Scanning Electron Microscopy of Murine Lenses

Eight- and 24-month-old B6-albino wild-type lenses were prepared for scanning electron microscopy (SEM) as previously described [50 (link), 61 (link), 62 (link)]. Briefly, freshly dissected lenses were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.3) at room temperature for 2–3 days. A sharp needle was used to bisect lenses along the visual axis, and lens halves were post-fixed in 1% aqueous OsO₄ for 1 hour at room temperature. Samples were dehydrated using ethanol and were critical point dried in a Samdri-795 critical point dryer (Tousimis Inc., Rockville, MD). Lens halves were mounted and coated with gold/palladium in a Hummer 6.2 sputter coater (Anatech Inc., Union City, CA). Images were acquired with a JEOL 820 scanning electron microscope at 10 kV (JEOL, Tokyo, Japan). Using the lens nucleus as a reference, images from different regions of the lens were compared between samples (i.e., comparable regions were located based on measurements from the center outward). At least four lenses from 2 different mice were examined for each age, and representative images are shown.