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© Research
Publication : IEEE International Symposium on Biomedical Imaging ISBI

Improving single particle localization with an empirically calibrated Gaussian kernel

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in IEEE International Symposium on Biomedical Imaging ISBI - 17 May 2008

Marcio Marim, Bo Zhang, Jean-Christophe Olivo-Marin and Christophe Zimmer

IEEE International Symposium on Biomedical Imaging ISBI, 2008

Accurate computational localization of single fluorescent particles is of interest to many biophysical studies and underlies recent approaches to high resolution microscopy using photo-switchable fluorophores. The position of individual particles is typically computed by least-squares fitting of aGaussian intensity profile to the image, whose band-width is either derived from an idealized theoretical model of the point spread function (PSF), or itself fitted to the image. However, the band-width best approximating the actual PSF may differ significantly from its theoretical value, while fitting it is expected to degrade localization accuracy. Here, instead, we measure the real PSF bandwidth using fluorescent beads as calibration probes, and use this new bandwidth in a Gaussian model fitting algorithm. We show on simulated and real images that this simple modification of the standardlocalization procedure results in significant improvement of the 3D accuracy in the nanometer range.