Systematic Method for the Kinetic Modeling of Temporally Resolved Hyperspectral Microscope Images of Fluorescently Labeled Cells

In this paper we report the application of a novel method for fitting kinetic models to temporally resolved hyperspectral images of fluorescently labeled cells to mathematically resolve pure-component spatial images, pure-component spectra, and pure-component reaction profiles. The method is demonstrated on one simulated image and two experimental cell images, including human embryonic kidney cells (HEK 293) and human A549 pulmonary type II epithelial cells. In both cell images, inhibitor kappa B kinase alpha (IKKα) and mitochondrial antiviral signaling protein (MAVS) were labeled with green and yellow fluorescent protein, respectively. Kinetic modeling was performed on the compressed images by using a separable least squares method. A combination of several first-order decays were needed to adequately model the photo-bleaching processes for each fluorophore observed in these images, consistent with the hypothesis that each fluorophore was found in several different environments within the cells. Numerous plausible mechanisms for kinetic modeling of the photo-bleaching processes in these images were tested and a method for selecting the most parsimonious and statistically sufficient model was used to prepare spatial maps of each fluorophore.