Axial sidelobe reduction in single-photon 4Pi microscopy by Toraldo filters

Full text: The 4Pi-confocal fluorescence microscope is a recently developed 3D imaging technique in which two opposing high-NA objectives are used for coherently illuminating and/or detecting the same point of the fluorescent sample. The interference process yields an intensity point spread function (PSF) with an extremely narrow axial core, but with very large axial sidelobes, which compromise the actual improvement in axial resolution. To overcome this problem we propose the use, in the illumination arm of the 4Pi-confocal microscope, of multiple-zones phase filters whose design is based on the Toraldo-design principle. Note that the Toraldo procedure allows to select at will the positions of the zeros of the PSF of an optical system. Then, what we propose here if to design a phase pupil filter such that the position of the first zero of the illumination axial PSF is close to the position of the maximum of the first axial sidelobe of the detection PSF. In the design procedure it is taken into account that: 1. The value of the parameter ε = λexc /λdet which, in a single-photon fluorescent process, is the responsible for the different scales of the illumination and detection PSFs. 2. The Toraldo procedure was originally designed to control the position of zeros of the transverse PSF. In this case the procedure is adapted to the aim of controlling the position of zeros of the axial PSF. 3. Since 4Pi-confocal microscopes are only useful when built with high-NA objectives, the Toraldo principle is reformulated in terms of the nonparaxial diffraction theory. We show that by using Toraldo filters in the illumination part of a 4Pi-confocal microscope it is possible to obtain up to a 60% reduction of height of the axial sidelobe of the whole-system axial PSF. This fact permits to fully benefit the axial resolution from the strong narrowness of the central peak of the axial PSF, inherent to the 4Pi principle. Copyright (2002) Australian Society for Electron Microscopy Inc