This work is funded by the NICHD/NIH (1R01HD053287) to Hukriede, NCI/NIH (P01 CA78039) to Vogt, and NHLBI/NIH (1R01HL088016) to Tsang.

1) Set up zebrafish matings and BCI treatment.
<ol>
<li> Two days prior to treatment, identify homozygous male Tg(dusp6:d2eGFP)pt6 zebrafish and place individually into mating tanks1. Add separator and a wild type AB* female fish and leave in mating cages overnight.</li>
<li> The next morning remove the separator, and collect embryos after 1 hour. Transfer embryos to 100mm dishes with E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), solution and incubate at 28&deg;C for 6 hours. Remove dead and unfertilized embryos, add fresh E3 solution, and incubate overnight.</li>
<li> Sort transgenic embryos for similar stage (24hpf) and uniform GFP expression. Array embryos individually into each well of a 96-well plate.</li>
<li> Remove excess E3 solution with a micropipette. Add 200&mu;l of E3 solution with a multi-channel pipettor into each well.</li>
<li> Add the following compounds as listed: 
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 <td>8</td>
 <td>9</td>
 <td>10</td>
 <td>11</td>
 <td>12</td>
 </tr>
 <tr >
 <td>A</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>B</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>C</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>D</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>E</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>F</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>G</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
 <tr >
 <td>H</td>
 <td>2&mu;l DMSO</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l BCl (0.1mM)</td>
 <td>2&mu;l BCl (0.5mM)</td>
 <td>2&mu;l BCl (1mM)</td>
 <td>2&mu;l BCl (2mM)</td>
 <td>2&mu;l BCl (2.5mM)</td>
 <td>2&mu;l DMSO</td>
 </tr>
</tbody>
</table>
Final concentrations are 1&mu;M, 5&mu;M, 10&mu;M, 20&mu;M, 25&mu;M BCI in 1% DMSO. Cover plate in aluminum foil and incubate for 6 hours at 28&deg;C.</li></ol>
2) Automated imaging of 96-well plates and analysis.
<ol>
<li> Add 10&mu;l of tricaine (0.61mM) to each well to anesthetize 30hpf embryos.</li>
<li> Load plate into Molecular Devices Imagexpress Ultra.</li>
<li> Open Metaxpress software. Choose a configuration with a 4x objective and maximum pinhole diameter, configure laser autofocus to detect plate bottom, and determine optimal Z-plane to detect regions of interest. Acquire images at excitation / emission wavelengths of 488/525 nm (GFP). Set scan area as a single site of 2000x2000 pixels (4 x 4 mm) with no binning. Configure laser power and gain such that brightest regions in positive control images do not saturate. Confirm proper image acquisition in several positive and negative control wells.</li>
<li> Start plate scan. The instrument will automatically acquire images from all wells and archive them in a SQL server database.</li>
<li> Upload images into Definiens Developer using the Cellenger application module and an import filter that recognizes image format and file structure generated by the Molecular Devices Imagexpress Ultra platform.</li>
<li> Process images with a custom designed Cognition Network Technology algorithm (also termed a ruleset). The algorithm, which we custom developed based on a recently published ruleset 2, is designed to automatically identify embryo, yolk, and head, and to quantify the brightness and area of GFP-expressing head structures. Wells in which no embryo is detected or where the head cannot be unambiguously assigned (for example, with high compound toxicity or out of focus images) are excluded from the analysis. Configure the algorithm using several negative control images (vehicle treated) and positive control images (BCI treated) such that head and yolk are correctly identified in both. Set a "head structures" detection threshold as multiples of mean yolk intensity until detection of bright head structures matches visual observation. Define parameters to export. In this example, the most informative parameter was the total integrated brightness of GFP-positive regions within the head, hereafter termed "total head structures brightness" (Figure 1). 
<img src="/files/ftp_upload/1900/1900fig2.jpg" alt="figure 1" /> 
Figure 1 Graph depicitng the dose dependent effects of BCI on GFP expression in transgenic embryos.</li>
<li> Run the algorithm on all plate images using the integrated job scheduler. The Definiens software uses distributed computing on several processors ("engines"), enabling simultaneous analysis of multiple images. The algorithm calculates numerical measures based on the results from the image analysis, which are saved in the database together with a copy of the processed image, with analysis applied.</li></ol>

