Image acquisition was performed in the LiM facility of iBiMED, a node of PPBI (Portuguese Platform of BioImaging): POCI-01-0145-FEDER-022122. This work was supported by FCT (EXPL/BTM-SAL/0902/2021) LCF (CI21-00276), a grant to DT from the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia of the Minist&#233;rio da Educa&#231;&#227;o e Ci&#234;ncia (2020.02006.CEECIND), a grant from ATG-The Gabba Alumni Association to VP, and the Institute for Biomedicine-iBiMED, University of Aveiro.

NOTE: This protocol has two main steps: a wet lab step, involving cell culture and live confocal microscopy to obtain images of live mitochondria (Figure 1) and an in silico step to analyze obtained images (Figure 2). For automated data analysis of 3D live imaged mitochondria, the MATLAB application Mitometer was used as provided by Lefebvre et al.9. The Routine optimization is written in MATLAB. The software, updated versions and processing ImageJ Macros are freely available online through GitHub, at&#160;https://github.com/JoseJoaoMV/Routine_Optimization_Mitometer_APP_MATLAB.&#160;
1. Live microscopy
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65452/65452fig01.jpg" /> 
Figure 1: Experimental protocol. SH-SY5Y cells were differentiated and treated with retinoids. (A) TMRM was used to live image healthy mitochondria in treated cells using a confocal microscope, capturing a time lapse z-stack of five visual fields. (B) Mitometer application MATLAB automatically segments and analyzes mitochondria images. In addition to analyzing, this software automatically discriminates mitochondria according to nuclear proximity. Blue dots are mitochondrial initial positions; red dots are final positions. Scale bar = 30 &#181;m. Abbreviation: TMRM = tetramethylrhodamine, methyl ester. <a href="https://www.jove.com/files/ftp_upload/65452/65452fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Cell culture
	<ol>
		<li>Incubate SH-SY5Y cells at 37 &#176;C in a humidified atmosphere of 5% CO2 and 95% air, cultured in equal parts of MEM (Minimal Essential Medium) and F12 (Ham&#39;s F12 Nutrient Mix) Medium, supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Plate SH-SY5Y cells in glass-bottom cell dishes at a density of 15 &#215; 104 cells/mL.</li>
		<li>Differentiate cells with 5 days of treatment with 10 &#181;M all-trans retinoic acid in 1% FBS-containing culture medium, followed by 2 days of treatment with 10 &#414;g/mL brain-derived neurotrophic factor (BDNF).</li>
	</ol>
	</li>
	<li>Cell treatment
	<ol>
		<li>Wash cells with sterile phosphate-buffered saline (PBS) and treat for 72 h with 10-7 M RAR-isoform agonists, in equal parts of MEM (Minimal Essential Medium) and F12 (Ham&#39;s F12 Nutrient Mix) Medium, supplemented with 1% FBS. 
		NOTE: The RAR&#945; agonist used was AM580; RAR&#946; agonist used was CD2314; Ch55 was used as agonist as RAR&#945; and &#946; co-agonist; atRA was used as a positive control; BMS493 was used as a RAR pan-antagonist.</li>
	</ol>
	</li>
	<li>Live confocal imaging
	<ol>
		<li>Replace culture medium with fresh 1% FBS-containing culture medium with 20 nM tetramethylrhodamine, methyl ester (TMRM) for 45 min. 
		NOTE: TMRM is a cell-permeant fluorescent dye, sequestered by active mitochondria, and this incubation period allows TMRM to reach an equilibrium and be taken up by polarized mitochondria11. Imaging should be started before equilibrium is established as TMRM signal intensity could artificially increase during imaging.</li>
