Diese Arbeit wurde zum Teil von der National Institutes of Health unterstützt Grants HL-085887, HL-092992, S10RR027535 und MOP-93676.

1. Fahrzeug Dissection und Imaging <ol><li> Ernte-Gewebe aus heimischen Jugend Schweine wie in Martens et al 27 beschrieben. Zeigen geerntet Schweine rechten Herzkammer in ein Polydimethylsiloxan (PDMS) unten Dissektion Schale mit HEPES-gepufferte physiologische Kochsalzlösung (PSS). <ol><li> Mit Hilfe eines Stereomikroskops, sezieren und entfernen ein Segment des linken vorderen absteigenden Koronararterie (~ 8 mm Länge, 0,5 mm Durchmesser) vom umgebenden Gewebe mit einer Pinzette und Schere Frühjahr durch die Gefäßsegment Entfernen aus den umliegenden Herzgewebeschichten. HINWEIS: Achten Sie auf die Gefäßwand nicht durchstechen. </li><li> Legen Sie ein PDMS-Block (1 x 0,5 x 0,5 cm) in die Dissektion Gericht, und verwenden Sie eine Nadel, um es auf den Boden zu fixieren. Schneiden Sie ein etwa 40 Mikrometer Durchmesser Wolframdraht in zwölf ~ 0,3 cm lange Segmente bilden Mikropins, und diese in die Schüssel. Mit einer Pinzette, sichern eine Ende des Gefäßsegments zu dem Block mit einem micropin. </li><li> Kleine Feder Schere vorsichtig in das Gefäßlumen und längs geschnitten auf einer Seite des Schiffes, um es vollständig zu öffnen. Richten Sie die geöffnet Gefäßsegment mit dem Endothel auf. </li><li> Verwenden Sie die restlichen Mikropins, um die Grenzen des geöffneten Gefäßsegment zu sichern, um zu blockieren, so dass das Schiff bildet ein flaches Rechteck. HINWEIS: Mikropinlösung Spitzen sollten bis 90 ° gebogen und bündig mit der Blockoberfläche eingesetzt werden. Es sollte darauf geachtet werden, um das Schiff Zubereitung ohne Überdehnung strecken; endgültige Breite sollte ~ 1,5 Mal die Start ungedehnten Breite werden. </li><li> Bereiten ein kleines Volumen (ca. 1 ml) von Fluo-4.00 Beladungslösung durch Mischen von Fluo-4 AM (10 &amp;mgr; M), gelöst in DMSO mit Pluronic (0,03%) in einer HEPES-gepufferten PSS (enthaltend in mM: 134 NaCl, KCl 6 , 1 MgCl, 10 HEPES, 10 Glucose) und stecken Sie den ganzen Block in die Ladelösung für etwa 40 Minuten bei Raumtemperatur in der Dunkelheit. </li><li> Nach dem Laden, waschen Sie den Block in HEPES gepuffertPSS für 5-10 min. </li><li> Montieren Sie den Block auf 50-100 um dicken Abstandshalter in einem Deckglas untere Kammer enthält HEPES gepuffert PSS. HINWEIS: Metallstifte können als Abstandhalter verwendet werden, und sicherzustellen, dass das Gefäßsegment nach unten und in die Abstandshalter nicht berühren. </li><li> Setzen Sie die Kammer auf der Stufe eines inversen Mikroskops für die konfokale Bildgebung ausgestattet ist, und sich auf die endotheliale Zellschicht. </li><li> Capture-Zeitbild Zeitraffer-Sequenzen der basalen relativen Fluoreszenz für ~ 3 min bei 20-facher Vergrößerung und einer Bildrate von ~ 8 Bilder pro Sekunde mit der konfokalen Bildsequenzerfassung Software. </li><li> Nach ca. 3 Min. Aufzeichnungs basale Fluoreszenz, ersetzen HEPES gepuffert PSS-Lösung mit dem gleichen Volumen (~ 1 ml) von Substanz P (100 pM) in HEPES gepuffert PSS und Rekord für eine addtional 3 min. </li></ol></li></ol> 2. Automatisierte Analyse <ol><li> Render-Bild-Sequenz (en) von fluoreszierenden Kalzium-Aktivität aus konfokalen Erfassungssoftware als 8-Bit-, grayscale. &quot;tif&quot;-Format-Dateien ohne angelegte Informations. </li><li> Offene ImageJ, klicken Sie auf &quot;Öffnen&quot; im Datei-Menü, und wählen Sie die entsprechende Bildsequenz in der Explorer-Fenster, um die Bildsequenz (en) in ImageJ anzuzeigen. </li><li> Bestimmen Sie einen entsprechenden ROI Durchmesser mit Hilfe der rechteckige ROI-Tool, um die oberen und unteren Grenzen der räumlichen Ausbreitung der Aktivität innerhalb der Bildsequenz (en) zu schätzen. HINWEIS: Die mittlere rechteckige Durchmesser ist die richtige Wahl für ROI Durchmesser. </li><li> Erstellen Sie einen neuen Ordner auf der Festplatte des Computers, und fügen Sie die Bildsequenz (en) in den Ordner-Verzeichnis. </li><li> In ImageJ, klicken Sie auf das Fenster &quot;Plugins&quot;, dann klicken Sie auf &quot;LC_Pro &#39;, um die Analyse zu starten. </li><li> Geben Sie den Durchmesserwert ROI und wählen Sie den Filter Schwellenwert (entweder 0,05 oder 0,01). </li><li> Klicken Sie auf die &quot;Drogenbehandlung&quot; Kästchen und unmittelbar vor und nach der Droge wurde hinzugefügt (in Sekunden) in die Zeitpunktwerte. </li><li> Klicken Sie auf &quot;OK&quot;, undgeben Sie die Bildsequenz-Verzeichnis in den Datei-Explorer-Fenster. </li></ol> 3. Grafische Ausgabe <ol><li> Laden R-Version 3.0.2 von <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li> Öffnen Sie die 32-Bit-Version von R. </li><li> Klicken Sie auf &quot;Datei&quot;, dann &quot;Open Skript&quot; und wählen Sie die &#39;traceplot.R &quot;R-Skript zur Erzeugung grafischer Versuchsberichte. </li><li> Installieren Sie die Kalibrierung, und gplots Pakete mit &#39;Pakete&#39;, &#39;Installieren von Paketen &quot;, und die Auswahl der zweckdienlichen Pakete. </li><li> Klicken Sie auf &quot;Datei&quot;, dann &quot;change directory&quot;-Befehl, und verwenden Sie das Explorer-Fenster auf das Verzeichnis, das auf die Analyse LC_Pro Ausgang entspricht wählen. </li><li> Klicken Sie auf das Skript-Fenster und dann die &#39;laufen alle &quot;zu befehlen, das Skript auszuführen. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Die Entschlüsselung komplexer Ca 2 +-Signale auf zellulärer Ebene und mehrzelligen strengen experimentellen und analytischen Ansätze erfordern. Hier wird ein Ansatz beschrieben, bei dem zeitaufgelöste konfokale Bildsequenzen von Ca 2 +-abhängige Fluoreszenz an einer automatisierten Analyse identifiziert und quantifiziert statistisch relevanten Ca2 +-Signale in intakten Zellfelder Im speziellen Fall dargestellt unterworfen, wurde isoliert eine Arterie Segment aus Schweineherz, merken...