Representative Results:
After all images are analyzed, data can be visualized within Cellenger or exported to third party software (Excel, GraphPad Prism) for further analysis and graphical representation. Figure 2 shows archived scan images from a vehicle treated and a BCI-treated well, with and without CNT analysis applied. Figure 1 documents a dose-dependent effect of BCI on GFP reporter gene expression in Tg(dusp6:d2EGFP)pt6 embryos. The concentration required to elicit a half maximal response (EC50) was approximately 12 &mu;M. 
<img src="/files/ftp_upload/1900/1900fig1.jpg" alt="figure 2" /> 
Figure 2 Automated image capture and analysis of transgenic zebrafish embryos. (A) DMSO vehicle treated Tg(dusp6:d2eGFP)pt6 embryo at 30hpf. (B) Transgenic embryos treated with 20&mu;M BCI show increases in GFP expression. (C) Image of embryo from (A) after running Definiens ruleset to find GFP expression in the head. Orange spots denote where the software measures GFP intensity. (D) Image of embryo from (B) after running Definiens ruleset to find GFP expression in the head. Orange spots denote where the software measures GFP intensity. Note that the ruleset was able to find an expanded area of GFP expression in the treated embryo.

<ol>
	<li>Molina, G. A., Watkins, S. C., Tsang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+FGF+reporter+transgenic+zebrafish+and+their+utility+in+chemical+screens.">Generation of FGF reporter transgenic zebrafish and their utility in chemical screens.</a> BMC Dev Biol. 7, 62-62 (2007).</li><li>Vogt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+image-based+phenotypic+analysis+in+zebrafish+embryos.">Automated image-based phenotypic analysis in zebrafish embryos.</a> Dev Dyn. 238, 656-663 (2009).</li><li>Molina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+chemical+screening+reveals+an+inhibitor+of+Dusp6+that+expands+cardiac+cell+lineages.">Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.</a> Nat Chem Biol. 5, 680-687 (2009).</li></ol>

A major factor limiting the throughput of zebrafish chemical screens is the lack of methodology to systematically process 96-well plates for imaging and analysis. Because images of multicellular organisms are complex, low contrast, and heterogeneous in nature, existing image analysis algorithms fail to detect and quantify specific structures within the organism. Most published zebrafish chemical screens therefore involve visual examination of individual wells by dedicated personnel throughout the procedure. This usually prevents the generation of numerical readouts and a complete archiving of screen images and data. Furthermore, manual evaluation eliminates several key advantages of image-based screening, namely the ability to conduct retrospective analyses of screen performance, to examine phenotypic changes that were not the primary focus of the screening campaign (e.g., toxicity or developmental defects) and to cross-reference screening data with past or future screens using the same compound library.
 
In this report we highlight methodology for automated imaging and analysis of zebrafish embryos in multiwell plates without user intervention. We automated image capture on an ImageXpress Ultra laser scanning confocal reader (Molecular Devices, Sunnyvale, CA) to image Tg(dusp6:d2EGFP)pt6 embryos in 96 well plates and developed an image analysis algorithm based on Definiens' Cognition Network Technology that quantified GFP expression in the heads of transgenic embryos. The method delivered graded responses and quantified the in vivo activity of a small molecule activator of FGF signaling. Similar results were obtained with the epifluorescence-based ArrayScan II (Cellomics Inc., Pittsburgh PA) documenting that it is possible to implement automated image capture of zebrafish embryos on a variety of commercially available plate readers 2. The development of methodology to automatically analyze multicellular organism images eliminated the need for visual scoring by a human observer and enabled the generation and archiving of numerical data and images for retrospective analysis and comparisons with future screens.

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<td>Tricaine (MS222)</td>
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development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Watch this Scientific Journal Video about Development of automated imaging and analysis for zebrafish chemical screens. at JoVE.com

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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11.3K Views.  University of Pittsburgh Drug Discovery Institute. We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Video: Development of automated imaging and analysis for zebrafish chemical screens.

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...