LeafJ wurde von JNM geschrieben, während er ein Sabbatical war Dr. Katherine Pollard Labor am Gladstone Institute. Diese Arbeit wurde durch ein Stipendium der National Science Foundation (Grant-Nummer IOS-0923752) unterstützt.

Ein. Pflanzenmaterialien Beachten Sie, dass diese Pflanze das Wachstum Protokoll zum Nachweis Vermeidung von Schatten zählen Antwort gerichtet ist. Sie können Pflanzen unter Ihrem Lieblings Zustand wachsen. <ol><li> Sprinkle Arabidopsis thaliana Samen auf Wasser eingeweicht Filterpapiere in 9 cm Petrischalen und möglich (Stratifizierung) sie bei 4 ° C für vier Tage in der Dunkelheit. </li><li> Übertragen Sie diese Petrischalen auf simulierte Sonnenbedingungen: FR-Verhältnis auf 1,86: 80-100 uE photosynthetisch aktive Strahlung (PAR) und weit-rote Ergänzung zu den R bringen. Verwenden Langtagsbedingungen (16 Stunden Licht / 8 Stunden Dunkelheit) und konstanter Temperatur von 22 ° C. Inkubieren in diesem Zustand für drei Tage, damit die Samen zu keimen. </li><li> Übertragen gekeimten Samen in den Boden und die Pflanzen unter der Sonne Zustand. Für Großversuche, empfehlen wir Herstellung kleiner Tags zur Kennzeichnung jedes Pflanzen mit Seriendruck-Manager in Microsoft Word 2004 (oder höher) für die Herstellung von Etiketten. </li><li> Elf Tage nach transfer den Boden, bewegen die Hälfte der Pflanzen, die Schatten Zustand: wie Sonne, aber mit zusätzlichem weit rotes Licht, um den R / FR-Verhältnis auf 0,52 zu bringen. </li><li> Nach weiteren 12 Tage, sind die Pflanzen bereit für Blatt Bildgebung. In diesem Stadium die älteren Blätter sind voll ausgereift während jüngere Blätter noch erweitern, so dass Sie eine Momentaufnahme der Entwicklung zu erfassen. Vielleicht möchten Sie eine andere Entwicklungszeit je nach Ihren Bedürfnissen zu wählen. </li></ol> 2. Capturing Dissected Blatt Images <ol><li> Bereiten Sie Transparentfolien mit pflanzlichen Genotyp und Wachstumsbedingungen mit fünf rechteckigen Rahmen gekennzeichnet. Ein Frame entspricht Blätter von einer Pflanze. Microsoft Excel kann verwendet werden, um eine konsistente Gitter mit Etiketten gedruckt werden. </li><li> Dissect Blätter 26 Tage alten Pflanzen. </li><li> Scan lässt bei 600 dpi auf einem Flachbett-Scanner. Beachten Sie, dass von einem Werk verlässt sollten vertikal in einem schwarzen Fenster platziert werden in einem Sandwich von transparenten Folien. Berühren Blätterzu einem schwarzen Fensterrahmen und überlappenden Blättern, die Fehler in folgenden Verfahren geben wird. </li></ol> 3. Blatt Image Analysis by LeafJ <ol><li> Herunterladen ImageJ Ziehen Sie den LeafJ.jar Datei in den Plugin-Ordner von ImageJ. </li><li> Öffnen Sie eine Bilddatei in ImageJ 1.45s oder später 14. </li><li> Split das Bild in drei Farbkanäle (rot, grün und blau) von &quot;Bild&gt; Farbe&gt; Split Channels&quot; und gelten Schwelle zum Bild im blauen Kanal. </li><li> Wählen Sie alle Blätter von einer Pflanze durch ein Rechteck-Werkzeug (Abb. 1A). </li><li> Wählen Sie &quot;LeafJ&quot; aus dem Plugin-Menü. </li><li> Wählen Anmerkungsinformationen für diese Anlage aus dem Dialogfeld, das erscheint. Sie könnenbearbeiten Sie die Standardwerte, die hier erscheinen, indem Sie auf &quot;Bearbeiten Sie diese Optionen&quot;. </li><li> Nach dem Ausführen LeafJ Plugin und bevor Sie auf &quot;OK&quot;-Taste, bearbeiten zurückverfolgt Zeilen aus der Region of Interest (ROI)-Fenster (falls erforderlich; Abbildung 1B). Ein Touchscreen-Tablet (z. B. ein iPad) ist nützlich für dieses Verfahren. iPads können an einen Computer wie einen externen Monitor mit Air Display Software angeschlossen werden. </li><li> Exportieren von Messergebnissen und damit verbundenen Informationen (Dateinamen, Blütezeit, zerschnitten von, gemessen, etc) in Microsoft Excel oder gleichwertige Software. </li></ol> 4. Blattzelle Image Analysis in ImageJ <ol><li> Fix seziert lässt, wie in Bezug 15 nach dem Scannen (Schritt 2) beschrieben. FAA festen Blätter können in 4 ° C für mindestens 6 Monate aufbewahrt werden. </li><li> Deaktivieren Sie die Blätter, indem FAA Fixativ Chloralhydratlösung und inkubieren Blätter für 1 bis 2 Stunden vor der mikroskopischen Beobachtung 15. </li><li> Berg verlässt auf mimikroskop gleitet mit Trichomen nach oben. Mit 40-facher Vergrößerung auf einem zusammengesetzten Mikroskop, Bild Mesophyll Schicht der Mitte jedes Blatt auf beiden Seiten der Hauptader, die Vermeidung Zellen in der Nähe Trichomen oder Venen. </li><li> Trace Blattzelle umreißt von ImageJ ROI-Manager-Tool mit Hilfe des Touchscreen-Tablet und einem Stift (wie in Schritt 3 beschrieben). Zelle Bildanalyse nutzt die integrierten Funktionen der ImageJ aber nicht erforderlich LeafJ. </li></ol>

