We study the use of coherent counterpropagating interfering waves to increase threefold to sevenfold the optical bandwidth and the resolution of fluorescence microscopy along the optic axis. Systematic comparison of the point-spread function and the optical transfer function (OTF) for the standing-wave microscope (SWM), the incoherent illumination interference image interference microscope (I5M), and the 4Pi confocal microscope reveals essential differences among their resolution capabilities. It is shown that the OTF's of these microscopes differ strongly in contiguity and amplitude within the enlarged range of transferred frequencies, and therefore they also differ in their ability to provide data from which interference artifacts can be removed. We demonstrate that for practical aperture angles the production of an interference pattern is insufficient for improving the axial resolution by the expected factor of 3-7. Conditions of the OTF for unambiguous improvement of axial resolution of arbitrary objects are fulfilled not at all in the SWM, partially in the I5M, and fully in the two-photon 4Pi confocal microscope.

We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse fibroblast cell. Comparison with two-photon confocal resolution reveals a fourfold better axial resolution in the 4Pi-confocal case. By combining 4Pi-confocal microscopy with Richardson-Lucy image restoration a further resolution increase is achieved. Featuring a threedimensional resolution in the range 100-150 nm, the 4Pi-confocal (restored) images are intrinsically more detailed than their confocal counterparts. Our images constitute what to our knowledge are the best-resolved three-dimensional images of entangled cellular microtubules obtained with light to date. Academic Press

We analyze the ability of nonlinear image restoration to remove interference artifacts in microscopes that enlarge the axial optical bandwidth through coherent counterpropagating waves. We calculate the images of an elaborate test object as produced by confocal, standing-wave, incoherent illumination interference image interference, and 4Pi confocal microscopes, and we subsequently investigate the extent to which the initial object can be restored by the information allowed by their optical transfer function. We find that nonlinear restoration is successful only if the transfer function is sufficiently contiguous and has amplitudes well above the noise level, as is mostly the case in a two-photon excitation 4Pi confocal microscope.

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.