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Near-field scanning optical microscopy (NSOM) is a scanning probe technique that enables optical measurements to be conducted with nanometric spatial resolution. In addition to high-resolution optical information, NSOM yields a topographic mapping of sample features that enables a direct comparison to be made between surface topography and the optical signal. For the biological sciences, where sophisticated fluorescence labeling protocols have been developed, the simultaneously collected fluorescence and topography information measured with NSOM offers a potentially powerful tool. The progress in implementing NSOM on unfixed, viable samples, however, has been slowed over concerns regarding sample heating and problems associated with damaging forces generated during imaging. Here, we discuss recent measurements that show sample heating to be modest and not limiting for NSOM applications on viable tissues. In addition, we highlight recent work on developing new NSOM probes that have been demonstrated to be amenable with imaging unfixed samples under buffered conditions. These developments now enable the high resolution of NSOM to be applied to areas in the biological sciences that were previously inaccessible. (JALA 2006;11:268–72)

Extending Near-Field Scanning Optical Microscopy for Biological Studies