BCECF (2',7' -bis-(Carboxyethyl)-5-(and-6)-carboxyfluorescein) is the most widely used fluorescent pH sensor. Because its pKa (6.97) is close to physiological pH, BCECF can detect cytosolic pH change with high sensitivity. At low pH, the dye is weakly fluorescent but becomes more fluorescent with increasing pH. The excitation spectrum of the dye undergoes a slight shift during pH change, while the wavelength of the emission maximum remains unchanged. The pH is determined ratiometrically by the relative fluorescent intensities at 535 nm when the dye is excited at 439 nm and505 nm respectively. BCECF, acid is membrane-impermeant and therefore can be loaded into cells via microinjection or scrape loading. Also see the membrane permeant AM ester form of BCECF.