		<li>Place the cells in an incubator attached to a laser-scanning confocal microscope at 37 &#176;C.</li>
		<li>Capture images using a 63x oil-immersion apochromat objective, with an image size of 512 x 512 pixels obtained with a pinhole aperture of 1 airy unit, capturing a time series of 15 frames from five different visual fields in each cell plate, and a z-stack of 8 equidistant z-planes. The resulting .lsm file is a time-, position-, and z-stack. 
		NOTE: Settings for gain, contrast, and brightness must be initially optimized using the minimum laser power necessary to use the entire dynamic range of the detector and kept constant throughout the study, ensuring that all imaging is performed under the same conditions. Up to nine different positions can be recorded in this hardware-software combination and the microscope cycles between imaging positions automatically.</li>
	</ol>
	</li>
</ol>
2. Image analysis
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65452/65452fig02.jpg" /> 
Figure 2: Routine optimization. (A) Representative code of the routine optimization. (B) Routine optimization Live-Script. (C) Routine optimization workflow. (D) Routine optimization result validation: representative image of mitochondria in untreated cells (left panel), treated with atRA (10-7&#160;M, 72 h, middle panel), and treated with RAR antagonist BMS493 (10-7 M, 72 h, right panel), imaged after incubation with TMRM (20 nM, 45 min incubation). Scale bar = 30 &#181;m. (E) TMRM Intensity in cell body mitochondria. Significant decrease with all-trans retinoic acid treatment (atRA, 10-7 M, 72 h), compared with control (p=0,0062), not observed when treated with RAR antagonist (BMS493, 10-7&#160;M, 72 hours). Five cells were quantified from each of three repetitions per condition. <a href="https://www.jove.com/files/ftp_upload/65452/65452fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Image processing
	<ol>
		<li>Open files in ImageJ 2.1.0 and separate position stacks by visual field: Open ImageJ and click Menu Bar | Image | Duplicate | Input slices/frame number | OK. 
		NOTE: To decrease repetitive inputs to the software, an ImageJ Macro was developed to facilitate duplication and saving of visual field images.</li>
	</ol>
	</li>
	<li>Macro protocol
	<ol>
		<li>Draw a region of interest (ROI) around the cell with the free selection tool.</li>
		<li>Run the macro by opening ImageJ and navigating to Menu Bar | Plugins | Macros | Run Clear the background and save images macro. 
		NOTE: Images can be randomized in this process, storing the solution key according to optimization requirements.</li>
		<li>Determine pixel size and voxel-depth: Open ImageJ | the Image and navigate to Menu Bar | Image | Properties.</li>
	</ol>
	</li>
	<li>Alternative direct protocol 
	NOTE: This alternative does not use Macro to process images
	<ol>
		<li>Find the pixel size and voxel depth: Open ImageJ | Open Image and navigate to Menu Bar | Image | Properties.</li>
		<li>Draw an ROI around the cell with the free selection tool, ensuring it encompasses the whole cell throughout the 15&#160;frames.</li>
		<li>Navigate to Menu Bar | Edit | Clear Outside.</li>
		<li>Navigate to Menu Bar | File | Save-as | Select Tiff.</li>
		<li>Select Saving Folder and click on Save. 
		NOTE: Images can be blinded/randomized manually at this point before continuing to analysis.</li>
	</ol>
	</li>
	<li>Automated image analysis
	<ol>
		<li>Prepare the folders for analysis files.
		<ol>
			<li>Create three main folders, titled &#34;All tracks&#34;, &#34;Perinuclear tracks&#34;, and &#34;Telenuclear tracks&#34;. 
