Visualization of Mitochondria within Intraepidermal Nerve Fibers

Begin with an epidermal skin tissue section containing intraepidermal nerve fibers or IENFs, thin sensory neuron endings. 

The IENFs are rich in mitochondria, providing energy for neuron signal transmission. 

The tissue has fluorescently labeled IENFs, mitochondria, and epidermal nuclei.

Using an inverted confocal microscope, sequentially acquire discrete fluorescent signals at appropriate wavelengths for the nuclei, IENFs, and mitochondria.

Activate a live scan to capture the IENF signal, encompassing the epidermal layer.

Then, perform sequential scanning for each signal to eliminate fluorescence signal crosstalk.

From the combined image, using the software, crop and isolate the blue-fluorescing epidermal cells.

Then, process the cropped area to enhance the resolution of green IENF signals and red mitochondrial signals.

Next, generate a 3D surface image to create a mask for IENFs, then overlay over the mitochondrial surface image to isolate nerve-specific mitochondria. 

In the final output, IENFs appear in cyan, while the mitochondria appear as tiny magenta-colored structures.

Select the 40x oil immersion objective on an inverted laser scanning confocal microscope. Select the appropriate lasers and detectors to image the nuclei, nerve fibers, and mitochondria. Enter the following scan parameters into the microscope software 12-bit intensity resolution. Scan rate of 600 Hz with two-frame averaging, and zoom of 2.2. Set the microscope software for optimized lateral resolution by selecting a scan resolution of 1024 by 1024. Optimize axial resolution and optical sectioning by selecting a confocal aperture of one airy unit with a z step size of 210 nanometers.

Scan each signal separately, and adjust the detector voltage and offset to remove any over and under saturated pixels. Activate a live scan for the nerve signal, and adjust the z focus control to find and set the upper and lower focal planes in the microscope software that encompass the nerve signal within the tissue section. Scan the final z series with sequential scanning to eliminate fluorescent signal crosstalk.

Isolate the epidermis from the stratum corneum and the dermis by drawing a region around the epidermis, using a selection tool on a duplicate image of the original image. Then crop the image to the selection. Calculate point spread functions for the green and red fluorescence confocal signals, using the calculate spread function feature. Then set the parameters for medium refractive index for oil at 1.515, and the numerical aperture for 40x oil objective at 1.25. Set the detector pinhole at one area, unit and choose a laser excitation wavelength.

Use iterative restoration set to 100% confidence, iteration limit of 10 cycles, and the green and red PSFs for the deconvolution of the green and red fluorescence signals. Use the Create surface tool to make a surface around the deconvolved nerve signals, selecting unchecked smooth feature, absolute intensity for thresholding, a lower threshold of 3,000, and an upper threshold of 65,535. Keep the surfaces above 10 voxels.

Remove the non-nerve surfaces by using the Edit tab in nerve surface to select single non-nerve surfaces, or hold down the Control key to select multiple non-nerve surfaces. Delete the selected non-nerve surfaces by pressing the Delete button. Use the Edit tab in nerve surface, and press the Mask All button in the Mask properties.

In the new window, select Channel 5 Mitochondria Deconvolved under the Channel selection, and then check duplicate channel before applying the mask. Press the radio button for constant inside outside, and check the set voxels outside surface to 0.0. And press the OK button.

Finally, create mitochondria specific surfaces using the Create surface tool to make a surface around the masked deconvolved mitochondrial signals by selecting unchecked Smooth feature, and using Background Subtraction for thresholding. Set the diameter of the largest sphere to 1.50 micrometers, the lower threshold to 2,000, and the upper threshold to maximum 65,535. Be sure to keep the surfaces above 1.0 voxels.