Name: Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice (Video) | JoVE Journal
Uploaded: 2026-04-10T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Learn to quantify mitochondrial morphology in skeletal muscle and terminal Schwann cells of mice using membrane potential-sensitive dyes and confocal microscopy. This method enables 3D analysis of mitochondrial networks in intact neuromuscular tissues without transgenic reporters.

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5 minutes

The authors thank Drs. Mariappan Muthuchamy, Mendell Rimer, and Peter Nghiem for generously providing the D2.mdx mice used to evaluate tSC mitochondria. This work was supported by the Sydney and J.L. Huffines Institute for Sports Medicine and Human Performance Student Research Grant and by funds from the College of Education and Human Development at Texas A&#38;M University. A.B.M. was also supported by the NIH LRP (NIAMS, 2L40AR077899-02A3).

Six-month-old (n=3) adult male DBA/2J [Wild-type (WT)] (RRID: IMSR_JAX:000671) and D2.B10-DMDD2.mdx/J [D2.mdx (mdx)] (RRID: IMSR_JAX:013141), while three to five-month-old (n=8) adult male and female B6; D2-Tg(S100-EGFP)1Wjt/J (S100B) (RRID: IMSR_JAX:005621) [also pooled with WT given no differences from DBA] were used from an ongoing colony of investigator-maintained mice. The overall experimental workflow for TA and GM injections, tissue processing, and imaging is summarized in Figure 1. An additional D2.mdx mouse was investigator-donated for tSC imaging in mdx (n=1). All animal procedures were approved by the Institutional Animal Care and Use Committee at Texas A&#38;M University (AUP 2022-0215) and were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (8th edition)30. Euthanasia was performed by cervical dislocation under an anesthetic overdose of isoflurane.
1. Preparation of fluorescent mitochondrial stain working solution of 2 &#181;M
<ol>
	<li>Suspend the mitochondrial stain (50 &#181;g) in 322 &#181;L of dimethyl sulfoxide (DMSO) according to the manufacturer&#8217;s instructions to prepare a 200 &#181;M stock solution.</li>
	<li>To prepare 30 mL of 2 &#181;M working solution, transfer 300 &#181;L of 200 &#181;M stock solution into 29.7 mL of sterile saline. Mix thoroughly.</li>
	<li>Aliquot 250 &#181;L of the 2 &#181;M working solution into microcentrifuge tubes and store at -80 &#176;C until use. 
	NOTE: No detectable signal degradation was observed after 3 years of storage at -80 &#176;C.</li>
</ol>
2. Dye and animal preparation
<ol>
	<li>Thaw one frozen aliquot 15 min prior to experimentation. Draw the solution into a 30 G, 1 cc insulin syringe.</li>
	<li>Anesthetize the animal with isoflurane. Apply ophthalmic ointment, shave the injection site, and maintain body temperature using a warming lamp with a temperature probe positioned between the animal and the heat source.</li>
	<li>Clean the shaved injection site with 70% isopropyl alcohol.</li>
</ol>
3. Tibialis anterior (TA) muscle mitochondrial dye administration and function
<ol>
	<li>TA dye administration
	<ol>
		<li>Under anesthesia, inject 50 &#181;L of 2 &#181;M mitochondrial stain into the Left or Right TA muscle of anesthetized WT and D2.mdx mice.</li>
		<li>Insert the needle at the distal tendon toward the knee and depress the plunger while withdrawing the needle along the injection path.</li>
		<li>Incubate the dye in vivo for 1 h prior to dissection.</li>
	</ol>
	</li>
	<li>TA muscle function measurements
	<ol>
		<li>After 45 min of dye incubation, anesthetize the mouse using isoflurane (2%&#8211;3% induction, 1.5%&#8211;2% maintenance in oxygen).</li>
		<li>Surgically expose the left TA muscle while preserving surrounding tissues. Tie the distal tendon to the load beam of the muscle testing system.</li>
		<li>Wrap the exposed muscle with a laboratory wipe and irrigate at 3 mL min&#8315;1 with 0.9% sterile saline warmed to 40 &#176;C. Maintain muscle temperature at 32 &#176;C using a heat lamp.</li>
		<li>Position platinum/iridium wire electrodes across the muscle belly for direct stimulation.</li>
