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 l