Dataset Information

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression.

ABSTRACT: Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105 Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains' average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

SUBMITTER: Sartori FMO

PROVIDER: S-EPMC8142558 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression.

Sartori Federica M O FMO Buzby Cassandra C Plavskin Yevgeniy Y Siegal Mark L ML

Journal of visualized experiments : JoVE 20210418 170

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 10<sup>5</sup> Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed ...[more]

PMID: 33938878

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Project description:BackgroundStreptomycetes are filamentous microorganisms of high biotechnological relevance, especially for the production of antibiotics. In submerged cultures, the productivity of these microorganisms is closely linked to their growth morphology. Microfluidic lab-on-a-chip cultivation systems, coupled with automated time-lapse imaging, generate spatio-temporal insights into the mycelium development of streptomycetes, therewith extending the biotechnological toolset by spatio-temporal screening under well-controlled and reproducible conditions. However, the analysis of the complex mycelial structure formation is limited by the extent of manual interventions required during processing of the acquired high-volume image data. These interventions typically lead to high evaluation times and, therewith, limit the analytic throughput and exploitation of microfluidic-based screenings.ResultsWe present the tool mycelyso (MYCElium anaLYsis SOftware), an image analysis system tailored to fully automated hyphae-level processing of image stacks generated by time-lapse microscopy. With mycelyso, the developing hyphal streptomycete network is automatically segmented and tracked over the cultivation period. Versatile key growth parameters such as mycelium network structure, its development over time, and tip growth rates are extracted. Results are presented in the web-based exploration tool mycelyso Inspector, allowing for user friendly quality control and downstream evaluation of the extracted information. In addition, 2D and 3D visualizations show temporal tracking for detailed inspection of morphological growth behaviors. For ease of getting started with mycelyso, bundled Windows packages as well as Docker images along with tutorial videos are available.Conclusionmycelyso is a well-documented, platform-independent open source toolkit for the automated end-to-end analysis of Streptomyces image stacks. The batch-analysis mode facilitates the rapid and reproducible processing of large microfluidic screenings, and easy extraction of morphological parameters. The objective evaluation of image stacks is possible by reproducible evaluation workflows, useful to unravel correlations between morphological, molecular and process parameters at the hyphae- and mycelium-levels with statistical power.

Dataset Information

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression.

Publications

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression.

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