Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Abstract

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Figure 1:. Schematics and setup of the 3D printed holder.

(A) Design of the 3D printed holder with annotated height and width dimensions. (B) Representative CAD file image from the 3D printer software. (C) Representative image of the holder with the attached cover containing a murine globe. (D) Sterile, single-use glue is applied to the bottom of the 3D printed holder. (E) The 3D printed holder is adhered to a glass-bottom p35 cell culture dish. (F) An enucleated globe is placed cornea-down into the holder using a sterile eye dropper. (G) The cover bar is adhered to the top of the 3D printed holder to ensure globe immobilization. (H) The glass-bottom plate with an adhered holder and globe is placed on the microscope stage.

Figure 2:. Using specialized 3D printed holders to stabilize the globe and yield higher-quality imaging data of multi-layered structures.

(A) and (B) represent typical live imaging data of a wounded ex vivo cornea stabilized without and with a 3D printed holder, respectively. Calcium signaling events (green) and cell counterstain (deep red plasma membrane stain) can clearly be identified in the apical, basal, and stromal layers of the cornea in (B) but not in (A).

Figure 3:. Z-stack of a cornea immobilized in the 3D printed holder.

Representative images of a z-stack taken through the layers of the cornea at a scratch wound (denoted with a white asterisk). The sample is stained with deep red plasma membrane stain to visualize the cell membranes and Fluo4-AM (green) to visualize calcium signaling. (A) The apical cell layer, (B) apical and basal cells, (C) basal cell layer, and (D) stroma can be seen in the wound bed.

Figure 4:. Representative 4 h time series experiment of a cornea revealing little movement of the globe in the x, y, or z directions.

The image is of a scratch wound injury (denoted with a white asterisk) at the central cornea of an ex vivo murine globe. The globe is stained with deep red plasma membrane stain to visualize the cell membranes and Fluo4-AM (green) to visualize calcium signaling events. The globe is immobilized using a 3D printed holder. The images were taken 1 h apart, beginning 5 min after injury. Little drift in the x, y, or z directions was observed using this imaging setup throughout the course of the experiment.

Figure 5:. Diagram of imaging locations on an intact globe.

The 3D printed holder permits imaging of an intact ex vivo globe at various locations. The holder was used to collect images from both the central and limbal regions of the cornea. Globes are stained with deep red plasma membrane stain to detect the cell membranes and Fluo4-AM (green) to detect calcium signaling. (A) Representative image of the central cornea at a scratch wound. (B) Representative image of the limbal region of the cornea after an injury to the central cornea.