Start by launching the open-source platform for biological image analysis. Open the BinDmito-stack and draw a rectangular region of interest with a 256-pixel width and five-micrometer height. Locate a central and a lateral ROI.
Obtain the plot profile of the ROIs using the shortcut Command K and transfer the data to a spreadsheet filling columns B and C.Fill column D by selecting the second cell in column D.Navigate to the Home menu, choose Fill, and click Series. Next, select columns and input the calculated delta sampling frequency as the step value and the calculated S as the stop value. Go to the Data menu, click Data Analysis and select Fourier Analysis.
Choose the range of data points in column C and the corresponding range in column E.Now, fill column F with the FFT Magnitude using the IMABS function to return the absolute value from the complex number in E, and multiply by 2 over N for normalization. Autofill column F with this formula. Plot the FFT spectrum using the magnitude in F as a function of FFT frequency in D until S.Finally, find the point of the maximum peak and its corresponding FFT frequency.
The plot profiles of the selected ROIs show differences in the fluorescents'distribution between the fibers derived from lean and obese rats, as well as ROI variation within the same fiber. In lateral ROIs, the frequency of mitochondrial longitudinal distribution was similar in the fibers derived from lean and obese rats, with higher amplitude in obese rat fiber. In contrast, the central ROI of the obese rat fiber is an example of a critical reduction of the FFT peak when an important alteration of the mitochondrial distribution is present.
