Ratiometric pH Imaging of Macrophage Lysosomes Using the Novel pH-sensitive Dye 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 (AD), 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 photobleaching. Herein, we describe the preparation, characterization, and use of a novel pH-sensitive probe, ApHID , a green-emitting dye with a pKa near 5, increasing brightness with acidity, and resistance to oxidation and photobleaching. We used ApHID to measure lysosomal pH in macrophages, yielding nearly identical results when compared with fluorescein and Oregon Green. ApHID circumvents limitations presented by most commercially available pH-sensitive probes, and it can be useful in demanding imaging applications such as intravital imaging of tissues. ### Competing Interest Statement The chemical synthesis and uses of ApHID have been included and described in a provisional patent application filing (63/526,386) entitled Ratiometric pH Imaging of Macrophage Lysosomes Using the Novel pH-sensitive Dye ApHID.