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Non-microfluidic methods for imaging live C. elegans.

ABSTRACT: There are many challenges to live Caenorhabditis elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluorescence without the use of microfluidic technologies. The methods present here use common microscopy consumables and equipment found in many imaging core facilities and can be easily adapted to fit on multiple microscopy systems.

SUBMITTER: Luke CJ

PROVIDER: S-EPMC4812562 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Non-microfluidic methods for imaging live C. elegans.

Luke Cliff J CJ Niehaus Jason Z JZ O'Reilly Linda P LP Watkins Simon C SC

Methods (San Diego, Calif.) 20140515 3

There are many challenges to live Caenorhabditis elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluoresce ...[more]

PMID: 24836996

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Project description:Live observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially complex and dynamic physiology of multicellular organisms can be a challenging environment to make observations with fluorescence microscopy. Due to the illumination of out-of-focus planes, confocal and particularly widefield fluorescence microscopy suffer from low signal-to-background ratio (SBR), photo toxicity and bleaching of fluorescent probes. In light-sheet microscopy (LSM), solely the focal plane of the detection objective is illuminated, minimising out-of-focus fluorescence and photobleaching, thereby enhancing SBR, allowing for low laser intensities and longer acquisition periods. Here we present a straightforward light-sheet microscope with a 1.0-NA detection objective and a fast sample-positioning stage that allows for four degrees of freedom. By imaging the sensory cilia and nervous system of living young adult C. elegans, we demonstrate that the instrument is well suited for relatively fast, volumetric imaging of larger (hundreds of micrometres cubed) living samples. These experiments demonstrate that such an instrument provides a valuable addition to commonly used widefield and confocal fluorescence microscopes. LAY DESCRIPTION: In fluorescence microscopy, sharp images can only be obtained when the light obtained from the section of the image that is in focus is not overwhelmed by light emerging from elsewhere. In this paper, we present a light-sheet fluorescence microscope, based on the OpenSPIM initiative, with a magnification of 90× and a sensitive sample positioning stage that allows for fast controlled linear movement and rotation. In a light-sheet microscope (LSM), the sample is illuminated from the side, compared to the direction of detection, limiting illumination only to the part of the sample that is imaged in the focal plane (general resources: Wikipedia or MicroscopyU). This does not only limit background noise, but also reduces damage to the sample due to phototoxicity. This makes a LSM particularly suitable for imaging living samples at high resolution, in three dimensions, over long periods of time. Our instrument was specifically designed for imaging adult C. elegans nematodes. We show here how the instrument compares to a standard epifluorescence microscope, imaging neuronal structures in the animals. The instrument proved well suited for fast volumetric imaging of larger cellular structures such as C. elegans neuronal cell bodies. Our experiments show that the instrument provides a valuable addition to widefield and confocal fluorescence microscopes commonly used to image adult C. elegans.

Project description:BackgroundReal-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures.ResultsIn this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment.ConclusionsWhile they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology.

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Non-microfluidic methods for imaging live C. elegans.

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Non-microfluidic methods for imaging live C. elegans.

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