High-throughput in vivo vertebrate screening

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.
Nature Methods (Impact Factor: 32.07). 08/2010; 7(8):634-6. DOI: 10.1038/nmeth.1481
Source: PubMed


We demonstrate a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae. The system automatically loads zebrafish from reservoirs or multiwell plates, and positions and rotates them for high-speed confocal imaging and laser manipulation of both superficial and deep organs within 19 s without damage. We performed small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays in combination with femtosecond laser microsurgery.

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    • "The use of microfluidic systems has emerged as a powerful approach to enable immobilization for high-resolution imaging, direct in vivo manipulation, and high-throughput screens of a variety of organisms including Drosophila melanogaster embryos and larvae1819202122, Caenorhabditis elegans21232425262728293031, and zebrafish (Danio rerio) embryos and larvae32333435. Immobilization of freshwater planarians, however, presents several unique challenges: Planarians are about an order of magnitude larger than C. elegans, have a squishy, readily deformed body, vary in size depending on the species, feeding state, last asexual reproduction event, and other factors36, and are significantly more photophobic than C. elegans or D. melanogaster larvae. These characteristics make it infeasible to apply existing microfluidic immobilization techniques directly to planarians, which prompted us to develop an alternative method. "
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    ABSTRACT: Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.
    Scientific Reports 09/2014; 4:6388. DOI:10.1038/srep06388 · 5.58 Impact Factor
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    • "a new biotech company with roots in academic automated zebrafish phenotyping , is developing network platforms for managing huge amounts of data from zebrafish phenotypic screens. Commercial imaging platforms, like the Vertebrate Automated Screening Technology marketed is based on academic innovations (Pardo-Martin et al., 2010b, 2013; Chang et al., 2012). When equipment is too expensive , academic bioinstrumentation laboratories are working to develop more affordable do-it-yourself kits (Alper, 2009; Marzullo and Gage, 2012). "
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    • "Functional toxicology in more complex whole organisms is not straightforward, but new technologies offer encouragement for the future. The zebrafish model is useful in large-scale in vivo genetic and chemical studies (Pardo-Martin et al., 2010). Forward chemical genetic screens in zebrafish have identified small molecules inhibiting the cell cycle (Murphey et al., 2006) and examined genes involved in copper-dependent hypopigmentation (Ishizaki et al., 2010). "
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