The authors would like to thank Drs. Heping Cheng, Huiliang Zhang and Stephen Kolwicz for their helpful comments and technical support in developing this method. This study was supported by NIH grants and the Scientist Development Grant from American Heart Association to WW.

1. Experiment Preparation
<ol>
	<li>Prepare the isotonic balanced salt solution (50 ml) containing: 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES (pH 7.2) for in vivo skeletal muscle imaging.</li>
	<li>Prepare 1 L of Krebs-Henseleit buffer (KHB) containing: 118 mM NaCl, 5.3 mM KCl, 1.2 mM MgSO4, 0.5 mM EDTA and 25 mM NaHCO3.</li>
	<li>Bubble the KHB with gas containing 95% O2/5% CO2 for 10-15 min prior to the addition of 2 mM C...

<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>

The authors declare that they have no competing financial interests.

Imaging single mitochondrial events in live animal or perfused organs has significant advantage over traditional methods for mitochondrial function evaluation17,19,21,22,24,25. The technique described here can achieve real-time in situ determination of mitochondrial function in a real physiological condition at the subcellular resolution. This is particularly useful, when combined with other measurements, to systemically study the role of mitochondria in the normal function of a particular organ or ce...

Mitochondria play a central role in cell bioenergetics, free radical signaling, redox homeostasis, ion regulation, and cell fate determination1,2. Mitochondria dysfunction often accompanies and underlies the pathogenesis of diseases3-6. Especially in the muscle systems such as the heart and skeletal muscles, mitochondrial respiration provides the majority of ATP to support timely regulation of intracellular calcium and robust force development7,8. These muscles possess a large number of mitochondria that often occupy up to 20-40% of the total cell volume and are &#34;fixed&#34; in between myofilaments2.
Despite numerous studies, our understanding of the mitochondrial function regulation, specifically in vivo and under physiologically relevant conditions, is limited. One of the reasons is that majority of the methods developed for evaluating mitochondrial function rely on in vitro or ex vivo approaches, such as monitoring the oxygen consumption of isolated mitochondria supplemented with artificial substrates, and the indirect determination of mitochondrial function through morphology (e.g.&#160;electron microscopy), enzyme activity (e.g.&#160;aconitase activity), or intracellular ATP levels9-11.
Recently, small molecule fluorescent indicators with relative mitochondrial enrichment have been applied to provide a glimpse of the mitochondrial signals, including membrane potential, calcium and reactive oxygen species (ROS), in intact cells11-13. Moreover, several green fluorescent protein (GFP) based redox and ROS indicators have been developed to achieve more specific evaluation of the compartmentalized intracellular redox or ROS signals14-16. Among this, we developed a genetically encoded superoxide indicator, the circular permuted yellow fluorescent protein, and targeted it into mitochondria (mt-cpYFP)17. mt-cpYFP can be excited at 405 or 488 nm with both emission peaks at 515 nm. The emission at 488 nm excitation is specifically responsive to superoxide as shown by previous in vitro and in vivo calibrations17,18. The emission at 405 nm excitation is used as internal control (please refer to Figure 1 of Ref 17 for detailed information on the emission and excitation spectra of mt-cpYFP under various conditions). With time-lapse confocal imaging, this indicator detects bursting superoxide production events, named superoxide flashes, in single mitochondria of intact cells. Superoxide flash serves as a composite function of mitochondrial respiration, accompanying transient mitochondrial membrane depolarization and ROS production17-20. Recently, we have generated the pan-tissue mt-cpYFP transgenic mice using the pUC-CAGGS-mt-cpYFP vector17,19 on C57/BL6 background and verified the strong expression of this indicator in the heart, skeletal muscles and other tissues (Figure 2). The transgenic mice will be available for interested academic investigators upon request and MTA approval by the University of Washington.
In this study, we describe in situ imaging of superoxide flashes in Langendorff perfused heart as well as in vivo imaging of flash events in skeletal muscles of anesthetized mt-cpYFP transgenic mice17,19. This technology allows real time monitoring of single mitochondrial ROS production events in a physiologically relevant condition or in vivo 21,22. It is also feasible to use the system to monitor other single mitochondrial parameters such as membrane potential and calcium with appropriate fluorescent indicators. Further, simultaneous or parallel evaluation of mitochondrial function with intracellular events (e.g.&#160;calcium transients) or heart function (e.g.&#160;ejection fraction) can be achieved. Pathological perturbations, such as ischemia and reperfusion, can be applied to the perfused heart to assess the impact of stress on single mitochondrial function in the intact myocardium.

<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

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

According to this protocol, in vivo imaging of single mitochondrial events can be done in skeletal muscles of anesthetized mice followed by in situ imaging in perfused heart (Figure 1). The optimal setting of the imaging conditions will ensure clear images of the intact muscle tissues and with single mitochondrion resolution (Figure 2). TMRM is often used to verify the location of mt-cpYFP and should show a complete overlapping pattern with the mt-cpYFP signal (...

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

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

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

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

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14.5K ビュー.  University of Washington. 共焦点走査顕微鏡は、生きた動物における灌流心臓または骨格筋​​におけるミトコンドリアの単一のイベントを画像化するために適用される。スーパーオキシドが点滅し、膜電位の変動などの単一ミトコンドリアのプロセスのリアルタイム監視は、生理学的に関連するコンテキストでかつ病理学的摂動の間、ミトコンドリア機能の評価が可能。 