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PLoS One. 2015 Jul 28;10(7):e0134112. doi: 10.1371/journal.pone.0134112. eCollection 2015.

Fluorescent Microspheres as Point Sources: A Localization Study.

Author information

1
Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America; Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, United States of America.
2
Department of Electrical Engineering, University of Texas at Dallas, Richardson, Texas, United States of America.
3
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, United States of America; Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, College Station, Texas, United States of America.

Abstract

The localization of fluorescent microspheres is often employed for drift correction and image registration in single molecule microscopy, and is commonly carried out by fitting a point spread function to the image of the given microsphere. The mismatch between the point spread function and the image of the microsphere, however, calls into question the suitability of this localization approach. To investigate this issue, we subject both simulated and experimental microsphere image data to a maximum likelihood estimator that localizes a microsphere by fitting an Airy pattern to its image, and assess the suitability of the approach by evaluating the ability of the estimator to recover the true location of the microsphere with the best possible accuracy as determined based on the Cramér-Rao lower bound. Assessing against criteria based on the standard errors of the mean and the variance for an ideal estimator of the microsphere's location, we find that microspheres up to 100 nm in diameter can in general be localized using a fixed width Airy pattern, and that microspheres as large as 1 μm in diameter can in general be localized using a floated width Airy pattern.

PMID:
26218251
PMCID:
PMC4517909
DOI:
10.1371/journal.pone.0134112
[Indexed for MEDLINE]
Free PMC Article

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