Point-spread sensitivity analysis for 3-D flourescence microscopy

Chrysanthe Preza; John M. Ollinger; James G. McNally; Lewis J. Thomas Jr.

doi:10.1117/12.59555

26 June 1992 Point-spread sensitivity analysis for 3-D flourescence microscopy

Chrysanthe Preza, John M. Ollinger, James G. McNally, Lewis J. Thomas Jr.

Proceedings Volume 1660, Biomedical Image Processing and Three-Dimensional Microscopy; (1992) https://doi.org/10.1117/12.59555
Event: SPIE/IS&T 1992 Symposium on Electronic Imaging: Science and Technology, 1992, San Jose, CA, United States

Abstract

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.

Citation Download Citation

Chrysanthe Preza, John M. Ollinger, James G. McNally, and Lewis J. Thomas Jr. "Point-spread sensitivity analysis for 3-D flourescence microscopy", Proc. SPIE 1660, Biomedical Image Processing and Three-Dimensional Microscopy, (26 June 1992); https://doi.org/10.1117/12.59555

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