PSICIC: Noise and Asymmetry in Bacterial Division Revealed by Computational Image Analysis at Sub-Pixel Resolution

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

Pinch position measurements in <i>E. coli</i>.

<p>(A) Schematic showing an asymmetrically dividing cell, indicating the geometric midpoint of the cell (solid line), the pinch position and width, the distance of the pinch position from midcell (double-headed solid arrow), and the maximal cell width. (B) Distribution of the distance of the pinch position from midcell, as a percentage of cell length, shown for wild-type (black line) and Δ<i>minC</i> (gray shading) strains. (C) Scatter plot of pinch position versus the depth of the pinch for wild type (green dots) and Δ<i>minC</i> (blue circles). Standard deviation is shown for wild type (solid green curve), Δ<i>minC</i> (solid blue curve), and the theoretical limits of PSICIC (dashed black curve, see also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000233#pcbi-1000233-g002" target="_blank">Figure 2C</a>).</p

Measurement of beads of known size.

<p>(A) Phase contrast image of 1 µm diameter beads (100× magnification) with PSICIC identification of outlines overlaid (blue lines). (B) Comparison of the size in microns of: a pixel in these images, mean bead size measured by PSICIC, mean bead size measured by electron microscopy, typical <i>E. coli</i> width, and typical <i>E. coli</i> length. (C) The distribution of bead sizes as measured by PSICIC (gray bars) compared to the expected distribution obtained from electron microscopy data (dashed curve).</p

Schematic view of the implementation of PSICIC.

<p>(A) The original image, prior to analysis. (B) The set of points (red dots) at which the image intensity crosses a given threshold is calculated, defining a contour for that cell. The given points are unevenly distributed. (C) The pair of points (stars) on the contour that are the greatest Euclidean distance apart are chosen as a first approximation of the poles. The choice of poles divides the contour into two curves (called “left” and “right” for simplicity). (D) An equal number of points (blue triangles) are evenly distributed along the left and the right curves, such that the distances between points on the left curve are all equal, but not necessarily equal to the distances between points on the right curve. (E) Each point on the left curve is paired with the corresponding point on the right curve, and a straight line, called a “width line” (blue lines) is drawn connecting the pair. (F) The midline (dotted green line) is drawn through the midpoint of each of the width lines. (G) Each pole is moved stepwise and the process described above iterated until the longest midline is identified (solid green line). (H) Using the resulting internal coordinate system of midline and width lines, measurements, such as cell width (dashed line) or fluorescence intensity (solid line), can be quantified.</p

<i>In silico</i> tests of PSICIC precision.

<p>(A) Symmetrical cell shapes were generated (first cell from left), rotated to different angles (not shown), blurred to simulate the point-spread function of our microscope (second cell), pixilated at a spatial density similar to that of the microscope (third cell), and measured by PSICIC (fourth cell). (B) Distribution of the difference between actual “cell” length and length measured by PSICIC, measured in pixels. The dashed line shows the mean deviation (+0.049 pixels, equivalent to 6.3 nm for the imaging apparatus used for the subsequent <i>E. coli</i> division experiments) and the two dotted lines show plus and minus one standard deviation (±0.094 pixels, equivalent to 12.2 nm). (C) The deviation of measured division-site location from midcell in symmetrically pinched “cell” images, as a percentage of cell length. Colored bars represent different pinch depths, measured by the thickness at the pinch as a fraction of cell thickness away from the pinch. Inset shows detailed data for the 5% of cell length closest to midcell.</p

SpoIIE-GFP is preferentially localized to the forespore in <i>B. subtilis</i>.

<p>(A) Schematic showing the displacement of SpoIIE-GFP (green) from FM4-64 (red), and the displacement measured by PSICIC (arrow). (B) A typical <i>B. subtilis</i> image, showing the GFP channel (top), FM4-64 channel (bottom), and merged image (middle). Highlighted are a SpoIIE-GFP peak (arrow) and an FM4-64 peak (arrowhead). (C) Histogram showing the magnitude and direction of SpoIIE-GFP displacement towards (positive) or away from (negative) the forespore.</p