Name: automated analysis of dynamic ca2+ signals in image sequences
Uploaded: 2014-06-16T15:00:00
Description: Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Dette arbejde blev støttet delvist af National Institutes of Health Tilskud HL-085.887, HL-092.992, S10RR027535 og MOP-93676.

1.. Vessel Dissektion og Imaging <ol><li> Høst væv fra indenlandske juvenile grise som beskrevet i Martens et al 27. Placer høstet svin højre ventrikler i en polydimethylsiloxan (PDMS) bund dissektion skål indeholdende HEPES bufferet fysiologisk saltopløsning (PSS). <ol><li> Ved hjælp af et stereomikroskop, dissekere og fjerne et segment af venstre forreste nedadgående kranspulsåre (~ 8 mm længde, 0,5 mm i diameter) fra det omgivende væv ved hjælp af pincet og foråret saks ved at fjern...

<ol>
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Afkodning komplekse Ca 2 +-signaler på det cellulære og flercellede niveau vil kræve strenge eksperimentelle og analytiske tilgange. Her er en tilgang, der er beskrevet i hvilken tid løst konfokal billedsekvenser af Ca 2 + afhængig fluorescens underkastes en automatiseret analyse, der identificerer og kvantificerer statistisk relevante Ca 2 + signaler inden intakte cellulære felter I den konkrete sag fremlagte en arterie segment blev isoleret fra gris hjerte, pinned åbnet for at b...

Ca2 + er en allestedsnærværende anden messenger signalmolekyle og cytosoliske Ca2 +-niveauer er meget reguleret. Intracellulære Ca 2 +-signaler er komplekse og omfatter isolerede transienter svingninger, og plantemateriale bølger 1 - 4. Rumlig og tidsmæssig kontrol af Ca 2 + menes at ligge til grund fysiologisk signal specificitet og dermed analysen af Ca 2 + signalmønstre er af betydelig interesse for efterforskere i flere felter 5. Ca2 + indikatorfarvestoffer såsom Fluo-4 og Fura-2, der almindeligvis anvendes til at måle intracellulære Ca 2 +-signaler med fluorescensmikroskopi 5-12. Typisk er timelige Ca 2 + signaler evalueret som tidsafhængige ændringer i den gennemsnitlige fluorescens inden for en brugerdefineret område eller region af interesse (ROI) 5,6,13 - 16. I øjeblikket analyse manuel ROI er både tidskrævende og arbejdskraft itensiv fordi det kræver, at brugerne til at identificere mange ROI&#39;er og udføre gentagne beregninger 17-19. Disse teknikker kan også være forbundet med en betydelig bruger fejl, herunder indførelse af kunstige signal modes og udelukkelse af lav amplitude eller diffuse signaler 18,20. Automatiserede ROI detekteringsalgoritmer er tidligere blevet gennemført ved anvendelse af forskellige statistiske metoder til at bestemme placeringen optimal ROI, men de har generelt været begrænset til analyse af liniescanning eller pseudo-line scan billeder, hvilket begrænser analysen til en enkelt rumlig dimension i tiden 17 19-22. Derudover har mange eksisterende algoritmer ikke er tilstrækkelige til at omfatte mangfoldigheden af Ca 2 release arrangementer +, der spænder fra periodiske, lokaliserede transienter til formerings bølger 23,24. Omfattende evaluering af fysiologiske Ca 2 +-signaler er ofte kompliceres yderligere af tilstedeværelsen af betydelige billede Artifhandle der forvirrer signal til diskrimination støj i mange eksperimentelle systemer. Tidligere en automatiseret påvisning ROI algoritme løsning på Ca 2 + signal forbigående afsløring, implementeret som et plugin til NIH ImageJ software (National Institutes of Health, Bethesda, MD), blev udviklet og valideret 25,26. Denne algoritme, kaldet LC_Pro, blev designet til at identificere og analysere ROIs omfatter Ca 2 + signal transienter i to-dimensionelle tidsudløbsdata billedsekvenser. Her er en praktisk forsøgsprotokol og repræsentativ demonstration af en ansøgning af algoritmen i svine kranspulsåren endotel er forudsat, med ekstra efterbehandlingen ved hjælp af open source statistisk bearbejdning software R til at generere brugbare grafiske output.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<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>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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			<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>
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		<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

Intracellulære Ca 2 +-signaler er almindeligt undersøgt med fluorescerende Ca2 + indikatorfarvestoffer og mikroskopi teknikker. Men kvantitativ analyse af Ca 2 + billeddannelse data er tidskrævende og underlagt bias. Automatiserede signal analyse algoritmer baseret på region af interesse (ROI) detektion er blevet implementeret for endimensional line scan-målinger, men der er ingen aktuelle algoritme, der integrerer optimeret identifikation og analyse af ROIs i to-dimensionelle billede sekvenser. Her beskrives en algoritme til hurtig indsamling og analyse af ROIs i billedsekvenser. Det udnytter ellipser passer til støj filtrerede signaler for at bestemme placeringen optimal ROI og beregner Ca 2 + signal parametre for amplitude, varighed og rumlig spredning. Denne algoritme blev gennemført som et frit tilgængeligt plugin til ImageJ (NIH) software. Sammen med analyse scripts skrevet til open source statistisk bearbejdning software R,Denne fremgangsmåde giver en høj kapacitet pipeline for at udføre hurtig statistisk analyse af eksperimentelle output. Forfatterne foreslår, at anvendelse af denne analyse protokol vil føre til en mere fuldstændig og objektiv karakterisering af fysiologisk Ca 2 +-signalering.

En brugerdefineret algoritme, LC_Pro, blev udviklet og implementeret for at udføre automatiseret analyse af Ca 2 + dynamik på konfokal billedsekvenser. Som vist i figur 1, algoritmen anvender sekventielle behandling moduler, A) registrere og spore sites dynamisk Ca 2 + skifte over statistisk (p &lt;0,01) støj, B) fastlægge områder af interesse (ROI) automatisk ved aktivt sted centre, og C) beregne gennemsnitlige fluorescensintensiteter på ROIs at fastlægge specifikke event p...

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

Automatiseret analyse af dynamiske Ca<sup&gt; 2 +</sup&gt; Signaler i billedsekvenser

Her er en roman region af interesse analyse protokol baseret på sortering bedst-fit ellipser tildelt regionerne positivt signal inden for to-dimensionelle tidsforskydningen billedsekvenser demonstreres. Denne algoritme kan gøre det muligt efterforskerne at omfattende analysere fysiologiske Ca 2 +-signaler med minimal bruger input og bias.

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

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Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

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