		<li>Attach the load beam to a micrometer and adjust optimal length (L0) using twitch contractions (Tw) at 1 Hz. Record L0 with a digital caliper.</li>
		<li>Induce maximum tetanic contractions (P0) three times at 140 Hz with 60 s rest between contractions. Record the highest force value (g). Acquire data using the muscle function system with analysis software.</li>
	</ol>
	</li>
	<li>TA tissue processing
	<ol>
		<li>After incubation, isolate and dissect the TA muscle prior to euthanasia.</li>
		<li>Place isolated muscle fiber bundles in 4% paraformaldehyde at room temperature for 15 min while gently teasing fibers under a stereomicroscope. 
		CAUTION: Paraformaldehyde is toxic and should be handled in a chemical fume hood while wearing appropriate personal protective equipment (lab coat, gloves, and eye protection).</li>
		<li>Add cell-permeant nucleic acid stain to a final concentration of 5&#8211;10 &#181;g/mL to label nuclei.</li>
		<li>Wash samples three times in phosphate-buffered saline (PBS) for 5 min each to remove excess dye.</li>
		<li>Dehydrate samples sequentially in 50%, 75%, and 100% ethanol for 5 min each.</li>
		<li>Incubate samples sequentially in tissue clearing solutions I and II for 30 min each at room temperature using sufficient volume to fully submerge tissue.</li>
		<li>Place cleared TA bundles on a coverslip and gently flatten using a glass block (2.5 cm &#215; 2 cm &#215; 1 cm; mass 7.8 g)26.</li>
	</ol>
	</li>
</ol>
4. Imaging of TA muscle samples
<ol>
	<li>Image muscle samples with a confocal microscope to obtain high-resolution three&#8211;dimensional (3D) optical sections.</li>
	<li>Acquire Z-stack images at a depth of approximately 100 &#181;m for muscle using a 63x magnification objective lens (N.A. 1.40).</li>
	<li>Use the imaging system&#8217;s confocal software with an excitation wavelength of 646 nm to provide the greatest clarity with the least background.</li>
	<li>Acquire images using deconvolution mode, employing &#8220;System Optimized&#8221; parameters with 1 &#181;m step sizes. 
	NOTE: To combat dehydration and photobleaching, transfer the tissues into tissue-clearing solution II and place them on a coverslip. Place two drops of clearing solution 2 directly upon the tissue prior to imaging.</li>
</ol>
5. GM muscle tSC dye loading and imaging
<ol>
	<li>Use S100&#946; mice for specific fluorescent identification of tSCs.</li>
	<li>Under anesthesia, perform a small skin incision above the GM muscle. Inject 75 &#956;L of a 2 &#956;M far-red fluorescent mitochondrial stain beneath the GM, prior to wound closure25.</li>
	<li>Close the wound and allow the dye to incubate for 45 minutes to selectively label tSCs without significant labeling of the underlying muscle fibers.</li>
	<li>Dissect the GM muscles and immediately place them in chilled 0.9% sterile saline. Rapidly trim excess fat and connective tissue.</li>
	<li>Transfer the dissected GMs onto a coverslip for imaging.</li>
	<li>Only if using D2.mdx mice, add fluorescent acetylcholine receptor (AChR) dye at a 1:500 dilution to the microdissection bath. Incubate the tissue for 30 min to visualize the postsynaptic components of the neuromuscular junction (NMJ).</li>
	<li>Use this dual-labeling approach to differentiate postsynaptic mitochondria from tSC-specific mitochondria. 
	CAUTION: Limit dissection and cleaning of the GM to no more than 10 minutes prior to placing onto a coverslip for imaging to minimize morphological changes following dissection. This time limit is critical to minimize post-dissection morphological changes and mitochondrial degradation.</li>
	<li>Perform confocal microscopy to obtain high-resolution three&#8211;dimensional (3D) optical sections.</li>
	<li>Acquire Z-stack images at a depth of approximately 16 &#181;m for tSCs using a 20x magnification objective lens (N.A. 0.75). Acquire high-resolution representative images of individual tSCs using a 63x magnification objective lens.</li>
	<li>Implement mounting procedures to minimize tissue dehydration and photobleaching during the imaging session. 
	CAUTION: Transfer tissues in saline onto a coverslip, ensuring the ventral side faces down for inverted microscopy.</li>
