Name: confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo
Uploaded: 2013-11-05T19:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Forfatterne vil gerne takke Drs. Heping Cheng, Huiliang Zhang og Stephen Kolwicz for deres nyttige kommentarer og teknisk støtte til at udvikle denne metode. Denne undersøgelse blev støttet af tilskud fra NIH og Scientist Development Grant fra American Heart Association til WW.

1.. Eksperiment Forberedelse <ol><li> Forbered isotonisk balanceret saltopløsning (50 ml) indeholdende: 140 mM NaCl, 5 mM KCI, 2,5 mM CaCl2, 2 mM MgCl2 og 10 mM HEPES (pH 7,2) til in vivo skeletmuskulatur billeddannelse. </li><li> Klargør 1 L af Krebs-Henseleit-puffer (KHB) indeholdende: 118 mM NaCl, 5,3 mM KCI, 1,2 mM MgSO4, 0,5 mM EDTA og 25 mM NaHCO3. </li><li> Bubble den KHB med gas indeholdende 95% O2 / 5% CO2 i 10-15 minutter før tils?...

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Imaging enkelt mitokondrie begivenheder i levende dyr eller perfusionerede organer har en betydelig fordel i forhold til traditionelle metoder til mitokondriefunktionen evaluering 17,19,21,22,24,25. Den her beskrevne teknik kan opnå real-time in situ bestemmelse af mitochondrial funktion i en reel fysiologisk tilstand ved subcellulær opløsning. Dette er især nyttigt, når den kombineres med andre målinger, at systemisk undersøge den rolle, mitokondrier i den normale funktion af et bestemt organ...

Mitokondrier spiller en central rolle i celle bioenergetik, frie radikaler signalering, redox homeostase, ion regulering, og celle skæbne beslutsomhed 1,2. Mitokondrier dysfunktion ofte ledsager og ligger til grund for patogenese af sygdomme 3-6. Især i musklen systemer såsom hjerte-og skeletmuskulatur, mitokondriel respiration giver hovedparten af ATP til at understøtte rettidig regulering af intracellulær calcium og robust styrke udvikling 7,8. Disse muskler besidder et stort antal af mitokondrier, der ofte optager op til 20-40% af det totale cellevolumen og &quot;faste&quot; imellem myofilaments 2. Trods talrige undersøgelser, vores forståelse af mitokondriefunktionen regulering, især in vivo og under fysiologisk relevante tilstande, er begrænset. En af grundene er, at flertallet af de metoder, der er udviklet for at vurdere mitokondriefunktionen stole på i vi troo eller ex vivo metoder, såsom overvågning iltforbrug af isolerede mitokondrier suppleret med kunstige substrater, og den indirekte bestemmelse af mitokondrie funktion gennem morfologi (fx elektronmikroskopi), enzym-aktivitet (fx aconitase aktivitet), eller intracellulære ATP-niveauet 9-11 . For nylig har små molekyle fluorescerende indikatorer med relativ mitokondrie berigelse blevet anvendt til at give et glimt af mitokondrie-signaler, herunder membran potentiale, calcium og reaktive ilt arter (ROS), i intakte celler 11-13. Desuden flere grønne fluorescerende protein (GFP) baseret redox og er blevet udviklet ROS indikatorer for at opnå mere specifik evaluering af opdelte intracellulære redox eller ROS signalerer 14-16. Blandt dette, har vi udviklet en genetisk kodet superoxid indikator, den cirkulære Permuted gult fluorescerende protein og targeted det i mitokondrier (mt-cpYFP) 17. mt-cpYFP kan blive anslået ved 405 eller 488 nm med både emission topper ved 515 nm. Emissionen ved 488 nm excitation er specielt lydhør over for superoxid som det fremgår af tidligere in vitro og in vivo kalibreringer 17,18. Emissionen ved 405 nm excitation bruges som intern kontrol (se figur 1 Ref 17 for detaljerede oplysninger om emissions-og excitationsspektre af mt-cpYFP under forskellige betingelser). Med time-lapse konfokal billedbehandling, registrerer denne indikator sprængfyldt superoxidproduktion begivenheder, opkaldt superoxid blinker, i enkelte mitokondrier intakte celler. Superoxid flash fungerer som en sammensat funktion af mitokondrie respiration, ledsagende forbigående mitochondriemembran depolarisering og ROS produktion 17-20. For nylig har vi skabt de pan-væv mt-cpYFP transgene mus ved hjælp af pUC-CAGGS-mt-cpYFP vektor 17,19 om C57/BL6 baggrund og verificeret de stærke udtrykdelse af denne indikator i hjertet, skeletmuskulatur og andre væv (figur 2). Den transgene mus vil være tilgængelige for interesserede akademiske efterforskere efter anmodning og MTA godkendt af University of Washington. I denne undersøgelse, vi beskriver i situ billeddannelse af superoxid blinker i Langendorff perfunderet hjerte såvel som in vivo billeddannelse af flash begivenheder i skelet muskler i bedøvede mt-cpYFP transgene mus 17,19. Denne teknologi giver tidstro overvågning af enkelte mitokondrie ROS produktions begivenheder i et fysiologisk relevant tilstand eller in vivo 21,22. Det er også muligt at bruge systemet til at overvåge andre enkelt mitokondrie parametre såsom membran potentiale og calcium med passende fluorescerende indikatorer. Endvidere samtidig eller parallel evaluering af mitokondriefunktion med intracellulære hændelser (f.eks calcium transienter) eller hjerte funktion (f.eks. Uddrivningsfraktion) kan opnås. Patologiske forstyrrelser, såsom iskæmi og reperfusion, kan anvendes på den perfunderede hjerte til at vurdere virkningen af ​​stress på enkelt mitokondrie funktion i den intakte myokardiet.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Toronto Research Chemicals</td>
			<td>B592500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros Organics</td>
			<td>AC34961-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7006-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1880-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256-25</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Invitrogen</td>
			<td>T-668</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &#34;Physiological Analysis&#34; module.</td>
		</tr>
	</tbody>
</table>

confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo

Mitokondrier er en kritisk intracellulær organel er ansvarlig for energiproduktion og intracellulær signalering i eukaryote systemer. Mitokondriel dysfunktion ofte ledsager og bidrager til sygdom hos mennesker. Størstedelen af de fremgangsmåder, der er blevet udviklet til at vurdere mitokondriefunktionen og dysfunktion er baseret på in vitro-eller ex vivo målinger. Resultater fra disse forsøg har begrænset evne til bestemmelse af mitochondrial funktion in vivo. Her beskriver vi en ny tilgang, der udnytter konfokal scanning mikroskopi til billeddannelse af intakte væv i levende aminaler, som tillader vurdering af enkelt mitokondriefunktion i en real-time måde in vivo. Først, vi frembringelsen af ​​transgene mus, der udtrykker mitokondrier målrettet superoxid indikator cirkulært permuteret gult fluorescerende protein (mt-cpYFP). Bedøvet mt-cpYFP mus er fastsat på et skræddersyet bordadapter og time-lapse billeder er taget fra de udsatte skelet muskler i bagbenet. Musen er efterfølgende aflivet, og hjertet er sat op til Langendorff perfusion med fysiologiske løsninger ved 37 ° C. Den perfunderet hjerte er placeret i en særlig afdeling på konfokal mikroskop scenen og blide tryk påføres immobilisere hjertet og undertrykke hjerteslag induceret bevægelsesartefakt. Superoxid blinker opdages af real-time 2D konfokal billedbehandling med en hyppighed på ét billede pr sekund. Perfusionsopløsningen kan modificeres til at indeholde forskellige åndedræt substrater eller andre fluorescerende indikatorer. Perfusion kan også justeres til at producere sygdomsmodeller såsom iskæmi og reperfusion. Denne teknik er en unik fremgangsmåde til at bestemme funktionen af en enkelt mitokondrie i intakte væv og in vivo.

Ifølge denne protokol, kan in vivo billeddannelse af enlige mitokondrie begivenheder ske i skelet muskler i bedøvede mus efterfulgt af in situ billeddannelse i perfunderet hjerte (Figur 1). Den optimale indstilling af de billeddannende betingelser vil sikre klare billeder af de intakte muskelvæv og med enkelt mitokondrier opløsning (Figur 2). TMRM bruges ofte til at kontrollere placeringen af mt-cpYFP og skal vise et komplet overlappende mønster med mt-cpYFP signa...

Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Konfokal scanning mikroskopi anvendes til billeddannelse enkelt mitokondrie begivenheder i perfunderet hjerte eller skeletmuskulatur i levende dyr. Real-time overvågning af enkelte mitokondrie processer såsom superoxid blinker og membran potentielle udsving muliggør evaluering af mitokondriefunktionen i en fysiologisk relevant kontekst og under patologiske forstyrrelser.

Konfokal Imaging af Single Mitochondriale Superoxide Blinker i intakte hjerte eller<em&gt; In Vivo</em

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Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

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A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Drosophila Forberedelse og Longitudinal Imaging of Heart Funktion in vivo Brug Optisk Kohærens mikroskopi (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Konfokal Imaging af neuropeptid Y-pHluorin: en teknik til at visualisere Insulin granulet exocytose i intakt Murine og menneskelige Holme

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Encellet opløsning tredimensionel billeddannelse af intakte organoider

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells (Video) | JoVE 

Fluorescensbaseret kvantificering af mitokondriemembranpotentiale og superoxidniveauer ved hjælp af levende billeddannelse i HeLa-celler

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...