Primary rat hippocampal neurons were supplied by Dr. George Lykotrafitis and Shiju Gu of the Biomedical Engineering Department at the University of Connecticut (Storrs, CT, USA). The Abberior STED instrument housed in the Advanced Light Microscopy Facility in the Center for Open Research Resources and Equipment was acquired with NIH grant S10OD023618 awarded to Christopher O&#39;Connell. This research was funded by NIH grant R01AG065879 awarded to Nathan N. Alder.

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

The authors have nothing to disclose.

This protocol presents the use of human neuroblastoma cell line SH-SY5Y and primary rat hippocampal neurons with the novel IMM-targeting PKMO dye for live cell STED imaging. Due to the novelty of PKMO, there is currently little published using this dye for live STED imaging. Using these cell types for STED imaging poses challenges, specifically because neuronal cells have narrower mitochondria. One limitation of this protocol is the PKMO dye used, as it can be toxic to cells. Different cells and cell lines respond differently to the dye, thus, adjustments to dye concentration and incubation time to optimize results for strong signal without harming cells may be required. A suggested solution is to lower the concentration and increase the staining time63; however, this may result in poorer staining without increasing cell viability.
Similarly to PKMO, the commercial dye Live Orange mito (Table of Materials) also exhibits some cell toxicity. This dye was used for a variety of cultured cells but was unable to exhibit comparable staining in RA-differentiated SH-SY5Y cells successfully with the same parameters as their undifferentiated counterparts (our unpublished observations). However, amenable staining protocols may be optimized for this probe and chosen cell types. With this dye, detector gating times of 1-1.05 to 7-7.05 ns were used, with all other parameters in Table 1 remaining the same. Generally, staining cells with 200-250 nM Live Orange mito for 45 min yielded comparable results as the PKMO results shown. Higher concentration staining for less time or lower concentration staining for the same amount of time or slightly longer can yield different results and may be favorable to other cell types or growth conditions.
Imaging primary rat hippocampal neurons differs from immortalized cells due to the nature of the axon and dendrite projections as well as mitochondrial distribution at the time of imaging. One difficulty in this part of the protocol is that seeding density determines whether the primary cultures will be able to adhere and grow healthily, and at higher densities, the projections tend to overgrow by DIV 10. Therefore, the mitochondria imaged from these primary neurons will likely come from the cell body and not the projections; however, successful growth from a lower starting cell density yields better imaging results at later growth times. The key is to ensure low background and out-of-focus light to have the best contrast for STED. To address concerns regarding cell population, culturing primary hippocampal cells in B27-supplemented neuron growth media prevents the growth of glial cells, and the source reports that &#60;5% of cells are astrocytes and the absence of NbActiv1 supplement in the growth media reduces the number of astrocytes in cultures to &#60;2%87. For both cultured SH-SY5Y cells and primary rat hippocampal neurons, the PDL coating used for growth contributes to background haze in images. Sufficient signal-to-noise is accomplished with the settings reported in (Table 1) and deconvolution removes most of the background observed.
In addition to the imaging covered here, it is also possible to add treatments or stress to cells before or during imaging. For example, adding tert-butyl hydrogen peroxide (tBHP) induces oxidative stress, and it is possible to monitor changes in mitochondria over time after addition. The addition of amyloid-&#946; (A&#946;) with a fluorescent tag allows monitoring of the distribution of this peptide in relation to mitochondria as well as the mitochondrial structure over time. Mitochondrial health has been heavily implicated in AD and is widely supported to play a role in A&#946; toxicity43,71,72. Notably, the differentiation status of SH-SY5Y cells affects A&#946; protein precursor (A&#946;PP) localization85, and experiments using A&#946;PP should be carefully constructed.
As an example of how this protocol can be adapted, it is shown that the fluorescent variant A&#946;(1-42)-HiLyte 647 can be added to PKMO-stained cells 15 min before imaging (Supplementary Figure 1). The imaging parameters are similar (Supplementary Table 2), with the main difference being that a smaller pinhole is needed when imaging narrower mitochondria. Imaging A&#946;-HiLyte647 with STED requires less overall excitation (6%-8%) and STED depletion (10%-12%) laser power and fewer accumulations (six). Detector gating is also extended from 0.1 to 10 ns. Although STED resolution of A&#946; is not necessary, the overall signal-to-noise ratio and A&#946; particle size of raw STED were better than those of the confocal images, and subsequent deconvolution can also be performed. Collecting STED images and deconvolving raw STED z-stack projections of A&#946; appears particularly useful when merging with raw STED or deconvolved STED images of the PKMO stain (Supplementary Figure 1B,C). Both channels were collected in a single frame step. Measurements of time-dependent localization, similar to those listed in Figure 2 and shown in Figure 5, where applicable, and cristae architecture differences can be obtained following stress treatment or other additions.
Other possible methods for dual-labeling in live cell STED of mitochondria not reported here but have been reported by others include the use of SNAP-tagged proteins93, Halo-tagged proteins, and the use of other cell-permeable dyes with generic targets, such as mtDNA63. Notably, the labeling strategy of SNAP and Halo tagging influences the resulting fluorescence signal intensity and longevity when imaging94. Additionally, while this protocol presents several examples of analyses that can be applied to segmented mitochondria, there are many other analyses that software packages can perform on these images.

