Near-field scanning optical microscopy probes: a comparison of pulled and double-etched bent NSOM probes for fluorescence imaging of biological samples.

Abstract

Bent near-field optical probes for biological applications have been fabricated using a combination of a two-step chemical etching method and focused ion beam milling to create a well-defined aperture. The transmission efficiencies have been evaluated as a function of laser wavelength (lambda) and aperture size (D) for both large and small core fibres. The probe transmission behaviour follows a (D/lambda)3 relationship. The double-etched probes are compared to pulled probes fabricated from highly GeO2-doped dispersion compensating fibre and a standard single-mode optical fibre. The transmission efficiencies of both types of pulled probes are approximately two orders of magnitude lower than double-etched probes with similar aperture sizes. To demonstrate the utility of the various probes, their imaging performance has been evaluated for samples of polymer beads and phase-separated phospholipid monolayers of dipalmitoylphosphatidylcholine or cholesterol/phosphatidylcholine/sphingomyelin mixtures. Both pulled and double-etched probes are suitable for fluorescence imaging of polymer spheres. However, pulled probes are rapidly damaged at the higher input laser intensities required for fluorescence imaging of monolayer samples doped with < 1% of a fluorescent dye-labelled lipid. The images obtained with the double-etched probes show excellent spatial resolution and signal/noise, illustrating the potential of such probes for imaging of biological samples.