Use of 1,4 diacetoxy-2,3 dicyanobenzol in microspectrofluorometry for determining the intracellular pH of isolated living cells

Microspectrofluorometry allows to obtain the fluorescence spectrum of an isolated living cell. When cells are preincubated with 1,4 diacetoxy-2,3 dicyanobenzol the cellular fluorescence spectrum can be resolved in its components i.e. the characteristic fluorescence spectrum of each ionized forms of the probe and the intrinsic cell fluorescence spectrum due to NAD(P)H. This allows the determination of the intracellular pH with good accuracy. Furthermore, comparison between the intensity of the intrinsic cell fluorescence and the probe fluorescence intensity offers us an opportunity to monitor the intracellular amount of the drug.