Mitochondria are eukaryotic organelles of endosymbiotic origin that are responsible for regulating several key cellular processes, including intermediary metabolism and ATP production, ion homeostasis, lipid biosynthesis, and programmed cell death (apoptosis). These organelles are topologically complex, containing a double membrane system that establishes multiple subcompartments1 (Figure 1A). The outer mitochondrial membrane (OMM) interfaces with the cytosol and establishes direct inter-organelle contacts2,3. The inner mitochondrial membrane (IMM) is an energy-conserving membrane that maintains ion gradients stored primarily as an electric membrane potential (&#916;&#936;m) to drive ATP synthesis and other energy-requiring processes4,5. The IMM is further subdivided into the inner boundary membrane (IBM), which is closely appressed to the OMM, and protruding structures called cristae that are bound by the cristae membrane (CM). This membrane delineates the innermost matrix compartment from the intracristal space (ICS) and the intermembrane space (IMS).
Mitochondria have a dynamic morphology based on continuous and balanced processes of fission and fusion that are governed by mechanoenzymes of the dynamin superfamily6. Fusion allows for increased connectivity and formation of reticular networks, whereas fission leads to mitochondrial fragmentation and enables the removal of damaged mitochondria by mitophagy7. Mitochondrial morphology varies by tissue type8 and developmental stage9 and is regulated to allow cells to adapt to factors including energetic needs10,11 and stressors12. Standard morphometric features of mitochondria, such as the extent of network formation (interconnected vs. fragmented), perimeter, area, volume, length (aspect ratio), roundness, and degree of branching, can be measured and quantified by standard optical microscopy because the sizes of these features are greater than the diffraction limit of light (~200 nm)13.
Cristae architecture defines the internal structure of mitochondria (Figure 1B). The diversity of cristae morphologies can be broadly categorized as flat (lamellar or discoidal) or tubular-vesicular14. All cristae attach at the IBM through tubular or slot-like structures termed cristae junctions (CJs) that can serve to compartmentalize the IMS from the ICS and the IBM from the CM15. Cristae morphology is regulated by key protein complexes of the IMM, including (1) the mitochondrial contact site and cristae organizing system (MICOS) that resides at CJs and stabilizes IMM-OMM contacts16, (2) the optic atrophy 1 (OPA1) GTPase that regulates cristae remodeling17,18,19, and (3) F1FO ATP synthase that forms stabilizing oligomeric assemblies at cristae tips (CTs)20,21. In addition, the IMM is enriched in nonbilayer phospholipids phosphatidylethanolamine and cardiolipin that stabilize the highly curved IMM22. Cristae are also dynamic, demonstrating morphological changes under various conditions, such as different metabolic states23,24, with different respiratory substrates25, under starvation and oxidative stress26,27, with apoptosis28,29, and with aging30. Recently it has been shown that cristae could undergo major remodeling events on a timescale of seconds, underscoring their dynamic nature31. Several features of cristae can be quantified, including dimensions of structures within individual cristae (e.g., CJ width, crista length, and width) and parameters that relate individual crista to other structures (e.g., intra-cristae spacing and cristae incident angle relative to the OMM)32. These quantifiable cristae parameters show a direct correlation with function. For instance, the extent of mitochondrial ATP production is positively related to the abundance of cristae, quantified as cristae density or cristae number normalized to another feature (e.g., cristae per OMM area)33,34,35. Because IMM morphology is defined by nanoscale features, it comprises mitochondrial ultrastructure, which requires imaging techniques that provide resolution greater than the light diffraction limit. As described below, such techniques include electron microscopy and super-resolution microscopy (nanoscopy).
