Quantitative confocal imaging along the crypt-to-surface axis of colonic crypts

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 mu m 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.