Gli autori desiderano ringraziare Drs. Heping Cheng, Huiliang Zhang e Stephen Kolwicz per i loro utili commenti e supporto tecnico nello sviluppo di questo metodo. Questo studio è stato sostenuto da sovvenzioni NIH e la Scientist Development Grant dalla American Heart Association a WW.

1. Esperimento Preparazione <ol><li> Preparare la soluzione isotonica salina bilanciata (50 ml) contenente: 140 mM NaCl, 5 mM KCl, 2,5 mM CaCl 2, 2 mM MgCl 2 e HEPES 10 mM (pH 7,2) per imaging in vivo muscolo scheletrico. </li><li> Preparare 1 L di tampone Krebs-Henseleit (KHB) contenente: 118 mM NaCl, 5,3 mM KCl, 1,2 MgSO mm 4, EDTA 0,5 mm e 25 NaHCO mm 3. </li><li> Bubble il KHB con gas contenente 95% O 2/5% di CO 2 per 10-15 minuti prima dell&#39;aggiunta di 2 mM CaCl 2. Aggiungi substrati metabolici (glucosio ad esempio 10 mm e piruvato 0,5 mm). </li><li> Preparare gli strumenti chirurgici sterilizzando la pinza, forbici e pinze in etanolo al 70% e poi risciacquo in sterilizzata DDH 2 O. </li><li> Accendere il sistema di perfusione cardiaca, regolare la temperatura su bagnomaria e circolatore, impostare la velocità della pompa peristaltica di 6 ml / min. </li><li> Bubble il KHB con il 95% O 2/ 5% CO 2 gas, e il controllo della portata e della temperatura (37 ° C, da una sonda di temperatura a fibre ottiche) dell&#39;acqua residua. Il sistema necessita di circa 20 minuti per il gas e temperatura di equilibrazione. </li></ol> 2. Imaging confocale dei muscoli scheletrici In Vivo <ol><li> Anestetizzare mouse con pentobarbital (80 mg / kg, ip). L&#39;animale raggiungerà anestesia chirurgica (nessuna risposta al pinch tep) entro 10-15 minuti e rimane in questo stato per 1-1,5 ore, sufficiente per l&#39;imaging in vivo dei muscoli scheletrici. </li><li> Rimuovere peli su una delle zampe posteriori e sterilizzare la pelle con etanolo al 70%. </li><li> Fare un&#39;incisione sulla pelle lungo il lato esterno dell&#39;arto per esporre i muscoli gastrocnemio. </li><li> Sollevare il epimisio delicatamente con una pinza taglienti e fare un&#39;incisione attraverso di essa con le forbici. Ulteriore sezionare per rimuovere il epimisio ed esporre le fibre muscolari sotto di esso. </li><li> Per il carico in vivo di TMRM nel scheletricomuscolare, includono TMRM (500 nM) in soluzione isotonica salina bilanciata di immergere muscolare per 30 minuti e poi lavare con una soluzione priva di indicatore. </li><li> Posizionare il mouse su un lato con il microscopio confocale (Zeiss LSM 510) stadio e trattenere l&#39;arto posteriore in una posizione che il muscolo scheletrico esposta sia rivolta contro il coprioggetto che forma il fondo della camera. Il vetrino è tra il tessuto muscolare e l&#39;obiettivo invertita (olio 40X). </li><li> Premere la gamba delicatamente verso il basso per rendere stretto contatto tra il tessuto e il vetrino. Immergere i muscoli esposti in soluzione isotonica salina bilanciata. </li><li> Record bidimensionali (2D, xy) le immagini confocale ad una frequenza di campionamento di un secondo per fotogramma. L&#39;intensità di ciascun pixel viene digitalizzato in profondità di 8-bit. Di solito, una scansione seriale contiene 100 fotogrammi. </li><li> Raccogliere immagini sequenziali di primo emozionante a 405 nm e la raccolta a&gt; 505 nm poi a 488 nm, mentre la raccolta a&gt; 505 nm per doppia lunghezza d&#39;onda excitation imaging mt-cpYFP. Utilizzare eccitazione sequenziale a 405, 488 e 543 nm, e raccogliere emissione a 505-545, 505-545 e&gt; 560 nm, rispettivamente, per tre lunghezze d&#39;onda di eccitazione imaging mt-cpYFP e TMRM. </li></ol> 3. Imaging confocale di cuore perfuso mouse <ol><li> Subito dopo imaging in vivo dei muscoli scheletrici, iniettare il mouse con 200 unità di eparina (ip). Dieci minuti dopo, eutanasia il mouse toracotomia e rapidamente rimuovere il cuore con i polmoni e timo collegato ad esso. </li><li> Rimuovere rapidamente il polmone in tampone ghiacciato. Identificare i lobi del timo e la buccia delicatamente per esporre l&#39;aorta ascendente. </li><li> Rimuovere il timo e isolare l&#39;aorta rimuovendo accuratamente qualsiasi tessuto