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Fluorescent objects closer than the diffraction resolution limit can be distinguished in far-field microscopy provided they feature different emission spectra. Utilizing the superior axial resolution of 4Pi-confocal microscopy of 100–150 nm, we investigate the precision with which fluorescence objects with subdiffraction axial distance can be measured in the far field. At a wavelength of 820 nm distances on the order of 60 nm between beads and a monomolecular Langmuir–Blodgett layer were determined with a precision of 1.2 nm within 3.2 s. The reduced spatial extent of the 4Pi-confocal point-spread-function improves the precision of colocalization measurements in double stained specimens and opens up the prospect on far-field fluorescence profilometry with (sub) nanometer height resolution.

Subresolution axial distance measurements in far-field fluorescence microscopy with precision of 1 nanometer