Point-spread sensitivity analysis for 3-D flourescence microscopy

Previous empirical results suggest that the use of an experimentally determined point-spread function (PSF) instead of a theoretical one improves reconstructions of three-dimensional (3- D) microscopic objects from optical sections. The microscope's PSF is usually measured by imaging a small fluorescent bead. There is a tradeoff in this measurement: very small beads are dim and bleach rapidly, while larger beads are a poorer approximation to a point source. We have simulated the effect of the bead on the shape of the PSF by convolving a theoretically determined PSF (of a 40 X 1.0 N.A. oil-immersion lens) with spheres of varying diameters. Simulated data were generated with a 3-D phantom and the theoretical PSF, which is defined to be the `true' PSF for the simulation. Reconstructions of the phantom were obtained with each of the theoretical PSFs obtained from the beads using a regularized linear least-squares method. Results show a significant drop (more than 50%) in the signal-to-noise ratio of the reconstructions for beads with diameter larger than 0.22 micrometers . These results suggest that the bead used in the PSF measurement should have a diameter less than 30% of the diameter of the first dark ring of the infocus two-dimensional (2-D) PSF. This study quantifies the tradeoff between the quality of the reconstructions and the bead size used in the PSF measurement.