circostante. </li><li> Effettuare un taglio all&#39;estremità superiore della aorta ascendente prima del primo ramo della dell&#39;arco aortico. </li><li> Tenere la parete dell&#39;aorta delicatamente con due pinze sutura micro per esporre il lume e posizionare accuratamente l&#39;aorta sulla cannula (PE50 tubo). L&#39;aorta è tenuto in posizione con un morsetto nave micro mentre suture vengono rapidamente legato intorno all&#39;aorta. </li><li> Rimuovere la fascetta, controllare attentamente la cannula con una pinza per assicurarsi che la punta della cannula è al di sopra della radice aortica. Legami vengono aggiunti se necessario per tenere il cuore a posto. </li><li> Accendere la pompa peristaltica e profumato cuore a 1 ml / min. Il cuore riprende a battere su di perfusione. </li><li> Regolare la posizione del sistema di perfusione e mettere il cuore sul palco microscopio. Lo stadio ha un adattatore che permette il riscaldamento della camera che contiene il cuore. </li><li> Aggiungere 1 ml di soluzione di perfusione KHB nella camera di immergere parzialmente cuore. Controllo della temperatura della camera a 37 ° C. Rimuovere effluenti dalla camera utilizzando una pompa peristaltica. </li><li> Aumentare la velocità della pompa peristaltica gradualmente per fornire un flusso sufficiente (circa 2 ml / min) al cuore. </li><li> Dopo 10 min di stabilizzazione, profumato il heart con 10 micron blebbistatin e 100-500 nM TMRM. Battito cardiaco rallenta dopo 10 min. </li><li> Applicare una leggera pressione sul cuore per assicurare stretto contatto del cuore con il coprioggetto sul fondo della camera e per sopprimere ulteriormente battito cardiaco. </li><li> Seguire la stessa procedura per l&#39;imaging confocale di miocardio intatto come descritto nel precedente punto 2.8. Regolare accuratamente il piano focale per rivelare l&#39;immagine più chiara possibile. </li></ol> 4. Image Processing e Analisi dei Dati <ol><li> Utilizzare gli strumenti forniti dal modulo &quot;Analisi fisiologica&quot; del software confocale per analizzare singoli mitocondriali lampeggia e membrana potenziali cambiamenti. Questo modulo è spesso incluso nel software di acquisizione di immagini del sistema confocale e permette la determinazione di certe aree dell&#39;immagine così come uscita di intensità di fluorescenza con etichette temporali. </li><li> Aprire il database e quindi il file immagine 2D di serie da analizzare. </li><li> Fare clic su &quot;Regioned&#39;interesse (ROI) significa &quot;per passare al&quot; media di ROI &quot;mode. Clicca sul&quot; regione di interesse (ROI) significare &quot;per passare alla modalità&quot; media del ROI &quot;. </li><li> Disattivare la visualizzazione di altri canali, tranne il canale di cpYFP 488 nm per la selezione dei lampi. </li><li> Zoom a immagine e spostare manualmente barra di scorrimento per riprodurre le immagini 2D seriali. </li><li> Identificare i singoli mitocondriali superossido lampeggia individuando il luogo in cui gli aumenti segnale fluorescente transitoriamente. Utilizzare lo strumento ROI adeguato per contrassegnare i flash. La traccia mostrando tempo-dipendente variazione di fluorescenza di ogni ROI apparirà accanto all&#39;immagine. </li><li> Dopo aver selezionato tutti i flash, attivare la visualizzazione di altri canali. Selezionare un ROI sull&#39;immagine all&#39;esterno della cellula per segnale sottrazione del fondo. Uscita la fluorescenza media di ogni ROI insieme alle etichette temporali. </li><li> Registrare il numero di lampeggi in ciascuno dei file immagine di scansione seriale con il tempo di scansione e l&#39;area dila cella. Utilizzare Excel per calcolare la frequenza flash come numero di lampi per 100 sec / 1,000 micron 2 superficie cellulare. </li><li> Utilizzare un programma personalizzato sviluppato scritto in Interactive Data Language (IDL, ResearchSystems) per calcolare i parametri di ogni flash (ampiezza, tempo al picco e tempo di decadimento). Software IDL è disponibile in commercio e il programma personalizzato sviluppato per analisi parametro flash può essere ottenuta dall&#39;autore su richiesta. </li></ol>