The neural and glial cells of the central nervous system (CNS) are particularly reliant on mitochondrial function. On average, the brain constitutes only 2% of the total body weight, but utilizes 25% of the total body glucose and accounts for 20% of body oxygen consumption, making it vulnerable to impairments in energy metabolism36. Progressive neurodegenerative diseases (NDs), including Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington&#39;s disease (HD), multiple sclerosis (MS), and Parkinson&#39;s disease (PD), are some of the most extensively studied pathologies to date, with research efforts ranging from understanding the molecular underpinnings of these diseases to seeking potential therapeutic prevention and interventions. NDs are associated with increased oxidative stress originating in part from reactive oxygen species (ROS) generated by the mitochondrial electron transport chain (ETC)37, as well as altered mitochondrial calcium handling38 and mitochondrial lipid metabolism39. These physiological alterations are accompanied by noted defects in mitochondrial morphology that are associated with AD40,41,42,43,44, ALS45,46, HD47,48,49, MS50, and PD51,52,53. These structural and functional defects can be coupled by complex cause-effect relationships. For example, given that cristae morphology stabilizes OXPHOS enzymes54, mitochondrial ROS are not only generated by the ETC, but they also act to damage the infrastructure in which the ETC resides, promoting a feed-forward ROS cycle that enhances susceptibility to oxidative damage. Furthermore, cristae disorganization has been shown to trigger processes such as mitochondrial DNA (mtDNA) release and inflammatory pathways connected to autoimmune, metabolic, and age-related disorders55. Therefore,&#160;analysis of&#160;mitochondrial structure is key to a full understanding of NDs and their molecular underpinnings.
Popular methods of viewing cristae, including transmission electron microscopy, electron tomography and cryo-electron tomography (cryo-ET), and X-ray tomography, in particular cryo-soft X-ray tomography, have revealed important findings and work with a variety of sample types56,57,58,59,60. Despite recent advancements toward better observation of organellar ultrastructure, these methods still come with the caveat of requiring sample fixation and, therefore, cannot capture real-time dynamics of cristae directly. Super-resolution fluorescence microscopy, particularly in the forms of structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), expansion microscopy (ExM), and stimulated emission depletion (STED) microscopy, have become popular ways of viewing structures requiring resolution below the diffraction limit that constrains classical methods of optical microscopy. When ExM is used in conjunction with another super-resolution technique, the results are impressive, but the sample must be fixed and stained in a gel61. By comparison, SIM, PALM/STORM, and STED have all been successfully used with live samples, and new and promising dyes that generally stain the IMM provide a novel and easy approach for live imaging of mitochondria cristae dynamics62,63,64,65,66. Recent advancements in live dyes for STED imaging have improved dye brightness and photostability, and these dyes target the IMM at a higher degree of specificity than their predecessors. These developments allow the collection of long-term timelapse and z-stack experiments with super-resolution imaging, opening the door to better live cell analysis of mitochondrial ultrastructure and dynamics.
Herein, protocols for live cell imaging of undifferentiated and differentiated SH-SY5Y cells stained with the PKmito Orange (PKMO) dye using STED63 are provided. The SH-SY5Y cell line is a thrice subcloned derivative from the parental cell line, SK-N-SH, generated from a bone marrow biopsy of metastatic neuroblastoma67,68,69,70. This cell line is a commonly used in vitro model in ND research, particularly with diseases such as AD, HD, and PD, in which mitochondrial dysfunction is heavily implicated10,43,71,72,73. The ability to differentiate SH-SY5Y cells into cells with a neuron-like phenotype through manipulating culture media has proven a suitable model for neuroscience research without relying on primary neuronal cells10,74. In this protocol, retinoic acid (RA) was added to the cell culture medium to induce the differentiation of SH-SY5Y cells. RA is a vitamin A derivative and has been shown to regulate the cell cycle and promote the expression of transcription factors that regulate neuronal differentiation75. A protocol for culturing and live cell imaging of neurons isolated from the rat hippocampus is also provided. The hippocampus has been shown to be affected by mitochondrial degeneration and, along with the cortex, plays an important role in aging and ND76,77,78,79,80.

