The device must be capable of keeping the decellularized tissue submerged in PBS solution for up to 72 hours while imaging using the multiphoton microscope. This is to mimic physiological conditions as well as the conditions in which the tissue’s mechanical properties are currently characterized and prevent the tissue from drying out. The bath is removable from the base to enable tissue attachment without interference from the loading arms. The bath was printed using PLA and has a central raised platform to prevent excessive tissue deformation when the microscope touches the surface of the tissue. There are two suture attachments that provide a stationary attachment point opposite the loading arms in each principal direction. The PBS bath walls are sufficiently elevated above the central tissue platform to both keep the tissue submerged and minimize the risk of spillage or leakage.

One of the design goals was to be able to stretch the tissue sample biaxially, which is done by attaching the sewn tissue with sutures to the stationary attachment in the PBS bath and to a movable loading arm along each principal direction. Two loading arms were positioned over adjacent sides of the PBS bath to enable biaxial movement. The back end of the loading arm has teeth to translate the rotational motion of the gear into linear motion using a rack and pinion system. The bottom of the arm has a groove that fits into the slot on the baseplate.

To precisely stretch the tissue, the device utilizes a rack and pinion mechanism to translate the movement of the driving gear into the linear motion of the loading arm. The gear has 0.543 mm +/- 0.05 mm teeth which facilitate the very small and precise displacements necessary for the scale of the tissue. There is a separate gear for each of the two loading arms to enable independent adjustments along both axes and tensioning of the sutures to bring the tissue into its “unloaded” state before stretching. The gear has an extruded center region that connects to the turning knob to facilitate synchronous movement which is described in later sections.

A key design goal of this device is to facilitate synchronous biaxial loading of the tissue sample. To do so, a removable gear connector piece can be placed in a slot on the baseplate that is positioned between the two rack and pinion gears. Once the tissue sutures have been tensioned independently as described in the previous section, the tissue can be biaxially stretched by inserting the connection piece and turning the gears using the turning knobs

The baseplate was designed to fit within the physical constraints of the multiphoton microscope at the La Jolla Institute of Immunology and the chosen objective lens. It was printed using PLA and has slots for the loading arms, overhangs, and gear connector pieces. It also has rod-shaped protrusions to position the gears and the locking mechanism. The wider circular base of the gear posts was included to minimize the friction between the gear and the baseplate and level the gear with the rack and pinion mechanism on the loading arm. The central hole has a lip and ledge to keep the PBS bath secure while minimizing the amount of material required to manufacture the baseplate.

While conducting SHG imaging, the tissue must remain in a stretched configuration for the duration of the multiple-hour imaging period. The locking mechanism utilizes a ratcheting system that keeps the gear in a stationary position after rotating into place. It pivots on a central post on the baseplate. The angle of the lock prevents the rack and pinion from slipping backward which would decrease the stretch on the tissue and interfere with the SHG imaging.