			NOTE: These match the main automated track options.</li>
			<li>In each main folder, create a sub-folder for each image to process, identified numerically from 1 upwards.</li>
			<li>Add a copy of the two routine optimization supplementary files (mitometer2table.m and getTXTfiles.m) to each image folder. 
			NOTE: These files help in data analysis and final format arrangement. The number of folders must match the number of elements in the randomized spreadsheet (.xlsx). After creating all numbered subfolders with supplementary files for one dataset, they can be batch-copied and pasted to the remaining datasets.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use the MATLAB application Mitometer to analyze images. 
	NOTE: This protocol was run on a mitometer, installed on MATLAB R2022a. Load images in batches of 30 for optimal time-running and output balance. Maximum MAT-file size is imposed by the native file system: by default, &#34;save&#34; operations can create a file &#60; 231 bytes (~2 GB); save format MAT-Files Version 7.3 can be used instead, as it allows maximum variable sizes larger than 2 GB.
	<ol>
		<li>Identify/alter the default MAT-file version: On the Home tab in the Environment section, click Preferences and select MATLAB | General | MAT-Files.</li>
		<li>Use MATLAB tool Mitometer to analyze: Open MATLAB and navigate to Menu Bar | APPS | Open Mitometer | Select Start 3D | Input data (Pixel Size (&#181;m): 0.1395089/Time between frames (s): 2/Number z-planes: 8/Axial distance between z-planes (&#181;m): 0.418809 | Select images to input.</li>
		<li>Go to Mitometer Side Menu, select Image, click on Select Highlighted, navigate to the mitometer Menu Bar | Select Tracks | Track views | select All-Tracks, Telenuclear or Perinuclear | mitometer Menu Bar | Select Analysis | Choose an element (i.e., Length) | select Save to &#34;.txt&#34;. 
		NOTE: More than one parameter can be downloaded at once, if selected concomitantly.</li>
		<li>Extract result files to the created folders (2.2.1).</li>
	</ol>
	</li>
	<li>Routine optimization and data analysis
	<ol>
		<li>Prepare/adapt the &#34;Randomization.xlsx&#34; file containing the key for image encoding.
		<ol>
			<li>Insert a list of consecutive integers, from 1 upwards, in column A. 
			NOTE: It is advisable to keep a duplicated folder with originally named images.</li>
			<li>Place the analytic variables in column B, composed of alphanumeric characters. 
			NOTE: The number of lines in the document must be consistent with the number of folders present in the main dataset. Copy and paste this &#34;Randomization.xlsx&#34; file into the other two main datasets.</li>
		</ol>
		</li>
		<li>Optimized data analysis
		<ol>
			<li>Double-click &#34;executable.mlx&#34;, input the number of folders, specify the folders directory (copy the directory from the top of the folder) | the save directory (copy the directory from the top of the folder) | the spreadsheet name in the output file and click Run.</li>
			<li>Perform statistical analysis as necessary. 
			NOTE: Optional creation of a .xlsx file in each folder with the .txt data and sound alert to the end of analysis may be selected. Live Script outputs a single spreadsheet file in a table format. In this output, columns represent analyzed parameters (e.g., &#34;Major Axis Length&#34;; &#34;Intensity&#34;) and lines represent visual fields of analytic variables (e.g., &#34;Control&#34;).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

MATLAB Optimization for Analyzing Mitochondrial Networks in Neuroscience

<ol><li>Trigo, D., Avelar, C., Fernandes, M., Sa, J., da Cruz, E. S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria%2C+energy%2C+and+metabolism+in+neuronal+health+and+disease%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">Mitochondria, energy, and metabolism in neuronal health and disease.</a> FEBS Letters. 596 (9), 1095-1110 (2022).</li>
<li>Zong, W. X., Rabinowitz, J. D., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+and+cancer%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">Mitochondria and cancer.</a> Molecular Cell. 61 (5), 667-676 (2016).</li>
<li>Clare, D. K., Saibil, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATP-driven+molecular+chaperone+machines%5BTitle%5D)+AND+%22Biopolymers%22%5BJournal%5D)">ATP-driven molecular chaperone machines.</a> Biopolymers. 99 (11), 846-859 (2013).</li>
<li>Tourniaire, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((All-trans+retinoic+acid+induces+oxidative+phosphorylation+and+mitochondria+biogenesis+in+adipocytes%5BTitle%5D)+AND+%22Journal+of+Lipid+Research%22%5BJournal%5D)">All-trans retinoic acid induces oxidative phosphorylation and mitochondria biogenesis in adipocytes.</a> Journal of Lipid Research. 56 (6), 1100-1109 (2015).</li>
<li>Psarra, A. M., Sekeris, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+receptors+and+other+nuclear+transcription+factors+in+mitochondria%3A+regulatory+molecules+in+a+new+environment%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Nuclear receptors and other nuclear transcription factors in mitochondria: regulatory molecules in a new environment.</a> Biochimica et Biophysica Acta. 1783 (1), 1-11 (2008).</li>
<li>Trigo, D., Goncalves, M. B., Corcoran, J. P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+regulation+of+mitochondrial+dynamics+in+neurite+outgrowth+by+retinoic+acid+receptor+beta+signaling%5BTitle%5D)+AND+%22FASEB+Journal%22%5BJournal%5D)">The regulation of mitochondrial dynamics in neurite outgrowth by retinoic acid receptor beta signaling.</a> FASEB Journal. 33 (6), 7225-7235 (2019).</li>
<li>Mitra, K., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+mitochondrial+dynamics+and+functions+using+imaging+approaches%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Analysis of mitochondrial dynamics and functions using imaging approaches.</a> Current Protocols in Cell Biology. Chapter 4 (Unit 4), 1-21 (2010).</li>
<li>Sajic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impulse+conduction+increases+mitochondrial+transport+in+adult+mammalian+peripheral+nerves+in+vivo%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">Impulse conduction increases mitochondrial transport in adult mammalian peripheral nerves in vivo.</a> PLoS Biology. 11 (12), e1001754(2013).</li>
<li>Lefebvre, A., Ma, D., Kessenbrock, K., Lawson, D. A., Digman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+segmentation+and+tracking+of+mitochondria+in+live-cell+time-lapse+images%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Automated segmentation and tracking of mitochondria in live-cell time-lapse images.</a> Nature Methods. 18 (9), 1091-1102 (2021).</li>
<li>Chu, C. -H., Tseng, W. -W., Hsu, C. -M., Wei, A. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image+analysis+of+the+mitochondrial+network+morphology+with+applications+in+cancer+research%5BTitle%5D)+AND+%22Frontiers+in+Physics%22%5BJournal%5D)">Image analysis of the mitochondrial network morphology with applications in cancer research.</a> Frontiers in Physics. 10, 855775(2022).</li>
<li>Creed, S., McKenzie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+mitochondrial+membrane+potential+with+the+fluorescent+dye+tetramethylrhodamine+methyl+ester+%28TMRM%29%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Measurement of mitochondrial membrane potential with the fluorescent dye tetramethylrhodamine methyl ester (TMRM).</a> Methods in Molecular Biology. 1928, 69-76 (2019).</li>
<li>Kovalevich, J., Langford, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+for+the+use+of+SH-SY5Y+neuroblastoma+cells+in+neurobiology%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology.</a> Methods in Molecular Biology. 1078, 9-21 (2013).</li>
<li>Sahin, M., Oncu, G., Yilmaz, M. A., Ozkan, D., Saybasili, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transformation+of+SH-SY5Y+cell+line+into+neuron-like+cells%3A+Investigation+of+electrophysiological+and+biomechanical+changes%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Transformation of SH-SY5Y cell line into neuron-like cells: Investigation of electrophysiological and biomechanical changes.</a> Neuroscience Letters. 745, 135628(2021).</li>
<li>Trigo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+dysfunction+and+impaired+response+to+oxidative+stress+promotes+proteostasis+disruption+in+aged+human+cells%5BTitle%5D)+AND+%22Mitochondrion%22%5BJournal%5D)">Mitochondria dysfunction and impaired response to oxidative stress promotes proteostasis disruption in aged human cells.</a> Mitochondrion. 69, 1-9 (2022).</li>
</ol>

The authors have no conflicts of interest to declare.

Live cell imaging produces large files that require serious computing processing, but even the most recent tools require extensive manual input to process. This routine optimization is focused on simplifying the process of mitochondria analysis on the Mitometer because this tool presents a very good balance between user input and data output. A comprehensive comparison between different tools for mitochondria image analysis has previously been reviewed10. While other pipelines are more focused on ...

Cellular mitochondria sit at the center of all physiological states, and a thorough understanding of their homeostasis (mitostasis) and behavior is paramount to assist in identifying pharmacological treatment for a wide range of illnesses, including cancer and Alzheimer&#39;s disease1,2.
Mitochondria play crucial cellular roles in energy homeostasis, ATP generation, calcium buffering, and ROS regulation, and mitostasis is essential for maintaining protein homeostasis as molecular chaperones are energy-dependent3. These require a constant and dynamic network modulation and adaptation to efficiently meet cellular needs, and mitochondria transport is regulated by different signaling pathways; previous work has described one such pathway, that of retinoic acid receptors (RARs)4,5. Retinoic acid (RA) promotes axonal and neurite outgrowth via RAR activation. In mouse primary cortical neurons, activation of RAR-&#946; encourages mitochondrial growth, speed, and mobility in the neurite6.
Considering the mitochondrial network adaptability and dynamics, the possibility of assessing mitostasis in &#34;real time&#34; is essential not only for investigating energy homeostasis, but also for proteostasis, cellular health, proliferation, or signaling. A commonly used method for evaluating mitostasis relies on confocal microscopy after highlighting mitochondria using a fluorescent dye or marker, as well as a specific microscopy setup allowing temperature and/or CO2 regulation7. This type of experimental setup entails that one experimental replicate be performed at a time. In addition to experimental repetition of different treatments, it should be considered that most experiments should have their technical replicates (where more than one position is imaged per plate), with a series of focal planes (z-stacks) being recorded in a series of time points. Thus, an experimental design with three repetitions of one control and two treatments, with five imaging positions per plate, and 15 time points, results in 225 stacks to be processed. Classically, videos of live mitochondria were analyzed by plotting kymographs, which would be individually analyzed8, in a time-consuming process requiring extensive manual input, even when relying on computer tools.
An algorithm was recently described9 that allows automated segmentation and tracking of mitochondria in live-cell 2-D and 3-D time-lapse files. Other quantification techniques are available, and all have their limitations10. Mitometer, an automated open-source application, is particularly adequate for time lapse and mitochondria dynamics analysis, requiring low user input. This application has a series of advantages over other existing MATLAB-based tools, namely allowing the automatic processing of individual TIF stacks, using up to 13 different parameters, particularly interesting for neurosciences, as it differentiates between peri- and tele-nuclear mitochondria.
However, for an experiment such as the above described, these 13 parameters applied to 225 stacks result in 2,925 individual output files. These require four individual computer inputs, which sum up to over 10,000 manual inputs being required to download all output files. For large experimental designs, this results in a needlessly extremely time-consuming analysis of each file and data integration. Herein we present a routine optimization that allows swift file analysis, greatly reducing document reading and data processing, analyzing data in a defined manner, consistent with the output from existing tools.

<table><tbody><tr><td>AM580</td><td>Sigma-Aldrich</td><td>A8843</td><td></td></tr><tr><td>BDNF&amp;nbsp;</td><td>Thermo-Fisher</td><td>RP8642</td><td></td></tr><tr><td>BMS493</td><td>Tocris Bioscience&amp;nbsp;</td><td>3409</td><td></td></tr><tr><td>CD2314</td><td>Tocris Bioscience</td><td>3824</td><td></td></tr><tr><td>Ch55</td><td>Tocris Bioscience&amp;nbsp;</td><td>2020</td><td></td></tr><tr><td>Foetal Bovine Serum</td><td>Thermo-Fisher</td><td>10270106</td><td></td></tr><tr><td>GraphPad Prism v4.0</td><td>GraphPad Software, La Jolla</td><td>n/a</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12 Nutrient Mix</td><td>Thermo-Fisher</td><td>21765029</td><td></td></tr><tr><td>MATLAB R2022a&amp;nbsp;</td><td>MathWorks</td><td>n/a</td><td></td></tr><tr><td>Minimal Essential Medium</td><td>Thermo-Fisher</td><td>31095</td><td></td></tr><tr><td>Nunc Glass Bottom Dishes</td><td>Thermo-Fisher</td><td>150680</td><td></td></tr><tr><td>Phosphate Buffer Saline Solution</td><td>Thermo-Fisher</td><td>28372</td><td></td></tr><tr><td>Retinoic acid</td><td>Sigma-Aldrich</td><td>&amp;nbsp;R2625</td><td></td></tr><tr><td>TMRM&amp;nbsp;</td><td>Thermo-Fisher</td><td>T668</td><td></td></tr><tr><td>Zeiss LSM 510</td><td>Carl Zeiss</td><td>n/a</td><td>Equiped with live-cell imaging culture chamber and 63x oil immersion objective&amp;nbsp;</td></tr></tbody></table>

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.

To enhance and accelerate the analysis of output files in .txt format, a routine optimization was coded that reads data consistent with Mitometer .txt output files, with columns representing a frame and lines representing identified mitochondria. The routine optimization produces data in a single value per parameter by averaging the frames for each identified mitochondria and then averaging the results of all mitochondria per visual field. The developed routine reads files from folders numbered from 1 upwards. The Live S...

Watch this Scientific Journal Video about Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria at JoVE.com

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.

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 ...

optimized-automated-analysis-live-neuronal-mitochondria-homeostasis

Research

JoVE Journal

Neuroscience

605 Views.  University of Aveiro. 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.

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

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

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

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

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

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

Bioengineering

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

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 ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Biology

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Stabilization of MAMs in a 3D Neural Model of Alzheimer

Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)

A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of Alzheimer&#39;s disease (AD) is presented here. To begin, fresh human neuro progenitor ReN cells expressing &#946;-amyloid precursor protein (APP) containing familial Alzheimer&#39;s disease (FAD) or na&#239;ve ReN cells are grown in thin (1:100) Matrigel-coated tissue culture plates. After the cells reach confluency, these are electroporated with expression plasmids encoding red fluorescence protein (RFP)-conjugated mitochondria-binding sequence of AKAP1(34-63) (Mito-RFP) that detects mitochondria or constitutive MAM stabilizers MAM 1X or MAM 9X that stabilize tight (6 nm &#177; 1 nm gap width) or loose (24 nm &#177; 3 nm gap width) MAMs, respectively. After 16-24 h, the cells are harvested and enriched by a fluorescence-activated cell sorter (FACS). An equal number of FACS-enriched cells are seeded in the 3-dimensional matrix (1:1 Matrigel) and allowed to differentiate into mature neurons for 10 days. Live cell images of the 10-day differentiated cells expressing the RFP-conjugated MAM stabilizers are captured under a fluorescent microscope equipped with a live-cell imaging culture chamber maintaining the CO2 (5%), temperature (37 &#176;C), and humidity (~90%). Toward this end, we performed live-cell imaging and kymographic analyses to measure the motility of free mitochondria labeled with Mito-RFP or ER-bound mitochondria of tight or loose gap widths stabilized by MAM 1X or MAM 9X, respectively, in the most extended neuronal process of each ReN GA neuron which is at least 500 nm long, considering these as axons.

Here, we describe measuring the axonal transport rate of constitutive stabilizers of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by increasing or maintaining&#160;neurotoxic &#946;-amyloid (A&#946;) generation from Alzheimer&#39;s disease (AD) neurons in real-time to serve as a direct and quantitative metric to measure MAM stabilization and aid the development of AD therapeutics.

A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Novel OCR Monitoring System for Differentiated RPE Cultures

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. 
Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. 
We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

Author Spotlight: Extended Oxygen Consumption Measurement in Retinal Pigment Epithelium Using Resipher

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for ...

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

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Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

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

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Live-imaging of Mitochondrial System in Cultured Astrocytes

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

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Cellular Redox Profiling Using High-content Microscopy

Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures