Intravital pH Imaging using the sensor ApHID Reveals Incomplete Lysosomal Acidification in Resident Meningeal Phagocytes

SUMMARY Tracking lysosomes and quantifying their pH in the living, intact brain has been challenging owing to the lack of suitable in vivo tools. Here we report the use of dextran polymers labeled with the pH sensor ApHID to visualize and quantify lysosomal pH in resident phagocytes of the meninges. ApHID-dextrans delivered systemically are rapidly endocytosed by mainly dural and subdural macrophages and accumulate in lysosomal compartments. Intravital pH imaging revealed a striking heterogeneity in lysosomal acidification, with only ∼5% of phagocytes reaching pH values below 5.0, in marked contrast with measurements in macrophage cell cultures. Photothrombotic ischemic lesions induced progressive lysosomal acidification in local phagocytes, while acute chloroquine treatment led to rapid alkalinization. Our methodology enables direct, quantitative pH measurements without genetic manipulations, and unveils a previously unappreciated heterogeneity in lysosomal acidification among phagocytic cells in the meninges.