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>0.4% Trypan blue</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>0.5% Trypsin-EDTA, no phenol red</td><td>Gibco</td><td>15400054</td><td></td></tr><tr><td>100 X antibiotic-antimycotic</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>100 X/1.40 UPlanSApo oil immersion lens</td><td>Olympus</td><td></td><td>Equipped in Olympus IX83 microscope for STED setup described in Section 4</td></tr><tr><td>All-trans-retinoic acid</td><td>Sigma</td><td>R2625</td><td></td></tr><tr><td>Amyloid-&amp;beta; (1-42, HiLyte Fluor647, 0.1 mg)</td><td>AnaSpec</td><td>AS-64161</td><td>Other fluorescent conjugates available</td></tr><tr><td>B27 supplement (50 X), serum free</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>Cell Counter (Countess II FL)</td><td>Life Technologies</td><td>AMQAF1000</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804-R</td><td></td></tr><tr><td>Counter slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>Conical tubes (15 mL)</td><td>Thermo Fisher Scientific</td><td>339650</td><td></td></tr><tr><td>Cuvettes (Quartz Cells)</td><td>Starna Cells, Inc.</td><td>9-Q-10</td><td>Used with Spectrometer as described in Section 1.3</td></tr><tr><td>DMEM (high glucose with sodium pyruvate)</td><td>Gibco</td><td>11995073</td><td>Used for SH-SY5Y cell materials as described in Section 1</td></tr><tr><td>DMEM (high glucose no sodium pyruvate)</td><td>Gibco</td><td>11965092</td><td>Used for primary cell materials as described in Section 2</td></tr><tr><td>DMEM (phenol red-free)</td><td>Gibco</td><td>31053028</td><td>Used for imaging as described in Section 3</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>DNAase I from bovine pancreas</td><td>Sigma</td><td>DN25</td><td>Used for primary cell materials as described in Section 2.2.1 and 2.2.2</td></tr><tr><td>DPBS (no calcium, no magnesium)</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>E18 Rat Hippocampus</td><td>Transnetyx Tissue</td><td>SDEHP</td><td></td></tr><tr><td>Ethanol (200 proof)</td><td>Fisher Bioreagents</td><td>BP28184</td><td></td></tr><tr><td>Fetal bovine serum (FBS), not heat-inactivated</td><td>Gibco</td><td>26140079</td><td>For cultured cells, in Section 1</td></tr><tr><td>Fetal bovine serum (heat inactivated)</td><td>Gibco</td><td>10082147</td><td>For primary cell culture, Section 2</td></tr><tr><td>Filter sterilization unit (0.1 &amp;micro;m, 500 mL)</td><td>Thermo Fisher Scientific</td><td>5660010</td><td></td></tr><tr><td>FIJI (Is Just ImageJ) and Trainable Weka Segmentation (TWS) plug-in</td><td>--</td><td>--</td><td>Free, open-source image analysis software that includes plug-ins including Trainable Weka Segmentation described in Section 5; TWS plug-in from ref. 90 of the main text</td></tr><tr><td>GlutaMAX supplement (100 X)</td><td>Gibco</td><td>35050061</td><td>Glutamine supplement used for primary cell materials described in Section 2.1.2</td></tr><tr><td>Hausser Scientific bright-Line and Hy-Lite Counting Chambers</td><td>Hausser Scientific</td><td>267110</td><td></td></tr><tr><td>HBSS (no calcium, no magnesium)</td><td>Gibco</td><td>14170120</td><td>Used for primary cell materials described in Section 2.2.1 and 2.2.2</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Huygens Professional deconvolution software (V. 20.10)</td><td>Scientific Volume Imaging (SVI)</td><td>--</td><td>The deconvolution software used in this protocol and described in Section 5</td></tr><tr><td>IX83 inverted microscope with Continuous Autofocus</td><td>Olympus</td><td>--</td><td>This paper uses a STED Infinity Line system built around an Olympus IX83 inverted microscope, described in Section 4</td></tr><tr><td>Lightbox software (V. 16.3.16118)</td><td>Abberior</td><td>--</td><td>Vendor software used for STED image acquisition, described in Section 4</td></tr><tr><td>Live Orange Mito dye</td><td>Abberior</td><td>LVORANGE-0146-30NMOL</td><td>Live cell imaging IMM-targeting dye described in Discussion</td></tr><tr><td>Neurobasal media</td><td>Gibco</td><td>21103049</td><td>Used for primary cell materials referred to in Section 2.1.2</td></tr><tr><td>Nunc Lab-Tek II 2-well chambered coverglass</td><td>Nunc</td><td>155379</td><td>Can purchase a variety of chambers but make sure the coverglass is #1.5</td></tr><tr><td>Pasteur Pipets (Fisherbrand)</td><td>Thermo Fisher Scientific</td><td>22183632</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>PKmito Orange dye</td><td>Spirochrome</td><td>SC053</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Gibco</td><td>A3890401</td><td></td></tr><tr><td>SH-SY5Y Cell line</td><td>ATCC</td><td>CRL2266</td><td></td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Gibco</td><td>11360070</td><td>Used for primary cell materials described in Section 2</td></tr><tr><td>Spectrometer (GENESYS 180 UV-Vis)</td><td>Thermo Fisher Scientific</td><td>840309000</td><td></td></tr><tr><td>STED Expert Line microscope</td><td>Abberior</td><td>--</td><td>STED setup can be customized, but at time of purchase instrument was considered Abberior&amp;rsquo;s Expert Line; brief description of setup is available in Section 4 of protocol</td></tr><tr><td>T25 flask (TC-treated, filter cap)</td><td>Thermo Fisher Scientific</td><td>156367</td><td>Other culture vessels and sizes available</td></tr></tbody></table>

