In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The aim of the following procedure is to study longitudinal dynamic changes in nerve endings in skin after selective lesion. For this, we anesthetize A YFP transgenic mouse and put it on the heating pad to maintain the body temperature. Put the hind paw on a plastic packaging material and place a drop of water on the skin to achieve better optical coupling.

Cover the hind pole with a glass cover slip firmly attached to a metal bar for stability. Put a drop of water for water immersion objective and perform twofold on microscopic imaging. Combined with laser induced lesion of the nerve endings, A variety of pathological conditions affect the dynamics of skin integrations.

One of the best ways to check this effect is to visualize pathological changes in cutaneous nerve endings directly within the skin of a living animal. Vivo to photo microscopy is an excellent Tool for this approach. Before the imaging weigh the mouse and induce anesthesia by injecting ketamine in the concentration of 80 microgram pergram of body weight en xylazine.

In the concentration of 10 microgram pergram body weight. Verify the anesthesia by slightly pinching the hind limb of the mouse. Use viscous eyedrops to prevent dehydration of the eyes of the mouse.

During imaging for stabilization, we use a proprietary design metal fixator with a ring. Put the small drop of glue on the ring and spread it uniformly. Then attach cover glass to the metal ring.

To facilitate an even coverage of the mouse skin for imaging, we use a commercially available two photon microscope equipped with a motorized stage. We fixed the metal ring with cover glass facing down to a solid metal bar that is firmly attached to the microscope stage. We maintain the body temperature of the animal at 37 degrees Celsius by keeping it on a heating pad.

Clean the hind pole with ethanol. Add a drop of water on the skin surface for immersion. Put the Hein pole between the glass cover slip and plastic packaging material.

Adjust the material to achieve the best positioning of the hind paw. Add a drop of water on top of the glass to ensure immersion of the objective. Drive the stage with a fixed hind paw under the objective.

First, use an epi fluorescence lamp to find the appropriate area for data acquisition. Locate the nerve ending of interest and write down its coordinates for repeated measurements in the same area. Once we focus on the appropriate nerve, we can switch to the two photon mode.

Set a suitable voltage on the photo multiplier tubes. Select the image size usually between 500 and 800 pixels, and set the excitation wavelength. For example, at 915 nanometers for YFP.

After acquiring the image stack, move on to the lesion procedure. We use the bleaching routine available in the Olympus software package and set the exposure time between 100 milliseconds and one second. Depending on the optical properties of each particular skin region.

Find a spot where you want to make the lesion and turn on the bleaching routine. After the lesion, track the changes using time-lapse twofold on imaging over minutes, hours or days. Usually duration of image session is about one hour, but it can be easily adjusted dependently on the task.

After the measurements, we drive the stage with the mouse away from the objective. Bring the mouse to a recovery box set at the temperature 37 degrees Celsius until it wakes up. The imaging procedure can then be repeated over a range of days and even months.

In the course of offline analysis, it is sometimes necessary to UNIX the signal of interest, such as YFP fluorescence from the overlapping order fluorescence. We perform spectral unmixing using Image J software. Open the image stack and split the channels.

Find the area with the nerve ending is clearly distinguishable from the hair or the other structures. You want to Unix it from. Use the plugin called the spectral and mixing to calculate the parameters of the unmixing matrix.

First, find the signal free area to measure the background noise level. Then outline carefully the part of the hair that can be distinguished from the nerve endings. The more precisely you do the selection, the better separation will be.

Then outline precisely a region of the nerve ending. Finally, use the same plugin and apply the obtained matrix to unmixed the data. The hair nerves should now appear in two different channels.

As a result, we can reconstruct three dimensional structures of nerve fibers and track the changes over time. You can use different timescales from several minutes up to several days and even months. Also, one could use other transgenic mouse lines, such as, for example, those expressing a fluorescent marker in mitochondria.

In vivo, multi photo microscopy of superficial structures combined with the proper transgenic model is a powerful tool to study physiological as well as pathophysiological processes and skin. The methodological approach we have demonstrated in this video protocol discloses some essential tips that allow performing multifold microscopic imaging and microsurgical manipulations on peripheral nerves in a rodent scheme in a simple, reliable and non-invasive fashion. Since this method could be easily applied to variety of available transgenic fluorescent models, we hope that this video will help other researchers who perform superficial imaging experiments.

Thank you for watching and good luck with your experiments.I.