Bh2 series light microscope

For optic nerve axon quantification, low and high magnification images were captured using an Olympus BH2 series light microscope with S Plan Apochromat objectives, an achromatic condenser (Olympus Corporation, Tokyo, Japan) and SPOT idea ™ camera (Diagnostic instruments, Inc., Sterling Heights, MI) running on SPOT Advanced software (Version 4.6). A low power image of each optic nerve was captured using a 20x objective to measure its cross sectional area and to divide the nerve into quadrants. A high power image of a subfield within each quadrant was then captured near its mid-point so that the images did not overlap or include the edges of the nerve, using a 100x oil immersion objective. The image of each subfield was overlain with a 4×6 grid of twenty-four 100 microns² counting boxes, and 2 boxes per row per grid randomly chosen for count axons. For each counting box, axons falling on the north and east lines of the grid were included, but axons on the south and west lines of the grid were not. The total number of axons counted was divided by the total box area counted to calculate an axon density for each quadrant, axon densities for the 4 quadrants were averaged, and the total number of axons estimated by multiplying the optic nerve cross sectional area by the axon density.