Ca 2 + ist ein allgegenwärtiges Signalmolekül second messenger und zytosolischen Ca 2 +-Spiegel sind stark reguliert. Die intrazelluläre Ca2 +-Signale sind komplex und umfassen isolierten Transienten, Schwingungen, Wellen-und Pflanzgut 1-4. Räumliche und zeitliche Steuerung von Ca 2 + wird gedacht, um physiologische Signal Spezifität zugrunde liegen und damit die Analyse von Ca 2 +-Signalmuster ist von großem Interesse für die Ermittler in mehreren Feldern fünf. Ca 2 +-Indikator-Farbstoffe, wie Fluo-4 und Fura-2 werden üblicherweise verwendet, um intrazelluläre Ca 2 +-Signale mit Fluoreszenzmikroskopie 5-12 messen. 16 - typischerweise werden zeitliche Ca 2 +-Signale als zeitabhängige Änderung der mittleren Fluoreszenz innerhalb einer benutzerdefinierten Bereich oder Region von Interesse (ROI) 5,6,13 ausgewertet. Derzeit ist die manuelle ROI-Analyse sowohl zeitaufwendig und arbeits inSpannender, weil es erfordert, dass Benutzer viele ROIs identifizieren und sich wiederholende Berechnungen 17-19. Diese Techniken können auch mit erheblichen Benutzerfehler, einschließlich Einführung der künstlichen Signalarten und den Ausschluss von niedriger Amplitude oder diffuse Signale 18,20. Automatisierte ROI Erkennungsalgorithmen sind zuvor unter Verwendung einer Vielzahl von statistischen Methoden, um eine optimale Platzierung ROI ermitteln implementiert worden, aber sie haben im Allgemeinen eine Analyse der Zeilen oder Pseudo-Zeilenbilder, die Analyse auf eine einzige räumliche Dimension in der Zeit 17 begrenzt begrenzt, 19-22. Darüber hinaus sind viele bestehende Algorithmen nicht aus, um die Vielfalt der Ca 2 +-Freisetzung aus Ereignissen, die periodisch, lokalisierten Transienten ausbreitende Wellen 23,24 Bereich umfassen. Umfassende Bewertung der physiologischen Ca2 +-Signale wird oft durch die Anwesenheit von signifikanten Bild Artif komplizierthandeln, dass verwechselt Signal-Rausch-Diskriminierung in vielen experimentellen Systemen. Zuvor eine automatisierte ROI-Erkennungsalgorithmus Lösung Ca 2 +-Signal Transientenerkennung, als Plugin für NIH ImageJ Software implementiert (National Institutes of Health, Bethesda, MD), wurde entwickelt und validiert 25,26. Dieser Algorithmus, genannt LC_Pro, wurde entwickelt, um zu erkennen und zu analysieren ROIs umfassenden Ca 2 +-Signal-Transienten in zweidimensionalen Zeitraffer-Bildsequenzen. Hier eine praktische Versuchsprotokoll und repräsentative Demonstration einer Anwendung des Algorithmus in Schweineherzkranzarterie Endothel vorgesehen ist, mit zusätzlichen Postprocessing mit der Open-Source-Software R statistische Verarbeitung zu brauchbaren grafischen Ausgabe zu erzeugen.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing">download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing</a></td>
		</tr>
	</tbody>
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automated analysis of dynamic ca2+ signals in image sequences

