Ratio imaging was performed with a stereo-zoom epifluorescence microscope (SMZ1500; Nikon, Tokyo, Japan) equipped with a tungsten halogen light source (X-Cite 120; EXFO, Mississauga, ON, Canada), 1.6× objective lens (numerical aperture, 0.21; working distance, 24 mm), cooled charge-coupled device (CCD) camera (EM-CCD; Hamamatsu, Bridgewater, NJ), and custom filter sets (Chroma, Rockingham, VT) for BAC, tetramethylrhodamine (TMR), Na+-sensitive, red fluorescent chromophore, Na+-insensitive, green fluorescent chromophore, and BCECF. For ratio image analysis, images (1000-ms acquisitions for pH; 1500-ms acquisitions for Cl−, Na+, and K+) were obtained from pairs of filter sets that were manually switched. Tear fluid fluorescence ratios were computed over at least three randomly selected regions after subtraction of background (determined from the same regions of the ocular surface imaged before the fluorescent indicator was added). Ion concentrations and pH were determined from fluorescence ratios using the calibrations described. Photobleaching, which was insignificant for the Na+, K+ and pH indicators (<1% in 2-minute continuous illumination), was minimized for the Cl− indicator by limiting BAC excitation to periods of image acquisition.