Perspective on Ionophore-Based Ion-Selective Nanosensors for pH-Independent Quantitative Lysosomal Calcium Imaging

Lysosomes, the acidic degradative hubs of cells, are increasingly recognized as critical regulators of calcium signaling, with dysregulation linked to neurodegenerative diseases and lysosomal storage disorders. However, quantifying lysosomal Ca²⁺ remains a formidable challenge due to the limitations of conventional fluorescent indicators, which suffer from pH interference in acidic environments. In this perspective, we critically evaluate emerging strategies for pH-independent Ca²⁺ sensing and advocate for ionophore-based nanosensors as a transformative solution. These nanosensors, featuring high Ca²⁺ selectivity, tunable dynamic range, and intrinsic pH insensitivity, leverage ion-exchange mechanisms coupled with solvatochromism or other transducers. While recent advances have demonstrated their utility for cation sensing in bulk systems, their application to quantitative lysosomal Ca²⁺ mapping remains underexplored. We highlight the unique advantages of these platforms, including endocytic uptake and compatibility with live-cell imaging, while identifying key challenges such as dye leakage, matrix stability under lysosomal conditions, and imaging-specific issues. By bridging gaps between nanosensor design and biological application, this discussion aims to catalyze the development of robust tools for elucidating lysosomal Ca²⁺ roles in health and disease.