<ol>
	<li>Brookes, P. S., Yoon, Y., Robotham, J. L., Anders, M. W., Calcium Sheu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium,+ATP,+and+ROS:+a+mitochondrial+love-hate+triangle.">Calcium, ATP, and ROS: a mitochondrial love-hate triangle.</a> Am. J. Physiol. Cell Physiol. 287, 817-833 (2004).</li><li>Scheffler, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondria. , (2008).</li><li>Rosca, M. G., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+heart+failure.">Mitochondria in heart failure.</a> Cardiovasc. Res. 88, 40-50 (2010).</li><li>Griffiths, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+heart+disease.">Mitochondria and heart disease.</a> Adv. Exp. Med. Biol. 942, 249-267 (2012).</li><li>Winklhofer, K. F., Haass, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+Parkinson's+disease.">Mitochondrial dysfunction in Parkinson's disease.</a> Biochim. Biophys. Acta. 1802, 29-44 (2010).</li><li>Pieczenik, S. R., Neustadt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+molecular+pathways+of+disease.">Mitochondrial dysfunction and molecular pathways of disease.</a> Exp. Mol. Pathol. 83, 84-92 (2007).</li><li>Szentesi, P., Zaremba, R., van Mechelen, W., Stienen, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+utilization+for+calcium+uptake+and+force+production+in+different+types+of+human+skeletal+muscle+fibres.">ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres.</a> J. Physiol. 531, 393-403 (2001).</li><li>Lemieux, H., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+the+human+heart.">Mitochondria in the human heart.</a> J. Bioenerg. Biomembr. 41, 99-106 (2009).</li><li>Chance, B., Williams, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+enzymes+in+oxidative+phosphorylation.+III.+The+steady+state.">Respiratory enzymes in oxidative phosphorylation. III. The steady state.</a> J. Biol. Chem. 217, 409-427 (1955).</li><li>Lambert, A. J., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+production+by+mitochondria.">Reactive oxygen species production by mitochondria.</a> Methods Mol Biol. 554, 165-181 (2009).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem. J. 435, 297-312 (2011).</li><li>Robinson, K. 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=Selective+fluorescent+imaging+of+superoxide+in+vivo+using+ethidium-based+probes.">Selective fluorescent imaging of superoxide in vivo using ethidium-based probes.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 15038-15043 (2006).</li><li>Dickinson, B. C., Srikun, D., Chang, 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=Mitochondrial-targeted+fluorescent+probes+for+reactive+oxygen+species.">Mitochondrial-targeted fluorescent probes for reactive oxygen species.</a> Curr. Opin. Chem. Biol. 14, 50-56 (2010).</li><li>Dooley, C. T., 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=Imaging+dynamic+redox+changes+in+mammalian+cells+with+green+fluorescent+protein+indicators.">Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.</a> J. Biol. Chem. 279, 22284-22293 (2004).</li><li>Hanson, G. T., 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=Investigating+mitochondrial+redox+potential+with+redox-sensitive+green+fluorescent+protein+indicators.">Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators.</a> J. Biol. Chem. 279, 13044-13053 (2004).</li><li>Belousov, V. V., 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=Genetically+encoded+fluorescent+indicator+for+intracellular+hydrogen+peroxide.">Genetically encoded fluorescent indicator for intracellular hydrogen peroxide.</a> Nat. Methods. 3, 281-286 (2006).</li><li>Wang, W., 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=Superoxide+Flashes+in+Single+Mitochondria.">Superoxide Flashes in Single Mitochondria.</a> Cell. 134, 279-290 (2008).</li><li>Wei-Lapierre, L., 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=Respective+Contribution+of+Mitochondrial+Superoxide+and+pH+to+Mitochondria-targeted+Circularly+Permuted+Yellow+Fluorescent+Protein+(mt-cpYFP)+Flash+Activity.">Respective Contribution of Mitochondrial Superoxide and pH to Mitochondria-targeted Circularly Permuted Yellow Fluorescent Protein (mt-cpYFP) Flash Activity.