Ommatidial histology

Jalmenus evagoras eyes were dissected under a stereomicroscope at room temperature (RT) and immersed in fixative solution (2.5% formaldehyde, 2.5% glutaraldehyde in 0.1 mol l⁻¹ cacodylate buffer pH 7.4, Electron Microscopy Sciences, Hatfield, PA, USA) for 1 h at RT then stored overnight at 4°C. Subsequently, the perfused fixed eye tissue was washed with 0.1 mol l⁻¹ cacodylate buffer, post-fixed for 1 h with 1% osmium tetroxide (OsO₄) containing 1.5% potassium ferrocyanide (KFeCN₆), washed in water 3 times and incubated in 1% aqueous uranyl acetate for 1 h. After two additional washes with water, the tissue was dehydrated for 10 min in increasing grades of alcohol (50%, 70%, 90%, 100%), placed in propylene oxide for 1 h, and infiltrated overnight in a 1:1 mixture of propylene oxide and TAAB Epon. The tissue was then embedded in TAAB Epon for polymerization at 60°C for 48 h. The eye tissue was excised with surrounding solid Epon, precisely realigned to 30 deg elevation in the dorsal region, prior to a second resin polymerization step.

Ultrathin 60 nm sections were cut with a diamond blade on a Reichert Ultracut-S microtome at the Harvard Medical School Electron Microscopy Facility and sections at recorded depths were placed onto copper grids. To increase contrast prior to examination with the electron microscope, the copper grids were stained at RT for 30 min with 2% uranyl acetate then for 4 min with Reynold's lead citrate. Images were obtained using a JEOL JEM 1400 Plus Transmission electron microscope (TEM) at the Lund Microscopy Facility. The principal rhabdomeric axes for contributing photoreceptor cells (R1-R8) were recorded in ommatidial arrays of 10–15 individual visual units across depth from the upper rhabdomeric tier to the equatorial zone.