<ol>
	<li>Furbank, R. T., Tester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenomics--technologies+to+relieve+the+phenotyping+bottleneck.">Phenomics--technologies to relieve the phenotyping bottleneck.</a> Trends Plant Sci. 16, 635-644 (2011).</li><li>Berger, B., Parent, B., Tester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+shoot+imaging+to+study+drought+responses.">High-throughput shoot imaging to study drought responses.</a> J. Exp. Bot. 61, 3519-3528 (2010).</li><li>Borevitz, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+genetic+variation+for+growth+and+development+revealed+by+high-throughput+phenotyping+in+Arabidopsis+thaliana.">Natural genetic variation for growth and development revealed by high-throughput phenotyping in Arabidopsis thaliana.</a> G3 (Bethesda). 2, 29-34 (2012).</li><li>Albrecht, D. R., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+behavioral+analysis+of+Caenorhabditis+elegans+in+precise+spatiotemporal+chemical+environments.">High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments.</a> Nat. Methods. 8, 599-605 (2011).</li><li>Chitwood, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+environment+modulates+leaf+size+and+response+to+simulated+foliar+shade+across+wild+tomato+species.">Native environment modulates leaf size and response to simulated foliar shade across wild tomato species.</a> PLoS ONE. 7, e29570 (2012).</li><li>Chitwood, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+trajectory+of+leaflet+morphology+in+wild+tomato+species.">The developmental trajectory of leaflet morphology in wild tomato species.</a> Plant Physiol. 158, 1230-1240 (2012).</li><li>Casal, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shade+Avoidance.">Shade Avoidance.</a> The Arabidopsis Book. , e0157 (2012).</li><li>Smith, H., Kendrick, R. E., Kronenberg, G. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photomorphogenesis in Plants. , 377-416 (1994).</li><li>Iwata, H., Ukai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE:+a+computer+program+package+for+quantitative+evaluation+of+biological+shapes+based+on+elliptic+Fourier+descriptors.">SHAPE: a computer program package for quantitative evaluation of biological shapes based on elliptic Fourier descriptors.</a> J. Hered. 93, 384-385 (2002).</li><li>Bylesjo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAMINA:+a+tool+for+rapid+quantification+of+leaf+size+and+shape+parameters.">LAMINA: a tool for rapid quantification of leaf size and shape parameters.</a> BMC Plant Biol. 8, 82 (2008).</li><li>Weight, C., Parnham, D., Waites, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LeafAnalyser:+a+computational+method+for+rapid+and+large-scale+analyses+of+leaf+shape+variation.">LeafAnalyser: a computational method for rapid and large-scale analyses of leaf shape variation.</a> Plant J. 53, 578-586 (2008).</li><li>Backhaus, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEAFPROCESSOR:+a+new+leaf+phenotyping+tool+using+contour+bending+energy+and+shape+cluster+analysis.">LEAFPROCESSOR: a new leaf phenotyping tool using contour bending energy and shape cluster analysis.</a> New Phytol. 187, 251-261 (2010).</li><li>Tsukaya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Leaf-shape+determination.">Mechanisms of Leaf-shape determination.</a> Annual Review of Plant Biology. 57, 477-496 (2006).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Horiguchi, G., Fujikura, U., Ferjani, A., Ishikawa, N., Tsukaya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+histological+analysis+of+leaf+mutants+using+two+simple+leaf+observation+methods:+identification+of+novel+genetic+pathways+governing+the+size+and+shape+of+leaves.">Large-scale histological analysis of leaf mutants using two simple leaf observation methods: identification of novel genetic pathways governing the size and shape of leaves.</a> Plant. J. 48, 638-644 (2006).</li><li>Horiguchi, G., Ferjani, A., Fujikura, U., Tsukaya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+cell+proliferation+and+cell+expansion+in+the+control+of+leaf+size+in+Arabidopsis+thaliana.">Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana.</a> J. Plant. Res. 119, 37-42 (2006).</li><li>Pierik, R., de Wit, M., Voesenek, L. A. C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-mediated+stress+escape:+convergence+of+signal+transduction+pathways+activated+upon+exposure+to+two+different+environmental+stresses.">Growth-mediated stress escape: convergence of signal transduction pathways activated upon exposure to two different environmental stresses.</a> New. Phytol. 189, 122-134 (2011).</li></ol>

