title: Three-Dimensional Spot Detection in Ratiometric Fluorescence Imaging For Measurement of Subcellular Organelles.
abstract: Lysosomes are subcellular organelles playing a vital role in the endocytosis process of the cell. Lysosomal acidity is an important factor in assuring proper functioning of the enzymes within the organelle, and can be assessed by labeling the lysosomes with pH-sensitive fluorescence probes. To enhance our understanding of the acidification mechanisms, the goal of this work is to develop a method that can accurately detect and characterize the acidity of each lysosome captured in ratiometric fluorescence images. We present an algorithm that utilizes theh-dome transformation and reconciles spots detected independently from two wavelength channels. We evaluated our algorithm using simulated images for which the exact locations were known. Theh-dome algorithm achieved anf-score as high as 0.890. We also computed the fluorescence ratios from lysosomes in live HeLa cell images with known lysosomal pHs. Using leave-one-out cross-validation, we demonstrated that the new algorithm was able to achieve much better pH prediction accuracy than the conventional method.
authors.affiliation: Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: William W
authors.initials: WW
authors.lastname: Lau
authors.affiliation: Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Calvin A
authors.initials: CA
authors.lastname: Johnson
authors.affiliation: Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Lioi
authors.affiliation: Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Mindell
keywords: Spot detection; h-dome transformation; lysosomes; microscopy; pH; ratiometric imaging