Abstract
Live-cell imaging by light microscopy has demonstrated that all cells are spatially and temporally organized. Quantitative, computational image analysis is an important part of cellular imaging, providing both enriched information about individual cell properties and the ability to analyze large datasets. However, such studies are often limited by the small size and variable shape of objects of interest. Here, we address two outstanding problems in bacterial cell division by developing a generally applicable, standardized, and modular software suite termed Projected System of Internal Coordinates from Interpolated Contours (PSICIC) that solves common problems in image quantitation. PSICIC implements interpolated-contour analysis for accurate and precise determination of cell borders and automatically generates internal coordinate systems that are superimposable regardless of cell geometry. We have used PSICIC to establish that the cell-fate determinant, SpoIIE, is asymmetrically localized during Bacillus subtilis sporulation, thereby demonstrating the ability of PSICIC to discern protein localization features at sub-pixel scales. We also used PSICIC to examine the accuracy of cell division in Esherichia coli and found a new role for the Min system in regulating division-site placement throughout the cell length, but only prior to the initiation of cell constriction. These results extend our understanding of the regulation of both asymmetry and accuracy in bacterial division while demonstrating the general applicability of PSICIC as a computational approach for quantitative, high-throughput analysis of cellular images.
Highlights
Biological light microscopy has been pushed to remarkable limits of resolution, speed, throughput, and ease by advances in protein-labeling methods, sample preparation, and microscopy hardware
Our method uses interpolation to find cell borders accurately and precisely. Using these contours as a starting point, we automated the construction of a general-purpose internal coordinate system for each cell to facilitate comparisons between differently shaped cells. We applied this new method to two unsolved problems in bacterial cell biology
We first showed that a Bacillus subtilis asymmetric-division regulator is itself asymmetrically localized, thereby demonstrating our ability to extract information previously thought inaccessible by light microscopy
Summary
Biological light microscopy has been pushed to remarkable limits of resolution, speed, throughput, and ease by advances in protein-labeling methods, sample preparation, and microscopy hardware. This imaging has revealed that the subcellular environments of cells from all kingdoms are exquisitely organized both spatially and temporally. Recent work has moved beyond this method, utilizing interpolation to increase spatial resolution and define more accurate cell borders [3,4,5] We have automated this method in a generally-applicable fashion, using interpolated contours to define cell borders with nearly an order of magnitude greater accuracy and precision than traditional approaches. The modularity, generality, and high-throughput automation of the PSICIC toolkit are such that it can be applied to virtually any type of cell in order to measure many different parameters of shape and localization
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