Keine Interessenskonflikte erklärt.

Unser &quot;LeafJ&quot; plugin ermöglicht die Messung von Blattstiel Länge halbautomatisch, Erhöhung des Durchsatzes fast 6 Mal über manuelle Messung. Blattstiel Länge ist ein wichtiger Index für SAS und ist auch ein Wahrzeichen von anderen Phänomenen wie Überflutung Widerstand und hyponastic Wachstum 17. Deshalb dieses Plugin kann nützlich sein, um eine breite Palette von pflanzlichen Forscher. Unser Plugin ist in einem gut etablierten java-basierte freie Software ImageJ...

<html><table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td></td></tr><tr><td> weit Rotlicht LED </td><td> Orbitec </td><td> Maßarbeit </td><td></td></tr><tr><td> Transparenz </td><td> IKON </td><td> HSCA / 5 </td><td></td></tr><tr><td> Scanner </td><td> Epson </td><td> Epson Perfection V700 PHOTO </td><td></td></tr><tr><td> Image J </td><td> NIH </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> LeafJ </td><td> Brauch </td><td></td><td> <a href="http://www.openwetware.org/wiki/Maloof_Lab" target="_blank">http://www.openwetware.org/wiki/Maloof_Lab</a> </td></tr><tr><td> Air Display </td><td> Avatron Software Inc. </td><td></td><td>blank &quot;&gt; http://avatron.com/ </td></tr><tr><td> iPad2 </td><td> Apple Inc. </td><td></td><td> <a href="http://www.apple.com/" target="_blank">http://www.apple.com/</a> </td></tr></tbody></table>

