Quantitative confocal microscopy methods are used to measure events along the crypt-to-surface axis in living mouse colonic mucosa. Experiments visualize carboxyseminaphthorhodofluor-1 (SNARF-1; a pH-sensitive fluorescent dye) in the extracellular fluid to measure extracellular pH within an intact epithelium. Lucifer yellow (LY; a pH-insensitive dye) is used to control for fidelity of the optical path. Light scatter from colonic tissue caused SNARF-1 or LY fluorescence to decrease 3% per micrometer focal distance into tissue at both 640- and 580-nm emission wavelengths. However, dual emission ratios of LY fluorescence were constant as a function of focal distance into tissue or in the presence of short-chain fatty acids (SCFA). SCFA, known to cause changes in extracellular pH, cause maximal changes in crypt luminal pH at 10 microns from the crypt base. Maximal changes of pH in lamina propria occur higher along the crypt-to-surface axis than maximal changes in luminal pH. Lateral intercellular spaces between colonocytes are a separate microdomain in which pH is constant in absence vs. presence of SCFA and along the base-to-apex axis of colonocytes.