Measurement of mitochondrial pH in situ

In this article, we describe the advantages adn disadvantages of procedures for monitoring mitochondrial pH in situ using optical microscopic techniqu es. The first method employs the combination of the fluorescent pH-sensitiv e indicator carboxy-SNARF an laser scanning confocal microscopy. Manipulati on of the loading and post-loading conditions enables relatively specific a ccumulation of carboxy-SNARF into mitochondria. With the use of a mitochond rial-specific marker, mitochondrial pH can be accurately monitored. More re cently, mitochondrial-targeted, pH-sensitive probes have been used to monit or mitochondrial pH. In particular mitochondrial targeting of the yellow fl uorescent protein (YFP) mutant of green fluorescent protein (GFP) combines the advantages of specific mitochondrial localization, high-fluorophore qua ntum yield, and extinction coefficient with an appropriate pKa for measurin g mitochondrial pH. The use of dual-excitation ratiometry with mitochondria lly targeted YFP increases the dynamic range of mitochondrial pH measuremen ts and corrects for differences in the amount of expression of mitochondria lly targeted YFP at the level of individual mitochondria.