	<li>Apply 2 &#181;L of general mounting medium onto the dorsal surface of the GM.</li>
	<li>Gently flatten the tissue using a calibrated glass block (2.5 cm &#215; 2 cm &#215; 1 cm; mass 7.8 g) to ensure a uniform imaging plane.</li>
</ol>
6. Focused ion beam microscopy
<ol>
	<li>Perform focused ion beam scanning electron microscopy (FIB-SEM) according to previously established protocols26.
	<ol>
		<li>Apply FIB-SEM specifically to confirm and visualize novel morphological features identified during confocal imaging.</li>
	</ol>
	</li>
</ol>
7. Image analysis
<ol>
	<li>TA muscle samples
	<ol>
		<li>Conduct quantitative analysis of confocal optical sections (1 &#956;m) of TA scans (approximately 75&#8211;112 &#956;m thick) using AI-driven image analysis software. 
		&#8203;NOTE: Perform multiple supervised training sessions prior to automated segmentation to enhance algorithm adaptation.</li>
		<li>Import single optical sections (Z-slices) to teach the software to retain pixelated information representing white mitochondrial dye while excluding the black background.</li>
		<li>Develop image masks for investigator review to ensure high-quality analysis and accuracy.</li>
		<li>Separate 3D image stacks into single optical sections following algorithm optimization.</li>
		<li>Select every fifth Z-slice from each 3D image for analysis, with each data point representing the average of these individual sections per sample.</li>
		<li>Quantify mitochondrial morphology to derive spatial distribution and network connectivity metrics.</li>
		<li>Standardize intensity thresholds, edge detection settings, and voxel size calibration across all samples to minimize experimental variability.</li>
	</ol>
	</li>
	<li>tSCs on GM samples
	<ol>
		<li>Assemble 1 &#956;m optical sections into Z-stacks (approximately 6&#8211;25 &#956;m thick) and separate images into individual color channels using 2D image analysis software.</li>
		<li>Calibrate software using scale bars and apply thresholding to optimize tSC mitochondria visualization with minimal background interference.</li>
		<li>Perform binarization and remove background noise to isolate tSC mitochondria or tSCs for the measurement of area and particle counts.</li>
		<li>Save binary images to ensure consistent processing and perform all imaging experiments under identical conditions to ensure reproducibility across biological replicates.</li>
	</ol>
	</li>
</ol>
8. Immunohistochemistry
<ol>
	<li>Embed left TA muscle samples in optimal cutting temperature (OCT) compound to evaluate cross-sectional area (CSA).
	<ol>
		<li>Section samples at 10 &#956;m thickness using a cryostat set to -17&#176;C and collect onto microscope slides.</li>
		<li>Stain muscle cross-sections for laminin to identify myofiber borders using rabbit anti-laminin primary antibody (1:400) and goat anti-rabbit Rhodamine secondary antibody (1:400).</li>
		<li>Mount slides using an appropriate mounting medium prior to imaging.</li>
	</ol>
	</li>
	<li>Image slides using a confocal microscope by randomly selecting 2&#8211;3 regions of interest (580 x 580 &#181;m) per sample with a 20x (N.A. 0.75) objective.</li>
	<li>Average values across regions per muscle and analyze approximately 400 myofibers per TA muscle section.</li>
	<li>Use semi-automatic muscle segmentation software to determine CSA (&#181;m2) by masking the myofibers.</li>
</ol>
9. Statistical analysis
<ol>
	<li>Report summary data as mean &#177; standard error of the mean (S.E.M.).</li>
	<li>Perform statistical analysis using a dedicated statistical software, utilizing parametric, unpaired Student&#8217;s t-tests to determine significance among group mean differences.</li>
	<li>Conduct simple linear regressions on CSA and muscle force from TA muscles, utilizing these as markers of DMD pathology to compare mitochondrial morphology from TA muscle and GM tSCs.</li>
	<li>Assess Pearson r correlation coefficients to determine the correlation between DMD pathology and mitochondrial pathology. Define statistical significance as P &#8804; 0.05.</li>
</ol>

