Development of transparent microwell arrays for optical monitoring and dissection of microbial communities

Halsted, Michelle C.; Wilmoth, John R.; Briggs, Paige A.; Hansen, Ryan R.; Briggs, Dayrl P.; Timm, Andrea C.; Retterer, Scott T.

doi:10.1116/1.4962739

Cited by 10 publications

(8 citation statements)

References 14 publications

(17 reference statements)

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“…Hansen and co-workers recently reported a microwell screening platform designed to probe microbe–microbe interactions. ,− Although this platform had the benefit of high-throughput measurement, it had limited characterization capabilities due to the lack of cell retrieval. Kim and co-workers recently addressed this problem using a manual capillary-driven bacteria retrieval strategy from 100 μm diameter wells .…”

Section: Introductionmentioning

confidence: 99%

On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes

Vlies

Barua

Nieves-Otero

et al. 2018

ACS Appl. Bio Mater.

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Microwell arrays are important tools for studying single cell behavior and cell−cell interactions, both in microbial and mammalian systems. However, retrieval of cells from microwell arrays with high spatial precision remains a major technical hurdle that prevents follow-up genetic and phenotypic characterization of cells within observed microwells. This work describes a new, material-based approach to grow and retrieve live bacterial cells from small (≥20 μm diameter) microwells in an array using the plant pathogen Agrobacterium tumefaciens as a model bacterium. Our approach uses a light-responsive, steppolymerized poly(ethylene glycol) hydrogel interface as a membrane that confines motile cells within microwells while allowing nutrient exchange and cell growth. The key design feature is the photodegradability of the membrane, as it enables individual wells of interest to be opened using patterned UV light for selective release and retrieval of cells. Extraction can occur in parallel from any number and combination of wells defined by the user. These advancements represent a new use for light-responsive hydrogels and the ability to retrieve cells from microwells with high spatial precision enables several applications that require the isolation and characterization of cells with rare phenotypes from heterogeneous populations.

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Section: Introductionmentioning

confidence: 99%

On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes

Vlies

Barua

Nieves-Otero

et al. 2018

ACS Appl. Bio Mater.

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“…This microwell fabrication and seeding process has opened the door to larger scale data-driven analysis of bacterial population interactions 6 . 7 Five snapshots of one of the microwell videos obtained by Timm et al 3 are shown in Fig. 1.…”

Section: Introductionmentioning

confidence: 99%

Performing Video Frame Prediction of Microbial Growth with a Recurrent Neural Network

Robertson¹,

Wilmoth²,

Retterer³

et al. 2022

Preprint

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A Recurrent Neural Network (RNN) was used to perform video frame prediction of microbial growth for a population of two mutants of Pseudomonas aeruginosa. The RNN was trained on videos of 20 frames that were acquired using fluorescence microscopy and microfluidics. The network predicted the last 10 frames of each video, and the accuracy's of the predictions was assessed by comparing raw images, population curves, and the number and size of individual colonies. Overall, we found the predictions to be accurate using this approach. The implications this result has on designing autonomous experiments in microbiology, and the steps that can be taken to make the predictions even more accurate, are discussed.

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“…Spatial organization has a strong influence on the development and dynamics of biological systems (Kreft et al, 1998 ; Lardon et al, 2011 ; Halsted et al, 2016 ; Hansen et al, 2016 ; McNally et al, 2017 ; Timm et al, 2017 ). The factors leading to changes in organization of multicellular assemblages at fine spatial scales are not well characterized or understood, however, they are expected to guide the succession of community development and function toward specific outcomes (Liu et al, 2009 ; Cline and Zak, 2015 ; Dini-Andreote et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

“…These results demonstrate the importance of spatial confinement and local organization on cell growth and survival. Thus, competition between neighboring microbial cells and spatial confinement are expected to drive changes in cell assemblage and organization (Borenstein et al, 2015 ; Halsted et al, 2016 ; Hansen et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

A Microfluidics and Agent-Based Modeling Framework for Investigating Spatial Organization in Bacterial Colonies: The Case of Pseudomonas Aeruginosa and H1-Type VI Secretion Interactions

et al. 2018

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The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P. aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.

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Development of transparent microwell arrays for optical monitoring and dissection of microbial communities

Cited by 10 publications

References 14 publications

On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes

On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes

Performing Video Frame Prediction of Microbial Growth with a Recurrent Neural Network

A Microfluidics and Agent-Based Modeling Framework for Investigating Spatial Organization in Bacterial Colonies: The Case of Pseudomonas Aeruginosa and H1-Type VI Secretion Interactions

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