4Pi microscopy deconvolution with a variable point-spread function

Abstract

To remove the axial sidelobes from 4Pi images, deconvolution forms an integral part of 4Pi microscopy. As a result of its high axial resolution, the 4Pi point spread function (PSF) is particularly susceptible to imperfect optical conditions within the sample. This is typically observed as a shift in the position of the maxima under the PSF envelope. A significantly varying phase shift renders deconvolution procedures based on a spatially invariant PSF essentially useless. We present a technique for computing the forward transformation in the case of a varying phase at a computational expense of the same order of magnitude as that of the shift invariant case, a method for the estimation of PSF phase from an acquired image, and a deconvolution procedure built on these techniques.