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<li>Pareyson, D., Piscosquito, G., Moroni, I., Salsano, E., Zeviani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+neuropathy+in+mitochondrial+disorders%5BTitle%5D)+AND+%22Lancet+Neurol%22%5BJournal%5D)">Peripheral neuropathy in mitochondrial disorders.</a> Lancet Neurol. 12 (10), 1011-1024 (2013).</li>
<li>Schröder, J. M., Sommer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+abnormalities+in+human+sural+nerves%3A+Fine+structural+evaluation+of+cases+with+mitochondrial+myopathy%2C+hereditary+and+non-hereditary+neuropathies%2C+and+review+of+the+literature%5BTitle%5D)+AND+%22Acta+Neuropathol%22%5BJournal%5D)">Mitochondrial abnormalities in human sural nerves: Fine structural evaluation of cases with mitochondrial myopathy, hereditary and non-hereditary neuropathies, and review of the literature.</a> Acta Neuropathol. 82 (6), 471-482 (1991).</li>
<li>Schröder, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropathy+associated+with+mitochondrial+disorders%5BTitle%5D)+AND+%22Brain+Pathol%22%5BJournal%5D)">Neuropathy associated with mitochondrial disorders.</a> Brain Pathol. 3 (2), 177-190 (1993).</li>
<li>Santosa, K. B., Keane, A. M., Jablonka-Shariff, A., Vannucci, B., Snyder-Warwick, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+relevance+of+terminal+Schwann+cells%3A+An+overlooked+component+of+the+neuromuscular+junction%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Clinical relevance of terminal Schwann cells: An overlooked component of the neuromuscular junction.</a> J Neurosci Res. 96 (7), 1125-1135 (2018).</li>
<li>Morton, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inducible+deletion+of+endothelial+cell+Efnb2+delays+capillary+regeneration+and+attenuates+myofibre+reinnervation+following+myotoxin+injury+in+mice%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.</a> J Physiol. 602 (19), 4907-4927 (2024).</li>
<li>Morton, A. B., Norton, C. E., Jacobsen, N. L., Fernando, C. A., Cornelison, D. D. W., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Barium+chloride+injures+myofibers+through+calcium-induced+proteolysis+with+fragmentation+of+motor+nerves+and+microvessels%5BTitle%5D)+AND+%22Skelet+Muscle%22%5BJournal%5D)">Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels.</a> Skelet Muscle. 9 (1), 27(2019).</li>
<li>Sukhorukov, V. M., Dikov, D., Reichert, A. S., Meyer-Hermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emergence+of+the+mitochondrial+reticulum+from+fission+and+fusion+dynamics%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">Emergence of the mitochondrial reticulum from fission and fusion dynamics.</a> PLoS Comput Biol. 8 (10), e1002745(2012).</li>
<li>Ferramosca, A., Zara, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+carriers+and+substrate+transport+network%3A+A+lesson+from+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Mitochondrial carriers and substrate transport network: A lesson from Saccharomyces cerevisiae.</a> Int J Mol Sci. 22 (16), (2021).</li>
</ol>