leafj: an imagej plugin for semi-automated leaf shape measurement

Hoher Durchsatz Phänotypisierung (phenomics) ist ein leistungsfähiges Werkzeug für die Verknüpfung von Genen, ihre Funktionen (siehe Beitrag 1 und jüngsten Beispiele 2-4). Blätter sind die primären photosynthetischen Organ, und ihre Größe und Form variieren entwicklungspolitisch und ökologisch innerhalb einer Anlage. Aus diesen Gründen Studien über Blattmorphologie erfordern Messung mehrerer Parameter aus zahlreichen Blättern, die am besten durch halbautomatische phenomics Werkzeuge 5,6 durchgeführt wird. Canopy Schatten ist ein wichtiger Umweltfaktor Cue das Pflanzenwachstum Architektur und das Leben der Geschichte betrifft, die Suite der Antworten wird zusammenfassend als Vermeidung von Schatten zählen Syndrom (SAS) 7. Unter SAS Reaktionen sind Schatten induzierten Blatt Blattstiel Dehnung und Veränderungen im Blade-Bereich besonders nützlich als Indizes 8. Bis heute kann Blattform Programme (zB SHAPE 9, LAMINA 10, LeafAnalyzer 11, LEAFPROCESSOR 12) zu messen Blatt Konturen und kategorisieren Blattformen, Kann jedoch nicht auszugeben Blattstiel Länge. Mangel an großen Messsysteme Blattstielen gehemmt hat phenomics Ansätze zur SAS Forschung. In diesem Papier beschreiben wir eine neu entwickelte ImageJ Plugin namens LeafJ, die schnell messen können Blattstiel Länge und Blattspreite Parameter der Modellpflanze Arabidopsis thaliana. Für das gelegentliche Blatt, das erforderliche manuelle Korrektur der Blattstiel / Blattspreite Grenze wir eine Touchscreen-Tablet verwendet. Ferner sind Blattzelle Form und Blatt Zellzahlen wichtige Determinanten der Blattgröße 13. Getrennt von LeafJ präsentieren wir auch ein Protokoll für die Verwendung eines Touchscreen-Tablet für Messzelle Form, Fläche und Größe. Unser Blatt trait Messsystem ist nicht auf Schatten-Vermeidungs-Forschung beschränkt und wird leaf Phänotypisierung von vielen Mutanten und Siebanlagen für Blatt Phänotypisierung beschleunigen.

Ein. Blatt Images Zeige Schätzungen der Blattstiel und Blattspreite Boundary, und ihre Messfenster Eines der nützlichsten Funktionen von LeafJ ist automatische Erkennung von Blattspreite / Blattstiel Grenze (Abbildung 1). Die LeafJ Algorithmus arbeitet wie folgt: die integrierte ImageJ ParticleAnalyzer Funktionalität wird verwendet, um herauszufinden und bestimmen die Orientierung der Blätter Innenseite der Benutzerauswahl. Für jedes Blatt die Breite des Blattes ist entlan...

Watch this Scientific Journal Video about LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement at JoVE.com

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

Demonstration der wichtigsten Methoden für hohen Durchsatz Blatt Messungen. Diese Methoden können verwendet werden, um Blatt Phänotypisierung zu beschleunigen, wenn das Studium vieler Pflanzen-Mutanten oder anderweitig Siebanlagen für Blatt Phänotyp werden.

LeafJ: Ein ImageJ Plugin für Semi-automatisierte Blattform Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

leafj-an-imagej-plugin-for-semi-automated-leaf-shape-measurement

Research

JoVE Journal

Biology

28.2K Aufrufe.  University of California Davis. Demonstration der wichtigsten Methoden für hohen Durchsatz Blatt Messungen. Diese Methoden können verwendet werden, um Blatt Phänotypisierung zu beschleunigen, wenn das Studium vieler Pflanzen-Mutanten oder anderweitig Siebanlagen für Blatt Phänotyp werden.

Serie: LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

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.

Semi-automatische optische Heartbeat-Analyse von kleinen Herzen

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

Forschung

Biologie

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secondary Structure Prediction Mit High-Throughput-SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Eine innovative Methode zur Quantifizierung und Exosom Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Ein verbessertes Verfahren für genaue und schnelle Messung der Flugleistung in<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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.

Semi-Automated Imaging von Gewebespezifische Fluoreszenz-in Zebrafischembryonen

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

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Bezug stomatäre Leitfähigkeit zu Blatt Funktionale Merkmale

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

Automatische Quantifizierung und Analyse von Zellzählung Schritte bei der Verwendung ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Clock Scan Protocol für Bildanalyse: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

Halbautomatische Isolierung von weißem Fettgewebe der Maus

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Autor im Rampenlicht: Halbautomatische Isolierung der stromalen vaskulären Fraktion aus weißem Fettgewebe der Maus mit einem Gewebedissoziator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

Halbautomatische Isolierung der stromalen vaskulären Fraktion aus murinem weißem Fettgewebe mit Hilfe eines Gewebedissoziators

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...