The 3D printed holder used in this protocol allows us to monitor short-term cell signaling and long-term cellular migration events in wounded corneas in real time. By keeping the globe intact, viable, and immobile, this protocol produces high quality live images of corneal cells, including the epithelium and nerves. The 3D printed holder could easily be modified for globes of various sizes and species.
We can also orient the eyes differently to image other regions or perform nano indention experiments. To begin, after removing the head of a euthanized mouse, place it immediately on ice to preserve the viability of the tissue. Then enucleate the globes by prop dozing it, using tweezers.
And clip the optic nerve using dissection scissors just below where it is held by the tweezers. Incubate the globes in two milliliters of the medium in a P-35 cell culture dish, including a calcium indicator and/or cell membrane stain for one hour in an incubator at 37 degrees Celsius and 5%carbon dioxide with low light conditions, ensuring that the globes are submerged in the stain medium for uniform staining. To adhere the holders to a clean glass bottom cover slip, wash the holder in 70%ethanol and place glue onto the holder.
Then, adhere the holder to the glass bottom cover slip, ensuring no glue is within the inner area of the holder, as glue can fluoresce, complicating imaging. After the glue solidifies, confirm that the holder is secure against the cover slip. Remove the globes from the staining solution using the sterile eyedroppers, taking care to prevent tissue damage to the region of interest.
Wash the globes for five minutes at room temperature, using sterile phosphate buffered saline to remove excess stain and place the globes in the medium for transport to the microscope. To wound the globes using a sterile 25 gauge needle in the region of interest, pick up and hold the globe from the back of the eye, using a sterile eyedropper to keep the globe stable and prevent it from rolling. For a scratch wound, gently move a sterile 25 gauge needle across the exposed cornea or gently press the needle directly into the central cornea for a puncture wound, ensuring the wound does not pierce the corneal basement membrane.
Place the cornea or limbal region onto the cover slip in the inner area of the holder, while confirming the globe is positioned correctly and that the site of interest is in contact with the glass cover slip. Stabilize using the 3D printed cover adhered to the holder, using glue. And do not try to remove the globe, as this may cause tissue damage.
Turn on the microscope and set the environmental chamber to 35 degrees Celsius and 5%carbon dioxide and verify the humidified chamber. Place the cover slip holder and stabilized globe on the microscope stage within the environmental chamber and image, using live cell imaging techniques. By vet additional growth media onto the cover slip to prevent dehydration and maintain tissue viability, ensuring there is enough medium in the well to cover the globe in the holder.
Begin experiments using live cell imaging techniques, while using low power laser settings to preserve the tissue and prevent tissue damage and appropriate objectives for long duration experiments. Record and save data in the preferred file format of CZI files for data recording. High quality imaging data of multi-layered structure was obtained for X vivo cornea, stabilized with a 3D printed holder, demonstrating calcium signaling events in the apical, basal, and stromal layers of the cornea.
Z-stack images of a cornea, immobilized in the 3D printed holder, at a scratch wound were obtained to visualize the cell membranes and calcium signaling throughout the layers of the cornea and calcium mobilization in different Z planes. A time series experiment of a cornea was performed to study the cellular migration into the wound bed during the healing response, revealing little movement of the globe in the X, Y, or Z directions. It was observed that by placing the globe in the holder, different regions of the cornea, both at the central and in the corneal limbal region can be visualized.
This protocol allows us to image eyes from disease models that exhibit differences in wound healing. We can characterize aberrations in cell signaling and motility in live tissue.