The authors have no competing interests.

The location of mitochondria within cellular structures, as well as their morphology, correlates with mitochondrial health and metabolic demand21. Given the clinical relevance of mitochondrial health, coupled with divergent respiration kinetics and morphologies of different cell types, an easily applied, confocal microscopy-based method for the evaluation of mitochondrial morphologies in tSCs was developed after first evaluating the method in skeletal muscle, providing information on mitochondrial...

Mitochondria are remarkably plastic organelles that act as the lungs of the cell, consuming oxygen during cellular respiration to support adenosine triphosphate (ATP) production in response to metabolic demand1. While once thought of as discrete organelles, it is now understood that mitochondria are highly integrated2,3,4, sharing energy substrate throughout their network in accord with local requirements. Integration provides a distinct advantage over isolation: connected mitochondria support energy deficits, maintaining electrochemical gradients for oxidative phosphorylation during elevated metabolic demand. Indeed, the distribution, volume, and morphology of mitochondria suit the energy needs in their vicinity5. In health, regular turnover of mitochondrial networks is maintained through mitochondrial biogenesis, fusion and fission events, and mitophagy2,3. Mitochondrial biogenesis is required to maintain and expand healthy mitochondria. Metabolic demand, such as physical exercise, increases expression of the peroxisome proliferator-activated receptor &#947; co-activator 1&#945; (PGC-1&#945;)5. This process occurs via primary endurance signals, specifically the exercise-induced elevation of AMP-activated protein kinase (AMPK), calcium, and reactive oxygen species6,7. Fusion expands networks, promoting survivability and complementing deficits, while fission mitigates further mitochondrial damage through the preferential exclusion of dysfunctional region2,3,8. Once removed, excluded regions must undergo mitophagy, preventing the accumulation of dysfunctional organelles and proteins6,9.
Skeletal muscle comprises about 40% of body mass and relies on mitochondria for aerobic metabolism and cellular signaling to maintain homeostasis10. Collectively, in conditions of skeletal muscle dysfunction, mitochondrial quality control mechanisms are impaired10. Investigations of muscle dysfunction typically measure mitochondrial respiration, volume, morphology, and reactive oxygen species (ROS) production to assess mitochondrial quality3,10. In that regard, during prolonged muscle dysfunction, mitochondrial respiration is reduced, while ROS are increased, and networks are fragmented3,10. Perturbations in mitochondrial morphology arise largely from activation of mitochondrial fission proteins, dynamin-related protein 1 (DRP1) and mitochondrial fission 1 (FIS1)2,8,11. In response to muscle atrophy, FIS1, residing on the surface of the outer mitochondrial membrane, recruits DRP1 to oligomerize with multiple DRP1 dimers to create a filament around the mitochondria, decreasing in size until a given section of mitochondria is &#8220;pinched&#8221; off, promoting fragmentation3,11. Together, overactivity of mitochondrial fission, reductions in fusion of newly formed mitochondria, and dysfunctional mitophagy are responsible for the fragmented appearance of dysfunctional mitochondrial networks, like those described in inactive or diseased muscle2,12.
The neuromuscular junction (NMJ) is the site of muscular excitation. Action potentials initiated in the soma propagate along axons, ensheathed by myelinating Schwann cells (SCs), and terminate at NMJs, which are encapsulated by terminal Schwann cells (tSC). NMJs can regenerate following injury. However, the likelihood and speed of regeneration are diminished as the distance between the site of injury and the NMJ increase13,14. Following nerve injury by transection, crush, or stretch, SCs dedifferentiate into a demyelinated progenitor-like state15,16. Demyelinated SCs proliferate and work in concert with macrophages to remodel the local environment for regeneration by clearing debris between the site of injury and the proximal axon stump through Wallerian degeneration17,18. Migrating SCs signal an inflammatory response that remodels the environment for successful nerve regeneration18. Nerve regeneration occurs inside the neural sheath formed by the progenitor-like SCs, which form bands of Bunger, through which the axons regrow19. The axon stump then extends filopodia to initiate reinnervation14. Following reinnervation, the presynapse can fully regenerate, provided tSCs reinvest at the NMJ20.
Mitochondrial morphology varies markedly between cell types, conforming to tissue function21. Reports from liver tissues suggest that mitochondria are more compact and spherical, whereas those in the white matter in the brain are more elongated and tubular22. Upon challenge, such as in conditions of aging and oxidative stress, mitochondria in these tissues exhibit a doughnut-like phenotype. However, in osteoblasts, mitochondrial doughnut formation and fragmentation were linked with increased secretion of mitochondria and mitochondrial-derived vesicles, and with osteoblast maturation23. While skeletal muscle mitochondrial networks have been extensively examined across multiple species, limitations remain in imaging mitochondrial networks from tSCs of the peripheral nervous system. These pose difficult&#160;to capture within their native environments, as cell ultrastructure is significantly altered following isolation, and these tissues are difficult to target with dye in vivo without also loading accompanying skeletal muscle.
Imaging techniques used to capture mitochondrial morphology in living systems can be categorized as either light or electron microscopy5. With traditional light capture microscopy, dyes can label live cells, fixed cells can be immunolabeled, and mitochondria can be genetically labeled5,24. Live-cell imaging typically requires incubating cells for 20&#8211;60 min prior to fluorescence analysis. Dyes are directed to mitochondria via membrane potential and, therefore, may be altered under conditions of altered membrane potential5. Capturing mitochondrial morphology of tSCs within skeletal muscle via dye loading is theoretically possible; however, it would require imaging of whole tissues and detailed labeling techniques. In particular, it captures mitochondria within intact structures, without interference from skeletal muscle labeling. To overcome these limitations, the mitochondrial fluorescent stain was used to label living skeletal muscle in healthy and dystrophic mice, followed by labeling tSC mitochondria for in vivo and in situ imaging. The tibialis anterior (TA) muscle was selected to optimize skeletal muscle imaging due to its superficial anatomical location, well-characterized injection volume and myofascial dispersion25,26,27, and suitability for concurrent functional measurements. The gluteus maximus (GM) muscle was selected for tSC imaging based on its established injection procedure28,29, its thin architecture, which minimizes background fluorescence, and the superficial ventral positioning of neuromuscular junctions between the GM and gluteus medius, enabling preferential labeling of tSCs prior to skeletal muscle fibers.

