Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One s ignificant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field f luorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The incre ase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the ti p interacts with the cell surface. By fitting a regression curve to the far -field fluorescence intensity as the illumination aperture approaches the c ell, it is possible to isolate near-field from far-field fluorescent signal s. We demonstrate the ability to resolve actin filaments in chemically fixe d, hydrated glial cells. A comparison of composite fluorescence signals wit h extracted near-field fluorescence demonstrates that this approach signifi cantly increases the ability to detect subcellular structures at subdiffrac tion resolution.