using live cell sted imaging to visualize mitochondrial inner membrane ultrastructure in neuronal cell models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

This protocol describes cell growth conditions for cultured and primary cells with a focus on live cell STED imaging and subsequent analyses of mitochondrial cristae. Projections made with ImageJ of mitochondria from undifferentiated SH-SY5Y (Figure 3A) and RA-differentiated SH-SY5Y (Figure 3B) cells can be collected as z-stacks with traditional confocal and STED, and the raw STED images can then be deconvolved. Timelapse imaging can also be performed and subsequently deconvolved (Figure 3C,D). Using slightly different imaging parameters for primary rat hippocampal neurons (Table 1), confocal and raw STED images can be acquired as z-stacks, and the raw STED images can be deconvolved (Figure 4A). Timelapse imaging of mitochondria from primary neurons is also possible (Figure 4B). In general, the time-lapse images should be able to show mitochondrial dynamic events.
When raw STED and deconvolved STED z-stack projections from the samples used for segmentation appear consistent, quantitative measurements are performed. The TWS plugin uses the deconvolved STED image to segment to make a probability mask, which is then used to create a binary mask of the cristae to obtain size and shape parameters (Figure 5A). The regions from this mask are saved in the ROI manager and can be applied to the raw STED image if desired to measure differences in relative intensity. The deconvolved STED projections can also be used to determine the cristae periodicity and density in a given area (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65561/65561fig01.jpg" /> 
Figure 1: Mitochondrial morphology. Mitochondria have a two-membrane system that defines different subcompartments (A). Cristae are infolds of the inner membrane with defined features (B). Abbreviations: OMM, outer mitochondrial membrane; ICS, intracristal space; IMS, intermembrane space; CM, cristae membrane; IBM, inner boundary membrane; IMM, inner mitochondrial membrane; CT, cristae tip; CJ, cristae junction. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65561/65561fig02.jpg" /> 
Figure 2: Schematic of workflow. SH-SY5Y cells or primary rat hippocampal neurons are grown on a PDL-coated coverglass. SH-SY5Y cells are grown in parallel to remain undifferentiated or subjected to RA differentiation over the course of six days. Primary rat hippocampal neurons were grown on a PDL-coated coverglass after being isolated from hippocampal sections for seven days. Once ready to be imaged, cells were stained with PKMO and imaged with STED. Raw STED images are then deconvolved, and the deconvolved images are processed in FIJI to obtain size and shape measurements, such as cristae density, area, perimeter, circularity, and aspect ratio. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65561/65561fig03.jpg" /> 
Figure 3: Imaging of mitochondria in SH-SY5Y cells. Representative confocal (left), raw STED (center), and Huygens deconvolved STED image z-stack projections (right) of mitochondria from non-differentiated (A) and&#160;RA-differentiated (B) SH-SY5Y cells with PKMO staining are shown. A timelapse with 30 s intervals and 5 iterations of RA-differentiated SH-SY5Y cells is shown (C) with selected regions (white boxes) expanded upon (D) using scaled images of those regions without interpolation. Scale bars: A,B, 250 nm; C,D, 1 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65561/65561fig04.jpg" /> 
Figure 4: Imaging of mitochondria in primary rat hippocampal neurons. Representative confocal (left), raw STED (center), and Huygens deconvolved STED (right) image z-stack projections of mitochondria from primary rat hippocampal neurons are shown (A). A timelapse with 25 s intervals and 5 iterations of mitochondria in these neurons is shown (B). Scale bars: A, 250 nm; B, 1 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65561/65561fig05.jpg" /> 
