µTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

This protocol allows observing the functions of taste cells in a natural microenvironment with neural connections and blood circulation remaining intact. Using this technique, cell-to-cell communication can be investigated in vivo. To begin, fill the reservoirs of the pressurized flow perfusion system with artificial saliva and tastants.

Connect the compressed air line to the regulator input and set the air pressure in the fluidic delivery system to 30 to 50 pounds per square inch. Set the output pressure of the regulator to 0.4 pounds per square inch and check if liquid starts flowing out from the tube. Connect the manifold from the reservoirs to the input port of the micro tongue, then connect the output port of the micro tongue to a syringe pump and withdraw liquid at approximately 300 microliters per minute to establish a steady state condition.

Confirm that the volume of the droplet hanging under the micro tongue remains constant and adjust the air pressure and flow rate to obtain the desired sample height. Once the microfluidic system has been set up, disconnect the compressed air line and switch off the syringe pump until the mouse has been prepared for in vivo imaging. After administering anesthesia to the mouse, intravenously inject TRITC-dextran through a retro-orbital route to observe blood circulation during imaging.

With the mouse placed in a supine position, spray 70%ethanol on its head. Use forceps to lift the head skin lightly, then use scissors to snip off approximately seven square millimeters of skin. After cleaning the hair around the scalp, remove the periosteum from under the skin and apply an instant adhesive to the skull.

Then attach the customized head fixer. Once the instant adhesive is hardened, apply dental glue around the head fixer and solidify it by illumination with a blue light. Using instant adhesive to glue the lower lip of the mouse to the bottom unit of the micro tongue, then placed the mouse on the mouse preparation board and secure the bottom unit at the micro tongue to the hold posts by aligning the holes at the edge of the micro tongue with the posts.

Tighten the mouse head fixer to the head fixer holder at the board and adjust the distance between the mouse head and the device. Then using the head fixer holder, rotate the mouse head smoothly by approximately 45 degrees. After securing the mouse to the board, use plastic tweezers to draw its tongue gently.

Then using instant adhesive, attach the ventral side of the tongue to the upper side of the bottom unit of the micro tongue. To keep the tongue moist, wipe the surface with a wet cotton swab, then place a piece of tissue soaked in artificial saliva on the exposed surface of the tongue. Place the curved washers on the posts, holding the bottom part of the micro tongue, then move the mouse preparation to the microscope stage.

Position the exposed tongue of the mouse under the approximate center of the microscope objective area, making sure not to deviate from the dynamic range of the stage, then tighten the screws to firmly attach the mouse board on the stage. Place a heating pad under the mouse to maintain its body temperature between 36.5 and 37.5 degrees Celsius. To prevent liquid from entering the mouse trachea, place a thin piece of twisted paper inside the mouse's mouth, then remove the wet tissue placed on the surface of the tongue and place the prepared micro tongue on the mouse tongue, such that the surface of the tongue is visible through the imaging window.

Secure the micro tongue by gently screwing both ends with minimal compressive pressure. To begin acquiring images, open the microscope software, then turn on the 920 nanometers two-photon laser. Mount the water immersion objective on the microscope, then lower the objective onto the imaging window of the micro tongue and immerse the objective.

In camera mode, illuminate the surface of the tongue by turning on the blue light from a mercury lamp. To find the approximate focal plane, adjust the Z-axis and search for an autofluorescence signal from the filiform papillae. Then using the X and Y adjustment knob, locate a taste bud.

Switch to the multi-photon mode, then set the excitation wavelength, emission filter set, scan mode, and frame size for image acquisition. With the taste bud at the center of the imaging window, locate the blood vessels surrounding the taste bud at about two thirds the height of the taste bud and visualize the blood circulation. If the blood flow is clogged, slightly loosen the fixing screws to resume blood flow.

Adjust the Z-axis to find the Z plane of a taste bud containing an adequate number of taste cells, then proceed with calcium imaging at two to six Hertz for 80 seconds. After imaging starts, switch on the reservoir of the fluidic system to provide a taste solution for 20 seconds. After 20 seconds of taste stimulation, switch the reservoir back to artificial saliva.

During imaging, wait for about three to four minutes between sessions consistently providing artificial saliva to keep the tongue moist and wash away the tastant remaining from the previous session. The tongue surface of Pirt-GCaMP6f-tdTomato mouse is covered with autofluorescent filiform papillae and sparsely spread taste buds. The filiform papillae in yellow are captured using a photo detector at 500 to 550 nanometers and can be observed from the very surface of the tongue up to approximately 25 micrometers in depth.

The GCaMP signals in green detected by the 500 to 550 nanometre filter and the tdTomato signals in red, detected by the 607 to 670 nanometer filter, represent the taste cells. The tdTomato signal is obtained for ratio metric analysis. The blood vessels that surround the taste bud are acquired using the 500 to 550 nanometer filter set in the collagen connective tissue, which structurally supports the taste bud, is acquired using the 447 to 460 nanometers filter set.

In this representative example of in-vivo taste screening using calcium imaging, each taste cell is demarcated by dashed lines. In this trial, cell 2 responded to both sweet and umami tastants. And cell 3 responded to both low and high salt tastants.

None of the cells in this taste bud responded to sour tastes. Since the purpose of this experiment is to observe tastes cell function in the natural environment, it is important to deliver the fluorescents into the blood vessels at the preparation step and confirm its circulation during the experiment.