Reconstructing the Forest of Lineage Trees of Diverse Bacterial Communities Using Bio-inspired Image Analysis

Cell segmentation and tracking allow us to extract a plethora of cell attributes from bacterial time-lapse cell movies, thus promoting computational modeling and simulation of biological processes down to the single-cell level. However, to analyze successfully complex cell movies, imaging multiple interacting bacterial clones as they grow and merge to generate overcrowded bacterial communities with thousands of cells in the field of view, segmentation results should be near perfect to warrant good tracking results. We introduce here a fully automated closed-loop bio-inspired computational strategy that exploits prior knowledge about the expected structure of a colony's lineage tree to locate and correct segmentation errors in analyzed movie frames. We show that this correction strategy is effective, resulting in improved cell tracking and consequently trustworthy deep colony lineage trees. Our image analysis approach has the unique capability to keep tracking cells even after clonal subpopulations merge in the movie. This enables the reconstruction of the complete Forest of Lineage Trees (FLT) representation of evolving multi-clonal bacterial communities. Moreover, the percentage of valid cell trajectories extracted from the image analysis almost doubles after segmentation correction. This plethora of trustworthy data extracted from a complex cell movie analysis enables single-cell analytics as a tool for addressing compelling questions for human health, such as understanding the role of single-cell stochasticity in antibiotics resistance without losing site of the inter-cellular interactions and microenvironment effects that may shape it

Active skeleton for bacteria modeling

The investigation of spatio-temporal dynamics of bacterial cells and their molecular components requires automated image analysis tools to track cell shape properties and molecular component locations inside the cells. In the study of bacteria aging, the molecular components of interest are protein aggregates accumulated near bacteria boundaries. This particular location makes very ambiguous the correspondence between aggregates and cells, since computing accurately bacteria boundaries in phase-contrast time-lapse imaging is a challenging task. This paper proposes an active skeleton formulation for bacteria modeling which provides several advantages: an easy computation of shape properties (perimeter, length, thickness, orientation), an improved boundary accuracy in noisy images, and a natural bacteria-centered coordinate system that permits the intrinsic location of molecular components inside the cell. Starting from an initial skeleton estimate, the medial axis of the bacterium is obtained by minimizing an energy function which incorporates bacteria shape constraints. Experimental results on biological images and comparative evaluation of the performances validate the proposed approach for modeling cigar-shaped bacteria like Escherichia coli. The Image-J plugin of the proposed method can be found online at http://fluobactracker.inrialpes.fr.Comment: Published in Computer Methods in Biomechanics and Biomedical Engineering: Imaging and Visualizationto appear i

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade-offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions

Particle detection and tracking in fluorescence time-lapse imaging: a contrario approach

This paper proposes a probabilistic approach for the detection and the tracking of particles in fluorescent time-lapse imaging. In the presence of a very noised and poor-quality data, particles and trajectories can be characterized by an a contrario model, that estimates the probability of observing the structures of interest in random data. This approach, first introduced in the modeling of human visual perception and then successfully applied in many image processing tasks, leads to algorithms that neither require a previous learning stage, nor a tedious parameter tuning and are very robust to noise. Comparative evaluations against a well-established baseline show that the proposed approach outperforms the state of the art.Comment: Published in Journal of Machine Vision and Application

Challenges of analysing stochastic gene expression in bacteria using single-cell time-lapse experiments.

Stochastic gene expression causes phenotypic heterogeneity in a population of genetically identical bacterial cells. Such non-genetic heterogeneity can have important consequences for the population fitness, and therefore cells implement regulation strategies to either suppress or exploit such heterogeneity to adapt to their circumstances. By employing time-lapse microscopy of single cells, the fluctuation dynamics of gene expression may be analysed, and their regulatory mechanisms thus deciphered. However, a careful consideration of the experimental design and data-analysis is needed to produce useful data for deriving meaningful insights from them. In the present paper, the individual steps and challenges involved in a time-lapse experiment are discussed, and a rigorous framework for designing, performing, and extracting single-cell gene expression dynamics data from such experiments is outlined

Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

Quantitative single-cell time-lapse microscopy is a powerful method for analyzing gene circuit dynamics and heterogeneous cell behavior. We describe the application of this method to imaging bacteria by using an automated microscopy system. This protocol has been used to analyze sporulation and competence differentiation in Bacillus subtilis, and to quantify gene regulation and its fluctuations in individual Escherichia coli cells. The protocol involves seeding and growing bacteria on small agarose pads and imaging the resulting microcolonies. Images are then reviewed and analyzed using our laboratory's custom MATLAB analysis code, which segments and tracks cells in a frame-to-frame method. This process yields quantitative expression data on cell lineages, which can illustrate dynamic expression profiles and facilitate mathematical models of gene circuits. With fast-growing bacteria, such as E. coli or B. subtilis, image acquisition can be completed in 1 d, with an additional 1–2 d for progressing through the analysis procedure