Probing bacterial cell biology using image cytometry

Abstract

Advances in automated fluorescence microscopy have made snapshot and time-lapse imaging of bacterial cells commonplace, yet fundamental challenges remain in analysis. The vast quantity of data collected in high-throughput experiments requires a fast and reliable automated method to analyze fluorescence intensity and localization, cell morphology and proliferation as well as other descriptors. Inspired by effective yet tractable methods of population-level analysis using flow cytometry, we have developed a framework and tools for facilitating analogous analyses in image cytometry. These tools can both visualize and gate (generate subpopulations) more than 70 cell descriptors, including cell size, age and fluorescence. The method is well suited to multi-well imaging, analysis of bacterial cultures with high cell density (thousands of cells per frame) and complete cell cycle imaging. We give a brief description of the analysis of four distinct applications to emphasize the broad applicability of the tool.

Fig. 1. Identification by morphology verses fluorescence. Panel A

A dot plot of long and short axes for the A. baylyi (blue) and E. coli (red) cells, imaged separately. Based on this data, appropriate gates for A. baylyi and E. coli based on morphology are selected (dotted). Panel B: A dot plot of long and short axes for the mixed A. baylyi and E. coli cells, with the A. baylyi and E. coli gates from Panel A applied. Panel C: Histogram of mean GFP fluorescence for known A. baylyi (solid blue) and E. coli (solid red) cells, as well as mixed cells sorted as A. baylyi (dotted blue) and E. coli (dotted red). We find that the fluorescence data for the mixed cells sorted by morphology closely match the fluorescence data for the known populations. Panel D: Bright field and fluorescence images of a sample field of view, with and without cell outlines determined by morphology and fluorescence gates.

Fig. 2. Conditional probability of cell age given a cell length

This bivariate histogram displays the cell length and age from over 56,000 measurements. The coloring of the points shows the resulting conditional probability of the cell age, given the cell’s length.

Fig. 3. Over-expression phenotypes. Panel A

KDE of cell length (at death) for 48 strains (including ftsZA fusion strains) and statistics: minimum to maximum (dotted), mean (circle) and standard deviation (solid line). Genes labeled as NA are currently not annotated genes in MG1655. Strains are ordered by mean length. Panel B: Analysis of high (+) and low (−) expression sub-populations of four strains. High-expression sub-populations of FtsA and FtsZ have long length. Panel C: Single cell tower and consensus images for FtsZ high and low expression sub-populations. The consensus image for the + sub-population shows a cloud around midcell, reflecting the averaging of tower images of z-rings with aberrant localization. Arrows have been included to point out the various clouds of higher intensity, localized away from cell center, resulting from misplaced z-rings in many cells.

Fig. 4. Effect of cell growth characteristics on cell age. Panel A-C

A sample field of view of a colony growing from a single progenitor cell, shown at t = 0, 60 and 300 minutes. The color of each cell outline indicates the old pole age: two (blue), greater than two (green), or inherited from progenitor/unknown (red). The lines are dashed (or solid) to represent cells at the edge (or inside) of the colony at birth. Panel D: A sample lineage tree from a single progenitor cell. The length of each line gives the age of the cell. The color and style of the lines again indicate the old pole age and position of the cell within the colony. Panel E: We compare the mean cell age for six different conditions of cell growth: (i) on the edge of a colony, (ii) on the inside of a colony (iii) inheriting the oldest poles (iv) inheriting the most new poles (v) longest at birth, and (vi) shortest at birth. Each cell descriptor is gated into three groups: low (−), medium, and high (+). It is clear from this figure that the mean cell cycle duration (or cell age) and standard error on the mean are consistent, independent of pole age.