Start by launching the image analysis software. Next, open the Z-stack file and adjust the orientation to place the fiber horizontally. Choose a rectangular region of interest or ROI covering the mitochondrial area.
Select sizes ranging from 65 to 90 micrometers in the X direction and appropriate sizes in the Y direction based on fiber width. Proceed to duplicate the Z-stack with the selected ROI. And save it as a new Z-stack in Tiff format.
Save the ROI position from the original stack using the ROI Manager tool. Open the threshold dialogue box with the shortcut, Command+Shift+T. Choose the Otsu threshold algorithm and select the black and white dark background option.
Observe the histogram of the fluorescence intensity distribution and the threshold value displayed in the dialogue box. Apply the threshold to the binary image stack, then choose Calculate threshold for each image and create new stack in the displayed dialogue box before clicking OK.Save the stack generated with three binary images in Tiff format. Next, access the Analyze menu and select Histogram.
In the displayed Histogram dialogue box, click Yes. And in the Histogram of stack dialogue box, click on List to obtain histogram data. Transfer histogram data to a spreadsheet and identify mito-pixels with a 255 value.
Calculate mitochondrial density by dividing the mito-pixels with the total pixels and multiplying with 100. The obese rat fiber presented a lower mitochondrial density. This was confirmed by the stack image analysis.
