Image segmentation and separation of spectrally similar dyes in fluorescence microscopy by dynamic mode decomposition of photobleaching kinetics

Abstract

Background: Image segmentation in fluorescence microscopy is often based on spectral separation of fluorescent probes (color-based segmentation) or on significant intensity differences in individual image regions (intensity-based segmentation). These approaches fail, if dye fluorescence shows large spectral overlap with other employed probes or with strong cellular autofluorescence. Results: Here, a novel model-free approach is presented which determines bleaching kinetics based on dynamic mode decomposition (DMD) and uses the inferred photobleaching kinetics to distinguish different probes or dye molecules from autofluorescence. DMD is a data-driven computational method for detecting and quantifying dynamic events in complex spatiotemporal data. Here, DMD is used to determine photobleaching characteristics of a fluorescent sterol probe, dehydroergosterol (DHE), compared to that of cellular autofluorescence in the nematode Caenorhabditis elegans. It is shown that decomposition of those dynamic modes allows for precise image segmentation, thereby separating probe from autofluorescence without invoking a particular model for the bleaching process. In a second application, DMD of dye-specific photobleaching is used to separate two green-fluorescent dyes, an NBD-tagged sphingolipid and Alexa488-transferrin, thereby assigning them to different cellular compartments. Conclusions: Data-based decomposition of dynamic modes can be employed to analyze spatially varying photobleaching of fluorescent probes in cells and tissues for image segmentation, discrimination of probe from autofluorescence and image denoising. The new method should find wide application in analysis of dynamic fluorescence imaging data.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells

“…DMD has been used to detect video shots or discriminate foreground from background in videos of natural scenes; it has also been used to segment images of human kidneys and detect functional brain states by magnetic resonance imaging image sequences [43][44][45][46]. In a recent study, the potential of DMD for use in the analysis of microscopy data was demonstrated by determining the photobleaching characteristics of fluorescent probes to distinguish probe fluorescence from cellular autofluorescence [47]. Here, DMD is applied to analyze simulated FLIP images and FLIP sequences of enhanced green fluorescent protein (eGFP) and of eGFP-tagged mtHtt (eGFP-mtHtt).…”