Figure 5: Processing of deconvolved STED images in ImageJ. Representative use of the Trainable Weka Segmentation plugin to measure cristae size and shape is shown (A). From left to right, the following images are shown: the deconvolved STED image, the probability map based on segmentation from the TWS plugin, the mask from thresholding in FIJI using the probability map as input, the mask with the ROIs outlined, and the ROIs overlayed onto the original deconvolved STED image. The resulting area, perimeter, circularity and aspect ratio measurements corresponding to these objects can be found in Supplementary Table 1.&#160;A line plot using the deconvolved STED image to measure peak-to-peak distances as a readout for cristae density is shown (B). Scale bars: 0.5 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Pixel size (nm)</td>
			<td>Dwell time (&#181;s)</td>
			<td>Line acc.</td>
			<td>561 nm excitation during STED acquisition (%)</td>
			<td>775 nm STED depletion power (%)</td>
			<td>Step size (nm)</td>
			<td>Pinhole (AU)</td>
			<td>Timelapse interval (s)</td>
			<td>Timelapse iterations</td>
		</tr>
		<tr>
			<td>Undiffer-entiated SH-SY5Y</td>
			<td>20</td>
			<td>4</td>
			<td>10</td>
			<td>15</td>
			<td>20</td>
			<td>200</td>
			<td>1.0</td>
			<td>30</td>
			<td>5</td>
		</tr>
		<tr>
			<td>RA-Differe-ntiated SH-SY5Y</td>
			<td>20-25</td>
			<td>4</td>
			<td>10-12</td>
			<td>15</td>
			<td>20-22</td>
			<td>150-200</td>
			<td>1.0</td>
			<td>30</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Primary Neurons</td>
			<td>20-25</td>
			<td>4</td>
			<td>10</td>
			<td>10</td>
			<td>25</td>
			<td>200</td>
			<td>0.7</td>
			<td>30</td>
			<td>5</td>
		</tr>
		<tr>
			<td colspan="10">NOTE: Pixel size can vary based on imaging requirements and intent to deconvolve images. Proper sampling is required for deconvolution. Pixel sizes for raw STED images without deconvolution can go up to 30 nm.</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of STED acquisition parameters. The settings used for 2D STED imaging for each cell type, undifferentiated SH-SY5Y, RA-differentiated SH-SY5Y, and primary rat hippocampal neurons, are displayed. For all time-lapses, 5 iterations were taken with varying intervals based on ROI size.
Supplementary Figure 1: Imaging of SH-SY5Y cells with amyloid-&#946; (A&#946;) addition. Representative confocal (left), raw STED (center), and deconvolved STED (right) images of RA-differentiated SH-SY5Y cells with PKMO stain (top) and A&#946;-HiLyte647 (bottom) are shown (A). Merged z-stack projections of raw PKMO STED (green) with raw&#160;A&#946; STED (magenta) (B) or deconvolved PKMO STED (green) with deconvolved A&#946; STED (magenta) (C) are shown. Scale bars: 0.5 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/Suppl Fig.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Size and shape measurements of segmented cristae. The size and shape measurements of area (&#181;m2), perimeter (&#181;m), circularity, and aspect ratio, corresponding to the objects outlined in Figure 5A from segmented mitochondria, are shown. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561_Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Summary of acquisition parameters with amyloid-&#946; samples. The settings used for 2D STED imaging of PKMO and A&#946;-HiLyte647 in undifferentiated and RA-differentiated SH-SY5Y cells are displayed. Confocal of A&#946;-HiLyte647 may be used alone as there is no specific structure to resolve; STED images of A&#946;-HiLyte647 are shown here for smaller particle sizes. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561_Supplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary File 1: Amyloid-&#946; treatment protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65561/65561_Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Decoding Mitochondrial Aging at JoVE.com

