title: Robust normalization protocols for multiplexed fluorescence bioimage analysis.
abstract: study of mapping and interaction of co-localized proteins at a sub-cellular level is important for understanding complex biological phenomena. One of the recent techniques to map co-localized proteins is to use the standard immuno-fluorescence microscopy in a cyclic manner (Nat Biotechnol 24:1270-8, 2006; Proc Natl Acad Sci 110:11982-7, 2013). Unfortunately, these techniques suffer from variability in intensity and positioning of signals from protein markers within a run and across different runs. Therefore, it is necessary to standardize protocols for preprocessing of the multiplexed bioimaging (MBI) data from multiple runs to a comparable scale before any further analysis can be performed on the data. In this paper, we compare various normalization protocols and propose on the basis of the obtained results, a robust normalization technique that produces consistent results on the MBI data collected from different runs using the Toponome Imaging System (TIS). Normalization results produced by the proposed method on a sample TIS data set for colorectal cancer patients were ranked favorably by two pathologists and two biologists. We show that the proposed method produces higher between class Kullback-Leibler (KL) divergence and lower within class KL divergence on a distribution of cell phenotypes from colorectal cancer and histologically normal samples.
authors.affiliation: Department of Computer Science, University of Warwick, Coventry, CV4 7AL UK.
authors.forename: Shan E
authors.initials: SE
authors.lastname: Ahmed Raza
authors.affiliation: Biodata Mining Group, Bielefeld University, Bielefeld, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Langenkämper
authors.affiliation: Department of Computer Science, University of Warwick, Coventry, CV4 7AL UK.
authors.forename: Korsuk
authors.initials: K
authors.lastname: Sirinukunwattana
authors.affiliation: Mathematics Institute, University of Warwick, Coventry, CV4 7AL UK.
authors.forename: David
authors.initials: D
authors.lastname: Epstein
authors.affiliation: Biodata Mining Group, Bielefeld University, Bielefeld, Germany.
authors.forename: Tim W
authors.initials: TW
authors.lastname: Nattkemper
authors.affiliation: Department of Computer Science, University of Warwick, Coventry, CV4 7AL UK ; Department of Computer Science and Engineering, Qatar University, Doha, Qatar.
authors.forename: Nasir M
authors.initials: NM
authors.lastname: Rajpoot
keywords: Bioimage informatics; Multiplexed fluorescence imaging; Normalization protocols; Protein signatures; Toponome imaging system