Tip diameter and transmission efficiency of a visible-wavelength near-field optic probe determine both the lateral spatial resolution and experimental utility of the near-field scanning optical microscope. The commonly used tip fabrication technique, laser-heated pulling of fused-silica optical fiber followed by aperture formation through aluminization, is a complex process governed by a large number of parameters. An extensive study of the pulling parameter space has revealed a time-dependent functionality between the various pulling parameters dominated by a photon-based heating mechanism. The photon-based heat source results in a temperature and viscosity dependence that is a complex function of time and fiber diameter. Changing the taper of the optical probe can affect transmission efficiency by an order of magnitude or more.

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Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.

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