<table><tbody><tr><td>3-in-1 Whole Animal System</td><td>Aurora Scientific, Ontario, Canada</td><td>1300A</td><td>Muscle funciton system with analysis software</td></tr><tr><td>AIVIA&amp;nbsp;</td><td>Leica Microsystems, Bellevue, WA, USA</td><td>15</td><td>AI driven image analysis software</td></tr><tr><td>anti-laminin&amp;nbsp;</td><td>MilliporeSigma</td><td>L9393-.2mL</td><td>myofiber boarder label</td></tr><tr><td>B6; D2-Tg(S100-EGFP)&lt;sup&gt;1Wjt&lt;/sup&gt;/J Mouse Strain</td><td>Jackson Labs, Bar Harbor, ME</td><td>JAX:005621</td><td>S100&amp;beta;</td></tr><tr><td>Cryostar NX50 Cryostat</td><td>Epredia, Kalamazoo, MI, USA</td><td>https://www.epredia.com/products/histology-instruments/cryotomy/cryostar-nx50</td><td>Cryostat</td></tr><tr><td>D2.B10-DMD&lt;sup&gt;D2.mdx&lt;/sup&gt;/J Mouse Strain</td><td>Jackson Labs, Bar Harbor, ME</td><td>JAX:13141</td><td>D2.mdx</td></tr><tr><td>DBA/2J Mouse Strain</td><td>Jackson Labs, Bar Harbor, ME</td><td>JAX:00671</td><td>WT</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Scientific; Hampton, NJ, USA</td><td>BP231-100</td><td>Mitochondrial stain reconstitution reagent</td></tr><tr><td>Hoechst 33342</td><td>Thermo Fisher Scientific</td><td>H1399</td><td>Cell-permeant nucleic acid stain</td></tr><tr><td>ImageJ/Fiji&amp;nbsp;</td><td>National Institutes of Health, Bethesda, MD, USA</td><td>SCR_002285</td><td>2D analysis software</td></tr><tr><td>Invitrogen ProLong Gold antifade reagent with DAPI&amp;nbsp;</td><td>Fisher Scientific, Hampton, NJ, USA</td><td>P36941</td><td>Mounting medium</td></tr><tr><td>KimWipe</td><td>Kimberly-Clark, Irving, TX, USA</td><td>06-666A</td><td>Laboratory wipes</td></tr><tr><td>Leica LAS_X software&amp;nbsp;</td><td>Leica Biosystems, Wetzlar, Germany</td><td>SCR_013673</td><td>Confocal software</td></tr><tr><td>MitoView Fix 640</td><td>Biotium</td><td>70075-50ug</td><td>Far-red fluorescent mitochondrial stain</td></tr><tr><td>NIS-Elements Advanced Research software&amp;nbsp;</td><td>Nikon, Melville, NY, USA</td><td>SCR_014329</td><td>Cross sectional area analysis software</td></tr><tr><td>Permount Mounting Medium&amp;nbsp;</td><td>Fisher Scientific, Waltham, MA 02454, USA</td><td>#SP15-100</td><td>General mounting media</td></tr><tr><td>Prism 9 software&amp;nbsp;</td><td>GraphPad Software, La Jolla, CA, USA</td><td>RRID: SCR_002798</td><td>Statistical analysis software</td></tr><tr><td>Rhodamine (TRITC)</td><td>Fisher Scientific; Hampton, NJ, USA</td><td>AP132RMI</td><td>Secondary antibody</td></tr><tr><td>Stellaris 5 White Light Confocal Microscope and corresponding deconvolution software</td><td>Leica Microsystems, Wetzlar, Germany</td><td>158301313:158201310:158004752:&lt;br/&gt; 158301312:158301314:158401100:&lt;br/&gt; 158209011:158301130:158204510:&lt;br/&gt; 158301120:158401140:158204511:&lt;br/&gt; 15500332:15506428:15506517:155&lt;br/&gt; 13859:15521522:15525226:155252&lt;br/&gt; 32:15525314:15555009:15555017:&lt;br/&gt; 15555046:27100022:155933660:&lt;br/&gt; 155933666:155933668:158000640&lt;br/&gt; :158002407:158003150:158004141&lt;br/&gt; :158004201:158004421:158004423&lt;br/&gt; :158200681:158202140:158203114:&lt;br/&gt; 158203200:158203220:158204201:&lt;br/&gt; 158204510:158204709:158301200:&lt;br/&gt; 9I_LL_STELLARIS_C:9T-CLSM-APP&lt;br/&gt; _CLASSC:9WE_LAS_405:9WE_&lt;br/&gt; LAS_WLL:9WE_LL_STELLARIS_C</td><td>Confocal microscope</td></tr><tr><td>SZ61</td><td>Olympus, Breinigsville, PA, USA</td><td>sz61</td><td>Stereo microscope</td></tr><tr><td>Tissue-Plus O.C.T. Compound&amp;nbsp;</td><td>Scigen, Fischer Scientific, Hampton, NJ, USA</td><td>23-730-571</td><td>Optimal cutting temperature compound</td></tr><tr><td>Visikol I</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>H1-30</td><td>Tissue clearing solution 1</td></tr><tr><td>Visikol II</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>H2-30</td><td>Tissue clearing solution 2</td></tr><tr><td>&amp;alpha;-bungarotoxin 552</td><td>Biotrend, Koln, Germany</td><td>00014</td><td>Acetylcholine receptor dye</td></tr></tbody></table>

