Technical details of in vivo microscopy of living mouse cornea will be reported elsewhere (Maurice D, Zhao J, Nagasaki T, manuscript in preparation). The microscope for the histology, a standard upright fluorescence microscope, as described earlier, was used with a 40× water-immersion (numerical aperture [NA], 0.8), a 10× dry (NA, 0.25), and a 4× dry (NA, 0.1) objective. Mice were anesthetized with isoflurane in oxygen. Anesthesia was maintained by supplying steady flow of anesthetic gas to the nose of the mouse through a nose cone. Before microscopic observation, topical xylazine (20 mg/mL in saline) was given to stimulate proptosis of the eye,
16 so that a wide area of the corneal surface, including the limbus, could be observed without forcefully opening the eyelid. In some mice, when the pupil became dilated, 1% pilocarpine was given topically to promote miosis. This was necessary because strong fluorescence from lens GFP interfered with the observation of corneal fluorescence.
To reduce motion blur introduced by the mouse’s breathing, its head was held and immobilized with an upper-jaw clamp, which was then magnetically secured to a mouse holder so that the cornea was positioned directly under, and facing toward, the objective lens. When the 40× objective was used, the cornea was lightly touched with a glass coverslip at the bottom of a conical spacer, which was secured on the microscope’s stage, to suppress eye movements in the z direction. The glass applanation also helped flatten the cornea so that a nearly entire field of observation (430 × 340 μm after digitization) was in focus. The mouse was placed on a platform attached to custom-made gimbals that could be rotated freely in all directions, which facilitated rapid scanning of a wide area of the cornea (Maurice D, Zhao J, Nagasaki T, manuscript in preparation).
Microscopic images were captured with a digital camera (Orca) through a relay zoom lens (0.4× to 2.0×; Carl Zeiss). To minimize phototoxicity to live cells, an illumination source (100-W Hg arc lamp) was operated at 50% or less of its full power. A narrow band-pass excitation filter (480 ± 20 nm; Chroma Technology, Brattleboro, VT) also helped to reduce unnecessary illumination. In addition, a mechanical shutter for the arc light (Uniblitz; Vincent Associates, Rochester, NY) was used to ensure that corneal exposure to light was minimal. The camera and the shutter were synchronously controlled by an image-processing package (Metamorph; Universal Imaging Corp., West Chester, PA), which was also used for postacquisition image processing.
For routine image acquisition, a few images from different angles were taken with a 4× objective with 0.5× zoom , and 5 to 15 overlapping microscopic fields with a 10× objective with 0.5× zoom. In some animals, 10 to 20 overlapping fields were recorded with a 40× objective with 0.5× zoom to document the details of the corneal surface, which allowed analysis of individual epithelial cells. However, the 40× objective was used only sparingly to minimize the possibility of illumination damage to epithelial cells. A wide-field view was reconstructed from the overlapping images (Photoshop; Adobe). With 10× images, only the central one half to one third of each image was used, because the peripheral zone was out of focus. This assured that a reconstructed image was suitable for measurement of actual corneal surface distance. Under these conditions, image resolution was approximately 3.4, 1.3, and 0.33 μm/pixel for images acquired with a 4×, 10×, and 40× objective, respectively.