A method of PSF generation for 3D brightfield deconvolution

Summary This paper addresses the problem of 3D deconvolution of through focus widefield microscope datasets ( Z ‐stacks). One of the most difficult stages in brightfield deconvolution is finding the point spread function. A theoretically calculated point spread function (called a ‘synthetic PSF’ in this paper) requires foreknowledge of many system parameters and still gives only approximate results. A point spread function measured from a sub‐resolution bead suffers from low signal‐to‐noise ratio, compounded in the brightfield setting (by contrast to fluorescence) by absorptive, refractive and dispersal effects. This paper describes a method of point spread function estimation based on measurements of a Z ‐stack through a thin sample. This Z ‐stack is deconvolved by an idealized point spread function derived from the same Z ‐stack to yield a point spread function of high signal‐to‐noise ratio that is also inherently tailored to the imaging system. The theory is validated by a practical experiment comparing the non‐blind 3D deconvolution of the yeast Saccharomyces cerevisiae with the point spread function generated using the method presented in this paper (called the ‘extracted PSF’) to a synthetic point spread function. Restoration of both high‐ and low‐contrast brightfield structures is achieved with fewer artefacts using the extracted point spread function obtained with this method. Furthermore the deconvolution progresses further (more iterations are allowed before the error function reaches its nadir) with the extracted point spread function compared to the synthetic point spread function indicating that the extracted point spread function is a better fit to the brightfield deconvolution model than the synthetic point spread function.