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.4K Views.  University of Connecticut. This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Video: Author Spotlight: Decoding Mitochondrial Aging 

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

Cancer Research

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

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

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.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

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 ...

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. 
To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. 
This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria

The mitochondrial network is extremely complex, making it very challenging to analyze. A novel MATLAB tool analyzes live confocal imaged mitochondria in timelapse images but results in a large output volume requiring individual manual attention. To address this issue, a routine optimization was developed, allowing for speedy file analysis.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in ...

Confocal Imaging of Mitochondrial Movement within Oligodendrocytes in Organotypic Brain Slice Cultures

<a href='https://appjove.unicartagenaproxy.elogim.com/v/56237/visualization-live-imaging-oligodendrocyte-organelles-organotypic'>Source: Kennedy, L. H., et. al. Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy. J. Vis. Exp. (2017)</a>This video demonstrates the imaging of oligodendrocyte mitochondria in organotypic mouse brain slices. The slice is placed inside a bath chamber with continuous fluid circulation and controlled temperature. After setting up various parameters of the confocal microscope, time-lapse images of mitochondria with red fluorescence are captured, followed by Z-stack images of the oligodendrocyte with green fluorescence.

Source: Kennedy, L. H., et. al. Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and ...

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

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Chapters in this video

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

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

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Live-imaging of Mitochondrial System in Cultured Astrocytes

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

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors

Confocal Imaging of Mitochondrial Movement within Oligodendrocytes in Organotypic Brain Slice Cultures