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We can resolve multiple discrete features within a focal region of m spatial dimensions by first isolating each on the basis of n >/= 1 unique optical characteristics and then measuring their relative spatial coordinates. The minimum acceptable separation between features depends on the point-spread function in the (m + n)d-dimensional space formed by the spatial coordinates and the optical parameters, whereas the absolute spatial resolution is determined by the accuracy to which the coordinates can be measured. Estimates of each suggest that near-field fluorescence excitation microscopy/spectroscopy with molecular sensitivity and spatial resolution is possible.
Commentary: Inspired by my earlier work (see below) in single molecule imaging and the isolation of multiple exciton recombination sites within a single probe volume, here I proposed the principle which would eventually lead to PALM. Indeed, all methods of localization microscopy, including PALM, fPALM, PALMIRA, STORM, dSTORM, PAINT, GSDIM, etc. are specific embodiments of the general principle of single molecule isolation and localization I introduced here.