Real-Time Ratiometric pH Imaging of Macrophage Lysosomes Using the Novel pH-sensitive Probe ApHID

Lysosomes actively regulate their lumenal pH, which is necessary for optimal enzymatic activity. Endocytic processes are involved in many diseases, including Alzheimer's disease, in which sub-optimal lysosomal function has been reported. To measure acidification, pH-sensitive probes can be delivered to endosomes and lysosomes using labeled dextran polymers or proteins. However, many commercially available probes have limited sensitivity in the acidic range of lysosomes, and their fluorescence is subject to enzymatic degradation and photobleaching. Herein, we describe the preparation, characterization, and use of a novel pH-sensitive probe, ApHID, a green-emitting fluorescent dye with optimal dynamic range within the acidity of endosomes and lysosomes. ApHID has a pKa near 5, increasing brightness with acidity, and it is robustly resistant to oxidation and photobleaching. We used ApHID ratiometric imaging to measure lysosomal pH in macrophages, yielding virtually identical results when compared with fluorescein and Oregon Green. Overall, ApHID circumvents limitations presented by most commercially available pH-sensitive probes and can be useful in demanding imaging applications such as intravital imaging of tissues.