A Tool for Alignment and Averaging of Sparse Fluorescence Signals in Rod-Shaped Bacteria

Fluorescence microscopy studies have shown that many proteins localize to highly specific subregions within bacterial cells. Analyzing the spatial distribution of low-abundance proteins within cells is highly challenging because information obtained from multiple cells needs to be combined to provide well-defined maps of protein locations. We present (to our knowledge) a novel tool for fast, automated, and user-impartial analysis of fluorescent protein distribution across the short axis of rod-shaped bacteria. To demonstrate the strength of our approach in extracting spatial distributions and visualizing dynamic intracellular processes, we analyzed sparse fluorescence signals from single-molecule time-lapse images of individual Escherichia coli cells. In principle, our tool can be used to provide information on the distribution of signal intensity across the short axis of any rod-shaped object.