pH sensing and imaging in living cells based on fluorescence lifetime of carbon dot nanosensors

The pH value is one of the most frequently measured chemical parameters, yet developing nanometric sensors capable of accurately mapping pH distribution and dynamics with high spatial and temporal resolution remains a significant challenge. Such sensors are vital for advancing our understanding of numerous physiological and pathological processes. Nanoparticle-based sensors, commonly referred to as nanosensors, represent a promising class of optical sensors, with fluorescence lifetime-based probes offering superior sensitivity and quantitative reliability. However, existing pH nanosensors relying on fluorescence lifetime are challenging to synthesize and often suffer from poor biocompatibility, narrow pH response ranges, low stability, and calibration-dependent performance. Here, we overcome these limitations by introducing a water-dispersible pH nanosensor based on fluorescence lifetime of colloidal carbon dots (CDs) derived via a one-step reaction from a single precursor Rhodamine B. These CDs are biocompatible, non-toxic, and stable in highly acidic/basic conditions, which makes them well-suited for intracellular applications. The intrinsic fluorescence lifetime of these CDs exhibits a pseudo-linear, self-referencing response across exceptionally broad pH range (1-11), driven by pH-induced transformations of their electronic structure occurring during protonation and deprotonation of CD surface. By applying micrometer-resolution, quantitative pH imaging via fluorescence lifetime imaging microscopy, we demonstrate how CDs are preferentially sequestered in lysosomes of human skin fibroblasts, enabling precise quantification of inhibitor-induced pH changes within these organelles. Our findings highlight a significant potential of the CD nanosensors for precise monitoring of lysosomal pH in living cells, offering broad utility in biomedical research and potential studies of pH-associated cellular dysfunction.