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Robust normalization protocols for multiplexed fluorescence bioimage analysis
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Date
2016-03-05
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Abstract
The 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.