</a> J. Biol. Chem. 288, 10567-10577 (2013).</li><li>Fang, 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=Imaging+superoxide+flash+and+metabolism-coupled+mitochondrial+permeability+transition+in+living+animals.">Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals.</a> Cell Res. 21, 1295-1304 (2011).</li><li>Wei, L., 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=Mitochondrial+superoxide+flashes:+metabolic+biomarkers+of+skeletal+muscle+activity+and+disease.">Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease.</a> FASEB J. 25, 3068-3078 (2011).</li><li>Sheu, S. S., Wang, W., Cheng, H., Dirksen, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+flashes:+illuminating+new+insights+into+cardiac+ischemia/reperfusion+injury.">Superoxide flashes: illuminating new insights into cardiac ischemia/reperfusion injury.</a> Future Cardiol. 4, 551-554 (2008).</li><li>Wei, L., Dirksen, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on:+SGP+symposium+on+mitochondrial+physiology+and+medicine:+mitochondrial+superoxide+flashes:+from+discovery+to+new+controversies.">Perspectives on: SGP symposium on mitochondrial physiology and medicine: mitochondrial superoxide flashes: from discovery to new controversies.</a> J. Gen. Physiol. 139, 425-434 (2012).</li><li>Johnson, I., Spence, M. T. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+12.2.">Ch. 12.2.</a> The Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies. , 510-511 (2013).</li><li>Wang, X., 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=Superoxide+flashes:+elemental+events+of+mitochondrial+ROS+signaling+in+the+heart.">Superoxide flashes: elemental events of mitochondrial ROS signaling in the heart.</a> J. Mol. Cell Cardiol. 52, 940-948 (2012).</li><li>Li, K., 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=Superoxide+flashes+reveal+novel+properties+of+mitochondrial+reactive+oxygen+species+excitability+in+cardiomyocytes.">Superoxide flashes reveal novel properties of mitochondrial reactive oxygen species excitability in cardiomyocytes.</a> Biophys. J. 102, 1011-1021 (2012).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+The+Langendorff+technique+of+isolated+heart+perfusion.">Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion.</a> J. Mol. Cell. Cardiol. 50, 940-950 (2011).</li><li>Pasdois, P., 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=Effect+of+diazoxide+on+flavoprotein+oxidation+and+reactive+oxygen+species+generation+during+ischemia-reperfusion:+a+study+on+Langendorff-perfused+rat+hearts+using+optic+fibers.">Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers.</a> Am. J. Physiol. 294, H2088-H2097 (2008).</li><li>Granville, D. J., 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=Reduction+of+ischemia+and+reperfusion-induced+myocardial+damage+by+cytochrome+P450+inhibitors.">Reduction of ischemia and reperfusion-induced myocardial damage by cytochrome P450 inhibitors.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 1321-1326 (2004).</li><li>Davidson, S. M., Yellon, D. M., Murphy, M. P., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+calcium+waves+and+redox+changes+precede+mitochondrial+permeability+transition+pore+opening+in+the+intact+heart+during+hypoxia+and+reoxygenation.">Slow calcium waves and redox changes precede mitochondrial permeability transition pore opening in the intact heart during hypoxia and reoxygenation.</a> Cardiovasc. Res. 93, 445-453 (2012).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Imaging eventi mitocondriali singoli organi irrorati di animali vivi o ha un vantaggio significativo rispetto ai metodi tradizionali di valutazione della funzione mitocondriale 17,19,21,22,24,25. La tecnica qui descritta può realizzare in tempo reale in situ determinazione della funzionalità mitocondriale in una condizione fisiologica reale alla risoluzione subcellulare. Ciò è particolarmente utile, quando combinato con altre misurazioni, per studiare sistematicamente il ruolo dei mitocondri nel ...

