FLUORESCENCE DIGITAL IMAGING AND PHI OF CELLULAR COMPARTMENTS

We have developed a setup for multipurpose digitally-enhanced videomic roscopy and studied various biological events involved in pH regulatio n of various cell compartments. The regulation of acid secretion by ga stric parietal cells in culture was followed by the uptake of the fluo rescent weak base 9-amino acridine in the closed apical secretory vacu ole. The method proved to be sensitive and accurate enough to provide reliable statistical data with the analysis of fewer than one hundred cells as compared with standard biochemical methods requiring thousand more cells. The variation of cytosolic pHi of NRK cells loaded with t he fluorescein derivative BCECF was studied next. The characterization of the various proton and anion exchangers present in NRK cells to re gulate the pHi provided evidence that the method was sensitive enough to detect pH variations in the 0.1 unit range. A similar methodology w as applied to study the acidic endocytic compartments of cultured cell s, using fluorescein-conjugated dextran that was internalized by fluid phase endocytosis. In breast cancer cells, the presence of large acid ic phagosomes was correlated with the invasive properties of the cells . The lumen of phagosomes was found very acidic with value reaching 4. 0 or lower, whereas that of endosomes and lysosomes present in the sam e cells was similar to what was described in normal cells.