An image analysis toolbox for high-throughput C. elegans assays

Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H; Riklin-Raviv, Tammy; Conery, Annie L.; O’Rourke, Eyleen J.; Sokolnicki, Katherine L; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E.; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M.; Carpenter, Anne E.

doi:10.1038/nmeth.1984

Cited by 160 publications

(173 citation statements)

References 21 publications

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“…The follow-up analysis of imaged worms relies heavily on a computational platform such as Cell Profiler using the WormToolbox 30 . For smaller numbers of images, a publicly available image analysis platform such as ImageJ, freely available from the NIH, may be used.…”

Section: Representative Resultsmentioning

confidence: 99%

Biochemical and High Throughput Microscopic Assessment of Fat Mass in <em>Caenorhabditis Elegans</em>

Pino

Webster

Carr

et al. 2013

JoVE

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The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

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Section: Representative Resultsmentioning

confidence: 99%

Biochemical and High Throughput Microscopic Assessment of Fat Mass in <em>Caenorhabditis Elegans</em>

Pino

Webster

Carr

et al. 2013

JoVE

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“…elegans has proven to be an effective model for systems-level analysis of in vivo phenotypes (Kamath et al, 2003;Rual et al, 2004;Fernandez et al, 2005;Gunsalus et al, 2005;Sönnichsen et al, 2005;Lehner et al, 2006;Piano et al, 2006;Byrne et al, 2007;Hunt-Newbury et al, 2007;Liu et al, 2009;Green et al, 2011). Automated image analysis and data mining methods are emerging to further facilitate such efforts (Dupuy et al, 2007;Long et al, 2009;White et al, 2010;Wählby et al, 2012). Early embryogenesis of C. elegans, where lineage patterning, gastrulation and extensive cell-cell signaling occur, is particularly amenable for systematic in vivo analysis because the entire cell lineage can be traced at minutelevel resolution (Schnabel et al, 1997;Hamahashi et al, 2005;Bao et al, 2006;Dzyubachyk et al, 2009;Hench et al, 2009;Santella et al, 2010;Giurumescu et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Systematic quantification of developmental phenotypes at single-cell resolution during embryogenesis

Moore

Bao

2013

Development

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SUMMARYCurrent imaging technology provides an experimental platform in which complex developmental processes can be observed at cellular resolution over an extended time frame. New computational tools are essential to achieve a systems-level understanding of this highcontent information. We have devised a structured approach to systematically analyze complex in vivo phenotypes at cellular resolution, which divides the task into a panel of statistical measurements of each cell in terms of cell differentiation, proliferation and morphogenesis, followed by their spatial and temporal organization in groups and the cohesion within the whole specimen. We demonstrate the approach to C. elegans embryogenesis with in toto imaging and automated cell lineage tracing. We define statistical distributions of the wild-type developmental behaviors at single-cell resolution based on over 50 embryos, cumulating in over 4000 distinct, developmentally based measurements per embryo. These methods enable statistical quantification of abnormalities in mutant or RNAi-treated embryos and a rigorous comparison of embryos by testing each measurement for the probability that it would occur in a wild-type embryo. We demonstrate the power of this structured approach by uncovering quantitative properties including subtle phenotypes in both wild-type and perturbed embryos, transient behaviors that lead to new insights into gene function and a previously undetected source of developmental noise and its subsequent correction.

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“…Moreover, emerging robotic and automated imaging technologies, together with powerful new analytic tools, are offering new possibilities for the higher throughput use of complex models in drug discovery, ranging from iPSCs to brain slices to C. elegans and D. melanogaster. For example, the Complex Object Parametric Analyzer and Sorter Biosort [143], which is able to automatically sort and dispense an accurate number of worms and Drosophila eggs and embryos into multiwell plates, and the WormToolbox [144], an automated image analysis tool for HTS of C. elegans phenotypes, are contributing considerably to increase the throughput of screens using model organisms. Together with recent and rapid progress in novel therapeutic modalities; disease and response biomarker development, including image-based biomarkers and target engagement assays; blood barrier penetrant antibodies; and inhibitory RNAs, peptides, and antisense oligonucleotides for protein misfolding and aggregation, there is much optimism that the polyQ diseases may soon make the transition from "fatal" to manageable.…”

Section: Discussionmentioning

confidence: 99%

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

Calamini

Kaltenbach

2013

Neurotherapeutics

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Huntington's disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, threedimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.

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An image analysis toolbox for high-throughput C. elegans assays

Cited by 160 publications

References 21 publications

Biochemical and High Throughput Microscopic Assessment of Fat Mass in <em>Caenorhabditis Elegans</em>

Biochemical and High Throughput Microscopic Assessment of Fat Mass in <em>Caenorhabditis Elegans</em>

Systematic quantification of developmental phenotypes at single-cell resolution during embryogenesis

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

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