Real-time pH imaging of macrophage lysosomes using the pH-sensitive probe ApHID

Active endolysosomal pH regulation is essential for optimal enzymatic activity. To measure acidification, pH sensors can be delivered to acidic compartments using labeled dextran polymers or proteins. However, commercial probes have limited sensitivity in the acidic endolysosomal range or their fluorescence undergoes degradation. Herein, we introduce the new pH-sensitive probe ApHID, a green-emitting sensor with optimal dynamic range matching the acidity of endosomes and lysosomes. Acid pH indicator dye (ApHID) has a pKa near 5, increasing brightness with acidity, and withstands oxidation and photobleaching. We used ApHID dextrans to measure endolysosomal pH in macrophages and compared it to other commercially available sensors. ApHID reported pH accurately and stably over time in cell culture and was sensitive to subtle variations in organelle acidification in real time. Overall, ApHID circumvents limitations of currently available commercial probes and can provide utility in demanding applications such as intravital imaging of tissues.