Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The remodeling of the mitochondrial network occurs in a physiological stress and pathological conditions. However, the effects of mitochondrial remodeling in a skeletal muscle remain incompletely understood. This protocol enables the quantification of circular changes of mitochondria in live skeletal muscle, enhancing our comprehension of the interplay between mitochondrial alterations and cellular function.

Working with live skeletal muscle present challenges due to fiber contraction during isolation and sensitivity to image during confocal scanning. This protocol describes an isolation process using a relaxation solution, enabling the analysis of viable muscle fibers for mitochondrial assessment via confocal microscopy. Our protocol addresses the lack of workflows to analyze quantitatively mitochondrial networks in skeletal muscle fibers.

The steps described here allow users to take more advantage of images obtained by confocal microscopy using image processing by thresholding and fast Fourier analysis to achieve quantification of mitochondrial density and distribution. We are interested in the study of skeletal muscle alteration, particularly mitochondrial dynamics and mitochondrial structural remodeling during adversity and how can they be modulated to intervention such as exercise and intermittent fasting. Begin by transferring the excised hind limb of a rat to a 60-milliliter Petri dish containing three milliliters of ice-cold relax solution.

Next, carefully lift the Achilles tendon with forceps. Using iris scissors, dissect the muscles away from the bone. With a stereo microscope, identify and separate the entire gastrocnemius muscle.

Using fine-tipped forceps, transfer the lateral muscle head to a new Petri dish with ice-cold relax solution. Gently hold the muscle with forceps on one end and separate the muscle into bundles using micro scissors. Finally, transfer the fiber bundles to a new Petri dish containing two milliliters of ice-cold relax solution.

Start by incubating the rat skeletal muscle fibers with TMRE at room temperature for 20 minutes. Next, launch the confocal microscope standard software and choose the configuration framework. Under the hardware configuration dialogue box, select laser and check the helium neon 543 option.

Access the acquisition dialogue box via the acquire framework and select the XYZ panel. Then select the 512-by-512 format with a 400 hertz speed. Under the displayed pinhole panel, select arbitrary units and input three airy for the pinhole.

In the beam path settings dialogue box, select a 20x water immersion objective with a 0.7 numerical aperture and an emission wavelength window of 576 to 700 nanometers. Choose DD 488/543 and set the laser power for the 543 laser at 15%After a 20-minute incubation, change the incubation medium twice. Next, prepare the recording chamber with a 0.15-millimeter borosilicate glass cover slip.

Transfer the fiber bundles to the chamber containing 200 microliters of relax solution. Utilize the confocal microscope's brightfield mode to identify viable fibers for the fluorescent recording of mitochondria. Adjust gain and offset by scanning the fluorescence using the live button.

Choose low fluorescent intensity values for a background of nearly zero arbitrary units and a mitochondrial signal between 100 and 200 arbitrary units. Select a pixel size between 150 to 190 nanometers and adjust the zoom factor in the XY dialogue box to acquire the fiber's full width. In the Z-stack dialogue box, set the Z distance starting at a fiber depth of 15 micrometers up to 22 micrometers and choose a Z-step size of three micrometers.

Click the start button to acquire the confocal images and obtain a Z-stack composed of three confocal images acquired at 15, 18, and 21 micrometers of depth. The confocal images of an exercised lean rat muscle fiber show an expected record of skeletal muscle fiber mitochondria with a consistent fiber pattern. In contrast, the obese rat muscle fiber shows substantial alterations in mitochondrial content and distribution.

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 TIF 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 TIF 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. 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 two 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 fluorescence 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.