methods for in situ quantification of mitochondrial morphology in muscle and terminal schwann cells of mice

Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues remains technically challenging. Terminal Schwann cells (tSCs), which are essential for the maintenance and regeneration of neuromuscular junctions, are particularly difficult to analyze in situ due to their anatomical location and sensitivity to tissue disruption. This protocol describes a reproducible approach for labeling and quantifying three&#8211;dimensional mitochondrial network morphology in whole-mount skeletal muscle and in tSCs in mice using standard confocal microscopy. The method employs in vivo delivery of a membrane potential&#8211;sensitive mitochondrial dye followed by rapid tissue processing and high-resolution confocal imaging. Image stacks are analyzed to quantify mitochondrial network connectivity, area, and fragmentation. The protocol is first validated in dystrophic and healthy skeletal muscle to confirm expected differences in mitochondrial morphology and is subsequently adapted to visualize and quantify mitochondrial networks in tSCs identified using S100&#946; reporter mice. This approach enables the analysis of mitochondrial morphology within intact neuromuscular tissues without requiring transgenic mitochondrial reporters or specialized imaging platforms. The protocol requires only equipment commonly available in university core facilities and can be adapted to other thin or superficially accessible tissues.

Mitochondrial networks displayed expected reductions in connectivity 
Representative 3D fluorescent images of TA muscle confocal scans in two groups, WT and mdx, are presented (Figure 2). In comparison with the WT group, mitochondrial connectivity appears reduced in mdx mice compared to the WT (Figure 2A–D). Greater numbers of small mitochondrial fragments were observed in mdx mice [&lt;100 µm2] (WT, 17.3 ± 3.5%; m...

Local injections of a mitochondrial dye label terminal Schwann cell mitochondria in vivo for confocal microscopy. This method utilizes proof of concept demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle to provide high-resolution mitochondrial visualization.

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues ...