I mitocondri svolgono un ruolo centrale nella bioenergetica di cellule, di segnalazione dei radicali liberi, redox omeostasi, regolazione ione, e il destino delle cellule determinazione 1,2. La disfunzione dei mitocondri spesso accompagna e sottende la patogenesi delle malattie 3-6. Soprattutto nei sistemi muscolari come il cuore e nei muscoli scheletrici, respirazione mitocondriale fornisce la maggior parte di ATP per sostenere regolamentazione tempestiva di calcio intracellulare e robusto 7,8 sviluppo della forza. Questi muscoli in possesso di un gran numero di mitocondri, che spesso occupano fino al 20-40% del volume cellulare totale e sono &quot;corretti&quot; tra miofilamenti 2. Nonostante i numerosi studi, la nostra comprensione della regolazione della funzione mitocondriale, in particolare in vivo e in condizioni fisiologicamente rilevanti, è limitata. Uno dei motivi è che la maggior parte dei metodi sviluppati per valutare la funzione mitocondriale contare su di Vitroo approcci ex vivo, quali il monitoraggio del consumo di ossigeno di mitocondri isolati, completata con substrati artificiali, e la determinazione indiretta della funzionalità mitocondriale mediante la morfologia (ad esempio microscopia elettronica), l&#39;attività enzimatica (ad esempio attività aconitasi), o livelli di ATP intracellulari 9-11 . Recentemente, piccoli indicatori fluorescenti molecola con relativa arricchimento mitocondriale sono stati applicati per fornire un assaggio dei segnali mitocondriali, tra cui il potenziale di membrana, di calcio e di specie reattive dell&#39;ossigeno (ROS), in cellule intatte 11-13. Inoltre, vari proteina fluorescente verde (GFP) basato redox e ROS indicatori sono stati sviluppati per ottenere una valutazione più specifica del redox intracellulare compartimenti o segnali ROS 14-16. Tra questo, abbiamo sviluppato un indicatore geneticamente codificato superossido, la proteina fluorescente gialla permutato circolare, e targeted in mitocondri (mt-cpYFP) 17. mt-cpYFP può essere eccitato a 405 o 488 nm con entrambi i picchi di emissione a 515 nm. L&#39;emissione a 488 nm di eccitazione è specificamente rispondenti a superossido come mostrato dalla precedente in vitro e in vivo calibrazioni 17,18. L&#39;emissione a 405 nm di eccitazione viene utilizzato come controllo interno (si veda la Figura 1 di Ref 17 per informazioni dettagliate sulle emissioni di eccitazione e spettri di mt-cpYFP in varie condizioni). Con il time-lapse imaging confocale, questo indicatore rileva scoppio eventi di produzione di superossido, chiamato superossido lampi, in singoli mitocondri delle cellule intatte. Superossido in flash serve come una funzione composta di respirazione mitocondriale, accompagnamento transitoria depolarizzazione della membrana mitocondriale e la produzione di ROS 17-20. Recentemente, abbiamo generato i pan-tessuto mt-cpYFP topi transgenici con il vettore pUC-CAGGS-mt-cpYFP 17,19 su C57/BL6 sfondo e verificato le forti espressionisione di questo indicatore nel cuore, muscoli scheletrici e altri tessuti (Figura 2). I topi transgenici sarà disponibile per ricercatori accademici interessati su richiesta e approvazione MTA dall&#39;Università di Washington. In questo studio, descriviamo in situ imaging lampeggia superossido in Langendorff cuore perfuso e immagini in vivo di eventi flash nella muscoli scheletrici di anestetizzati mt-cpYFP topi transgenici 17,19. Questa tecnologia consente il monitoraggio in tempo reale dei singoli eventi mitocondriali di produzione di ROS in una condizione fisiologicamente rilevanti o in vivo 21,22. È anche possibile utilizzare il sistema per monitorare altri parametri singoli mitocondriali come potenziale di membrana e calcio con indicatori fluorescenti appropriati. Valutazione ulteriore, simultanea o parallela della funzione mitocondriale con eventi intracellulari (ad esempio transienti di calcio) o funzione cardiaca (ad esempio. Frazione di eiezione) può essere raggiunto. Perturbazioni patologiche, quali ischemia e riperfusione, possono essere applicati al cuore perfuso per valutare l&#39;impatto dello stress sulla singola funzione mitocondriale nel miocardio intatta.

