On-Demand Isolation and Manipulation of C. elegans by In Vitro Maskless Photopatterning.

C Ryan Oliver,Nikos Chronis,Daphne Bazopoulou,A John Hart,Eleni Gourgou,David T Eddington

doi:10.1371/journal.pone.0145935

C Ryan Oliver, Nikos Chronis + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0145935

Copy DOI

Abstract

Caenorhabditis elegans (C. elegans) is a model organism for understanding aging and studying animal behavior. Microfluidic assay techniques have brought widespread advances in C. elegans research; however, traditional microfluidic assays such as those based on soft lithography require time-consuming design and fabrication cycles and offer limited flexibility in changing the geometric environment during experimentation. We present a technique for maskless photopatterning of a biocompatible hydrogel on an NGM (Agar) substrate, enabling dynamic manipulation of the C. elegans culture environment in vitro. Maskless photopatterning is performed using a projector-based microscope system largely built from off-the-shelf components. We demonstrate the capabilities of this technique by building micropillar arrays during C. elegans observation, by fabricating free-floating mechanisms that can be actuated by C. elegans motion, by using freehand drawing to isolate individual C. elegans in real time, and by patterning arrays of mazes for isolation and fitness testing of C. elegans populations. In vitro photopatterning enables rapid and flexible design of experiment geometry as well as real-time interaction between the researcher and the assay such as by sequential isolation of individual organisms. Future adoption of image analysis and machine learning techniques could be used to acquire large datasets and automatically adapt the assay geometry.

Highlights

Capabilities for digital design and microfabrication of synthetic environments have advanced the study of model organisms such as C. elegans, Drosophila sp. and zebrafish
We demonstrate a new approach for experimentation with C. elegans, via photopatterning of hydrogel microstructures directly on NGM plates, which can be performed before or during culture of C. elegans on NGM
We show that maskless photopatterning of PEG-DA enables rapid fabrication of simple microstructured assays on NGM plates, and in situ modification of the environment during live culture

Summary

Introduction

Capabilities for digital design and microfabrication of synthetic environments have advanced the study of model organisms such as C. elegans, Drosophila sp. and zebrafish. Capabilities for digital design and microfabrication of synthetic environments have advanced the study of model organisms such as C. elegans, Drosophila sp. In the case of small organisms such as C. elegans, microfluidic assays have been used for immobilization and imaging, behavioral studies, laser microsurgery, and high throughput drug screening[1,2,3,4]. Microfluidic devices can enable modification of the synthetic environment during use via pressurization (e.g., valving) or by application of chemical, mechanical, or thermal stimuli to the specimen[3,5,6]. Despite advances in fabrication and capabilities of microfluidic assays, NGM plates remain a standard and widely accessible tool for C. elegans culture in biology and neuroscience laboratories. Even though microfluidic assays present elegant and effective.

Objectives

Methods

Results

Conclusion