Our research investigates mitochondrial morphology of terminal Schwann cells at the neuromuscular junction for the first time. Our approach enables quantification of mitochondrial morphology in otherwise hard-to-obtain tissues, such as capillary and lymphatic beds. To begin, obtain a wild type and a D2-mdx mouse.After confirming the anesthetic depth by toe-pinch reflex, insert the needle at the distal tendon toward the right knee. Inject 50 microliters of two-micromolar far red fluorescent mitochondrial stain on the tibialis interior, or the TA, muscle while withdrawing the needle along the injection path, and incubate the dye in vivo for one hour. While maintaining the animal under anesthesia, use scissors and forceps to dissect and isolate the TA muscle prior to euthanasia.Place the isolated TA muscle in 4%paraformaldehyde at room temperature for 15 minutes. Then, under a stereo microscope, gently separate the fibers by teasing. Add cell-permeant nucleic acid stain to the 4%paraformaldehyde to obtain a final concentration of five to 10 micrograms per milliliter for nuclear labeling.Then, wash the TA muscle three times in PBS for five minutes each to remove excess dye. Dehydrate the muscle sequentially in 50%75%and 100%ethanol for five minutes each. Then, incubate the sample sequentially in tissue-clearing solutions one and two for 30 minutes each, ensuring they are fully submerged.Place the muscle on a coverslip. Use a 2.5-by-2-by-1-centimeter glass block weighing 7.8 grams to gently flatten it. Obtain an S100 beta mouse.After confirming the anesthetic depth by toe-pinch reflex, make a small skin incision above the gluteus maximus, or GM, muscle. Inject 75 microliters of two-micromolar far red fluorescent mitochondrial stain beneath the GM muscle. Close the wound and let the dye incubate for 45 minutes to selectively label terminal Schwann cells.While maintaining the animal under anesthesia, dissect the GM muscle within 10 minutes, and immediately place it in chilled 0.9%sterile saline. Only if using D2-mdx mice, add fluorescent acetylcholine receptor dye at a one-to-500 dilution to the microdissection bath, and incubate the tissue for 30 minutes. Trim the excess fat and connective tissue, and transfer the GM muscle onto a coverslip with the ventral side facing down for inverted microscopy.Add two microliters of general mounting medium onto the dorsal surface of the GM, and flatten the tissue using glass block before imaging. In the software, set the excitation wavelength to 646 nanometers. After adding two drops of clearing solution two, select the 63X objective lens to image the TA muscle, and set the Z stack depth close to 100 micrometers.Then, select deconvolution mode, and apply system optimized parameters. Set the step size to one micrometer and acquire images. To acquire terminal Schwann cells'images, select the 20 times magnification objective lens, set the Z stack depth to approximately 17 micrometers, and acquire images.Then, select the 63 times magnification objective lens and acquire images of individual terminal Schwann cells. Import individual Z slices to train the software to recognize the mitochondrial signal. And conduct quantitative analysis using AI-driven software.Develop image masks for investigator review. Separate 3D image stacks into individual optical sections following algorithm optimization. Select every fifth Z slice from each 3D image, and calculate the average per sample.Standardize imaging parameters across all samples, and quantify mitochondrial morphology. Perform immunohistochemistry and statistical analysis to assess group differences and correlations. Mitochondrial connectivity was reduced in mdx muscle compared to the wild type mice.A greater number of small mitochondrial fragments and a reduced number of large interconnected mitochondrial networks were observed in mdx mice. In wild type mice, mitochondria in terminal Schwann cells exhibited interconnected loop structures near bifurcations. In contrast, in mdx mice, mitochondria appeared fragmented and lacked loop formations near bifurcations.Confocal and electron microscopy confirmed similar donut-shaped mitochondrial structures in terminal Schwann cells. Postsynaptic analysis showed sub-sarcolemmal mitochondria clustered around acetylcholine receptors, lacking the characteristic pretzel-like structure. Image analysis revealed that terminal Schwann cell mitochondria in mdx muscle showed increased area and fragmentation compared to wild type muscle, with no difference in relative particle size.Muscle force measurements showed reduced twitch with greater variability and decreased peak force in mdx muscle. Force production across increasing stimulation frequencies was also significantly reduced in mdx muscles. The mdx muscle showed a leftward shift in fiber size, indicating approximately 60%smaller myofibers compared to wild type.This protocol allows researchers to study and quantify mitochondrial morphology in skeletal muscle and terminal Schwann cells. Future studies can adapt this approach to investigate mitochondrial morphology in micro-circulation and lymphatic vessels.

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Research

JoVE Journal

Biology

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Skeletal muscle is essential for locomotion and is the bodies’ main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.



Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in C. elegans to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Learn to quantify mitochondrial morphology in skeletal muscle and terminal Schwann cells of mice using membrane potential-sensitive dyes and confocal microscopy. This method enables 3D analysis of mitochondrial networks in intact neuromuscular tissues without transgenic reporters.

Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

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