<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

Mitocondrio è un organello intracellulare critico responsabile per la produzione di energia e di segnalazione intracellulare nei sistemi eucarioti. Disfunzione mitocondriale spesso accompagna e contribuisce alla malattia umana. La maggior parte dei metodi che sono stati sviluppati per valutare la funzione mitocondriale e la disfunzione si basano su in vitro o ex vivo misurazioni. I risultati di questi esperimenti hanno limitato la capacità di determinare la funzione mitocondriale in vivo. Qui, descriviamo un nuovo approccio che utilizza la microscopia confocale a scansione per l&#39;imaging di tessuti intatti in aminals vivi, che permette di valutare singola funzione mitocondriale in maniera tempo reale in vivo. In primo luogo, generiamo topi transgenici che esprimono la mitocondriale mirato indicatore superossido, proteina fluorescente giallo circolarmente permutato (mt-cpYFP). Mt-cpYFP anestetizzato mouse è fissato su una scheda palcoscenico su misura e time-lapse immagini sono prese fRom muscoli scheletrici esposte del arti posteriori. Il mouse viene successivamente sacrificato e il cuore è impostato per Langendorff perfusione con soluzione fisiologica a 37 ° C. Il cuore perfuso è posizionato in una camera speciale sul palco microscopio confocale e leggera pressione viene applicata per immobilizzare il cuore e sopprimere battito cardiaco indotto artefatti da movimento. Superossido lampi vengono rilevati mediante real-time confocale 2D immagini ad una frequenza di un fotogramma al secondo. La soluzione di perfusione può essere modificata per contenere diversi substrati respiratori o altri indicatori fluorescenti. La perfusione può anche essere regolata per produrre modelli di malattie come l&#39;ischemia e riperfusione. Questa tecnica è un approccio unico per la determinazione della funzione di singolo mitocondrio in tessuti normali e in vivo.

Secondo questo protocollo, imaging in vivo di eventi mitocondriali singoli può essere realizzata in muscoli scheletrici di topi anestetizzati, seguita da in situ imaging cuore perfuso (Figura 1). La regolazione ottimale delle condizioni di imaging garantisce immagini chiare dei tessuti muscolari intatte e con una risoluzione singolo mitocondrio (Figura 2). TMRM viene spesso utilizzato per verificare la posizione del mt-cpYFP e dovrebbe mostrare un pattern completa sov...

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

La microscopia confocale a scansione viene applicato per l&#39;imaging eventi mitocondriali singoli in cuore perfuso o muscoli scheletrici in animale vivo. Monitoraggio in tempo reale dei processi mitocondriali singoli, come vampate di superossido e di membrana potenziali fluttuazioni consente la valutazione della funzione mitocondriale in un contesto fisiologicamente rilevanti e durante le perturbazioni patologiche.

Imaging confocale delle singole mitocondriali superossido illumina di cuore intatto o<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

confocal-imaging-single-mitochondrial-superoxide-flashes-intact-heart

Research

JoVE Journal

Biology

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

14.5K Views.  University of Washington.  La microscopia confocale a scansione viene applicato per l'imaging eventi mitocondriali singoli in cuore perfuso o muscoli scheletrici in animale vivo. Monitoraggio in tempo reale dei processi mitocondriali singoli, come vampate di superossido e di membrana potenziali fluttuazioni consente la valutazione della funzione mitocondriale in un contesto fisiologicamente rilevanti e durante le perturbazioni patologiche. 

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

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Cella singola elettroporazione in vivo all&#39;interno del cervello intatto in via di sviluppo

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Ricerca

Biologia

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Valutazione del calcio Sparks in intatte fibre muscolari scheletriche

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

Ad alta risoluzione respirometria mitocondriale per valutare la funzione in permeabilizzate e cellule intatte

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)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

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

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila preparazione e imaging longitudinale della funzione cardiaca in vivo utilizzando Optical Coherence Microscopy (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

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Confocale Imaging di Neuropeptide Y-pHluorin: una tecnica per visualizzare esocitosi di granuli di insulina in isolotti intatti Murine ed umani

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

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Risoluzione a una cellula di immagini tridimensionali di organoidi intatti

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

Quantificazione del potenziale di membrana mitocondriale e dei livelli di superossido

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

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Autore in primo piano: Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa

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