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 CaCl2. Add metabolic substrates (e.g.&#160;10 mM glucose and 0.5 mM pyruvate).</li>
	<li>Prepare the surgical instruments by sterilizing the clamp, scissors, and forceps in 70% ethanol and then rinsing in sterilized ddH2O.</li>
	<li>Turn on the heart perfusion system, adjust the temperature on the water bath and circulator, set the speed of peristaltic pump to 6 ml/min.</li>
	<li>Bubble the KHB with 95% O2/5% CO2 gas, and monitor the flow rate and temperature (37 &#176;C, by a fiber optic temperature probe) of the effluent. The system needs about 20 min for gas and temperature equilibration.</li>
</ol>
2. Confocal Imaging of Skeletal Muscles In Vivo
<ol>
	<li>Anesthetize mouse with pentobarbital (80 mg/kg, i.p.). The animal will reach surgical anesthesia (no response to toe pinch) within 10-15 min and remain in this status for 1-1.5 hr, sufficient for the in vivo imaging of skeletal muscles.</li>
	<li>Remove hairs on one of the hindlimbs and sterilize the skin with 70% ethanol.</li>
	<li>Make an incision on the skin along the outer side of the limb to expose the gastrocnemius muscles.</li>
	<li>Pick up the epimysium gently with a sharp forceps and make an incision through it with scissors. Further dissect to remove the epimysium and expose the muscle fibers beneath it.</li>
	<li>For in vivo loading of TMRM into the skeletal muscle, include TMRM (500 nM) in the isotonic balanced salt solution to immerse muscle for 30 min and then wash out by indicator-free solution.</li>
	<li>Put the mouse on its side on the confocal microscope (Zeiss LSM 510) stage and restrain the rear limb in a position that the exposed skeletal muscle is facing against the coverslip that forms the bottom of the chamber. The coverslip is in between the muscle tissue and the inverted objective (40X oil).</li>
	<li>Press the leg down gently to make tight contact between the tissue and the coverslip. Immerse the exposed muscles in isotonic balanced salt solution.</li>
	<li>Record two dimensional (2D, xy) confocal images at a sampling rate of one second per frame. The intensity of each pixel is digitized at 8-bit depth. Usually, a serial scan contains 100 frames.</li>
	<li>Collect sequential images by first exciting at 405 nm and collecting at &#62;505 nm then at 488 nm while collecting at &#62;505 nm for dual wavelength excitation imaging of mt-cpYFP. Use sequential excitation at 405, 488 and 543 nm, and collect emission at 505-545, 505-545, and &#62;560 nm, respectively, for triple wavelength excitation imaging of mt-cpYFP and TMRM.</li>
</ol>
3. Confocal Imaging of Perfused Mouse Heart
<ol>
	<li>Immediately after the in vivo imaging of skeletal muscles, inject the mouse with 200 units of heparin (i.p.). Ten minutes later, euthanize the mouse by thoracotomy and quickly remove the heart with lungs and thymus attached to it.</li>
	<li>Quickly remove the lung in ice-cold buffer. Identify the lobes of the thymus and gently peel back to expose the ascending aorta.</li>
	<li>Remove the thymus and isolate the aorta by carefully removing any surrounding tissue.</li>
	<li>Make a cut at the upper end of ascending aorta before the first branch of aortic arch.</li>
	<li>Hold the wall of the aorta gently with two micro suturing forceps to expose the lumen and carefully place the aorta onto the cannula (PE50 tube). The aorta is held in place with a micro vessel clamp while sutures are quickly tied around the aorta.</li>
	<li>Remove the clamp, carefully check the cannula with forceps to make sure the tip of the cannula is above aortic root. Additional ties are added as necessary to hold the heart in place.</li>
	<li>Turn on the peristaltic pump and perfuse the heart at 1 ml/min. The heart will resume beating upon perfusion.</li>
	<li>Adjust the position of the perfusion system and put the heart on the microscope stage. The stage has an adaptor that allows heating of the chamber that holds the heart.</li>
	<li>Add 1 ml of KHB perfusion solution in the chamber to partially submerge the heart. Monitor temperature of the chamber at 37 &#176;C. Remove effluent from the chamber by using a peristaltic pump.</li>
	<li>Increase the speed of peristaltic pump gradually to provide sufficient flow (approximately 2 ml/min) to the heart.</li>
	<li>After 10 min of stabilization, perfuse the heart with 10 &#956;M blebbistatin and 100-500 nM TMRM. Heart beat will slow down after 10 min.</li>
	<li>Apply gentle pressure on the heart to ensure tight contact of the heart with the coverslip at the bottom of the chamber and to further suppress heartbeat.</li>
	<li>Follow the same procedure for confocal imaging of intact myocardium as described in step 2.8 above. Carefully adjust the focal plane to reveal the clearest image possible.</li>
</ol>
4. Image Processing and Data Analysis
<ol>
	<li>Use tools provided by the &#34;Physiological Analysis&#34; module of the confocal software to analyze single mitochondrial flashes and membrane potential changes. This module is often included in the image acquiring software of the confocal system and allows determination of certain areas in the image as well as output of fluorescence intensity with time labels.</li>
	<li>Open the database and then the serial 2D image file to be analyzed.</li>
	<li>Click the &#34;Region of Interest (ROI) mean&#34; to switch to the &#34;mean of ROIs&#34; mode. Click the &#34;Region of Interest (ROI) mean&#34; to switch to the &#34;mean of ROIs&#34; mode.</li>
	<li>Turn off the display of other channels except the channel of cpYFP 488 nm for selecting the flashes.</li>
	<li>Zoom in the image and manually move slide bar to play the serial 2D images.</li>
	<li>Identify single mitochondrial superoxide flashes by locating the site where fluorescent signal increases transiently. Use the appropriate ROI tool to mark the flashes. The trace showing time-dependent fluorescence change of each ROI will show up beside the image.</li>
	<li>After selecting all the flashes, turn on the display of other channels. Select an ROI on the image outside of the cell for background signal subtraction. Output the average fluorescence of each ROI together with the time labels.</li>
	<li>Record the number of flashes in each of the serial scanning image file together with the scan time and area of the cell. Use Excel to calculate flash frequency as number of flashes per 100 sec/1,000 &#956;m2 cell area.</li>
	<li>Use a custom-developed program written in Interactive Data Language (IDL, ResearchSystems) to calculate the parameters of each flash (amplitude, time to peak and decay time). IDL software is commercially available and the custom-developed program for flash parameter analysis can be obtained from the author upon request.</li>
</ol>

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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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Blebbistatin</td><td>Toronto Research Chemicals</td><td>B592500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</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>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8282-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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 colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td colspan="4">Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &quot;Physiological Analysis&quot; 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 (...

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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 ...

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14.5K Views.  University of Washington. 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.

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

Bioengineering

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Medicine

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Mitochondrial fusion was measured by tracking the equilibration of photoconverted matrix-targeted GFP across the mitochondrial network over time. Thus far, only one cell could be subjected to an hour long kinetic analysis at a time. We present a method that simultaneously measures multiple cells, thereby speeding up the data collection process.

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Neuroscience

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

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.

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

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.

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

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

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

Summary

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Chapters in this video

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Visualization of Vascular Ca²⁺ Signaling Triggered by Paracrine Derived ROS

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Live-imaging of Mitochondrial System in Cultured Astrocytes

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Studying Mitochondrial Structure and Function in Drosophila Ovaries

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

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells