Begin by opening the time-lapse mitochondrial images in Image J2.1.0. Separate position stacks based on the visual field by navigating to the Menu bar and clicking on Image. Then, go to Duplicate, Enter Slice, or Frame Number, and click OK.Next, draw a region of interest around the cell using the Free Selection Tool.
To run the Macro, open Image J, navigate to Menu bar and click on Plugins, Macros, and Run. Then, select Clear the Background and Save the image's MACRO. To determine pixel size and voxel depth, open Image J and select Image.
Access the Menu bar, select Image, and click on Properties. After determining the pixel size and voxel depth, draw a region of interest encompassing the entire cell using the Free Selection Tool to include the whole cell throughout all 15 frames. Navigate to the Menu bar, click on Plugins, Macros, and select Clear the Background, and Save the image's MACRO.
Choose the desired saving folder, and click Save to store the file in TIFF Format. Then, create three main folders titled All Tracks, Perinuclear Tracks, and Telenuclear Tracks. Create a numbered sub folder for each image to process within each main folder, starting from one.
Add a copy of the two routine optimization supplementary files into each image folder. To identify or alter the default mat file version, navigate to the Home tab in the Environment section and click on Preferences. Then, select MATLAB.
Open MATLAB software on the computer, navigate to the Menu bar, select apps, and open Mitometer. Select Start 3D and set the pixel size to 0.1395089 micrometers, time between frames to 15 seconds, number of z-planes to 8, and axial distance between z-planes to 0.418809 micrometers. Select images to input.
Now, go to the Mitometer side menu, select Image, and click Select Highlighted. Navigate to the Mitometer Menu bar, choose Select Tracks followed by Track Views. Then, choose from All Tracks, Telenuclear, or Perinuclear Options.
Save the results to a text file by selecting Save to txt, and extract the result files to the created folders. Prepare the randomization xlsx file, which contains the key for image encoding. Insert a sequence of consecutive integers, starting from one, in column A, populate column B with alphanumeric characters, which constitute the analytic variables.
Double click on the executable mlx, input the number of folders, click Specify the folder's directory, and select Save Directory. Select the spreadsheet name in the output file and click Run. Following this, perform the necessary statistical analysis.
Representative images of mitochondria after treatment, respective surface plots, and automated segmentation are presented. A significant decrease in average mitochondrial length was observed in cells treated with AM580. The mitochondrial surface area significantly decreased in cells treated with AM580 and BMS493.
TMRM intensity normalized for mitochondrial volume showed a significant decrease in cells treated with retinoic acid and CH55.
