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

Fluorescent objects closer than the diffraction resolution limit can be dis tinguished in far-field microscopy provided they feature different emission spectra. Utilizing the superior axial resolution of 4Pi-confocal microscop y of 100-150 nm, we investigate the precision with which fluorescence objec ts 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-sp read-function improves the precision of colocalization measurements in doub le stained specimens and opens up the prospect on far-field fluorescence pr ofilometry with (sub) nanometer height resolution. (C) 2000 American Instit ute of Physics. [S0034-6748(00)04007-7].