A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

This protocol uses a multi-filter microscope for live imaging of the entire structure of mouse ocular surface in the dual fluorescent transgenic mouse model. The combination of a custom multiphoton microscopic platform and dual fluorescent transgenic mice enables the real-time visualization of the and structure of the ocular surface. To set up the multiphoton microscope, select the water immersion 20X, 1.00 NA objective and set the Titanium Sapphire laser as the excitation source on an upright microscope.

Set the laser output wavelength to 880 nanometers for EGFP and 940 nanometers for tdTomato. Include two dichroic mirrors for the separation of SHG EGFP and EGFP tdTomato and use 434-17, 510-84 and 585-40 nanometers band pass filters to spectrally separate the SHG EGFP and tdTomato signals. To set up the eye holder, after confirming a lack of response to pedal reflex in an anesthetized 8-12 week old mouse, place the mouse on the heated microscope stage and insert a temperature monitoring probe.

Place the mouse into a custom stereotaxic mouse holder and insert ear bars into the external auditory meatus. Use three point fixation to secure the head holder and apply a 0.4%Oxybuprocaine Hydrochloride and saline solution to the ocular surface. Cover the tips of a pair of number five Dumont forceps with the PE tube loop.

After three minutes perform a manual eyelid retraction to confirm eyeball protrusion and carefully place a PE holder tube loop along the eyelid margin to expose the ocular surface. And use the knob of the holder to stabilize the eyeball with the forceps. Then, cover the corneal surface with an eye gel with a refractive index of 1.338.

For Z serial imaging of the corneal surface, use a mercury light source to image the targeting field and open the microscope software. Select the appropriate photo multiplier and digital gains for visualizing the cellular structure in the ocular surface and set the first and last slide to allow the acquisition of an image stack. Set the image resolution to 512 by 512 pixels and the Z step to one micrometer.

Then, click start to collect Z serial images acquiring one live image with an 880 nanometer excitation for SHG EGFP signal collection, and one live image at a 940 nanometer excitation for EGFP tdTomato signal collection within each area. Throughout the image, use the stepper motorized stage holder to rotate the eyeball to allow imaging across the entire corneal surface. When all of the images have been acquired, load the Z serial images into Fiji and select the Median 3D filter plugin.

After removing the background noise, select the unsharp mask filter package Fiji to sharpen the images and click auto brightness contrast to automatically optimize the quality of the images. After processing, save the images as a serial image sequence and export the image sequence into an appropriate 3D reconstruction software program. In all of the multiphoton microscopy images, present the EGFP tdTomato and SHG signals in pseudo green, red, and cyan respectively before capturing the 3D structure images by snapshot.

Multiphoton microscopy allows the visualization of superficial Wing and Basal cells in the corneal epithelium of dual fluorescent transgenic mice. Single cells from the basal layer can be mapped to the superficial layer as well as single hexagonal shaped superficial cells. As revealed by the cytoplasmic expression of the tdTomato signal, the membrane protein rich intracellular vesicular system, including the golgi apparatus and endoplasmic reticulum is scattered within the wing cells.

Within the collagenous stroma, the stellate shaped coratesites are outlined by the membrane targeting EGFP fluorescents in these mice. The coratesites embedded in the collegen stroma are more loosely spaced than within the endothelial cells. In addition, thin branching nerves within the corneal stroma can also be visualized by membrane targeting tdTomato signals and corneal endothelial cell monolayers demonstrate a relatively homogenous hexagonal shape connected in a honeycombed pattern.

The Limbal epithelium consists of one to two layers of epithelial cells. The dual fluorescent reporter transgenic strain also enables the imaging of capillaries within the conjunctiva, facilitating the reconstruction of the 3D architecture of the capillaries outlining the vascular endothelium. Stabilizing the eyeball with moderate pressure without injury is critical for acquiring good quality images as insufficient or excessive pressure can compromise the image quality.

This NVivo image imaging platform can be modified for visualization of structures beneath the ocular surface and can be applied to in-situ studies of various ophthalmic diseases.