
