Intracellular pH (pHi) Measurement in Rat Duodenal Epithelium In Vivo Using SNARF-1

Abstract

Our laboratory has previously used BCECF to measure the pHi of the duodenal epithelium [1–3]. BCECF loads into epithelial cells and fluoresces well, but since BCECF is a dual excitation-single emission compound, a ratio pair cannot be obtained simultaneously, but requires sequential acquisition of image pairs. Measurement of pHi in vivo is impaired, due to the constant motion of epithelial cells which are affected by respiratory and cardiac movement. Exact registration of image pairs is not possible and ratiometric pHi calculations must be therefore be performed manually. 5-(and 6)-Carboxyseminaphthorhodafluor-1 (SNARF-1) is a single excitation dual emission fluorescent compound. The emission ratios calculated from the two peaks of 640 and 580 nm are a sensitive indicator of pH; furthermore, image pairs at two different wavelengths can be obtained simultaneously. It is thought that this specific feature can be used for more accurate determination of pHi especially in vivo. In the present paper, we used SNARF-1 for measurement of pHi in rat duodenal epithelium in vivo.