Intrazelluläre Ca 2 +-Signale sind allgemein mit fluoreszierenden Ca 2 +-Indikatorfarbstoffe, und Mikroskopie-Techniken untersucht. Ist die quantitative Analyse von Ca 2 +-Bilddaten jedoch zeitaufwendig und je nach Vorspannung. Automatisierte Signalanalyse-Algorithmen basieren auf Region von Interesse (ROI) Detektion wurden für eindimensionale Zeilen Messungen durchgeführt worden, aber es gibt keine aktuelle Algorithmus optimiert die Identifizierung und Analyse von ROIs in zweidimensionale Bildsequenzen integriert. Hier wird ein Algorithmus für die schnelle Erfassung und Analyse von ROIs in Bildfolgen beschrieben. Es nutzt Ellipsen passen Rauschen gefilterten Signale, um eine optimale Platzierung ROI zu ermitteln, und berechnet Ca 2 +-Signalparameter der Amplitude, Dauer und räumliche Ausbreitung. Dieser Algorithmus wurde als frei verfügbare Software-Plugin für ImageJ (NIH) durchgeführt. Zusammen mit der Analyse Skripte für die Open-Source-Software R statistische Verarbeitung geschrieben,Dieser Ansatz bietet eine Hochleistungs-Pipeline für die Durchführung schnelle statistische Analyse der experimentellen ausgegeben. Die Autoren schlagen vor, dass die Verwendung dieser Analyseprotokoll wird zu einem vollständigen und unvoreingenommene Charakterisierung der physiologischen Ca 2 +-Signal führen.

Eine benutzerdefinierte Algorithmus, LC_Pro, wurde entwickelt und umgesetzt, um die automatisierte Analyse von Ca 2 + durchführen Dynamik auf konfokalen Bildsequenzen. Wie in Abbildung 1 dargestellt ist, nutzt sequentielle Verarbeitungsmodule, die A) erkennen und von dynamischen Websites Spur Ca 2 + der Algorithmus ändern statistischen oben (p &lt;0,01) Lärm, B) definieren Regionen von Interesse (ROI) automatisch am aktiven Zentrum Zentren und C) Berechnen Sie die durchschnittli...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Hier ein neuer Bereich von Interesse Analyseprotokoll auf Basis von Sortier Best-Fit-Ellipsen zu Regionen positives Signal innerhalb von zwei-dimensionalen Zeitbild Zeitraffer-Sequenzen zugeordnet wird demonstriert. Dieser Algorithmus kann Forschern ermöglichen, umfassend zu analysieren physiologischen Ca 2 +-Signale mit minimalen Benutzereingaben und Bias.

Automatisierte Analyse von dynamischen Ca<sup&gt; 2 +</sup&gt; Signale in Bildsequenzen

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K Aufrufe.  University of South Alabama.  Hier ein neuer Bereich von Interesse Analyseprotokoll auf Basis von Sortier Best-Fit-Ellipsen zu Regionen positives Signal innerhalb von zwei-dimensionalen Zeitbild Zeitraffer-Sequenzen zugeordnet wird demonstriert. Dieser Algorithmus kann Forschern ermöglichen, umfassend zu analysieren physiologischen Ca 2 +-Signale mit minimalen Benutzereingaben und Bias. 

Serie: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Anwendung von Licht-gehärteten Dental Adhesive Resin für Montage-Elektroden oder Mikrodialysesonden in Chronic Experimente

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Forschung

Biologie

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 ...

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Mosaic Zebrafisch Transgenese zur Beurteilung Enhancer-Sequenzen

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotik und Dynamic Image Analysis für Studies of Gene Expression in pflanzlichen Geweben

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques for Imaging Ca 2 + Signaling in menschlichen Spermien

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Labeling und Bildanalyse von Tetracysteinmotiv Sensor Proteinen in Zellen

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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 ...

Methods Article

Automatisierte Analyse von dynamischen Ca