Integrated two-photon imaging platform for spatiotemporal tracking of pH and viscosity in the lipid droplet...

Pathological alterations in intracellular viscosity, acidity, and lipid droplet (LD) dynamics are critical biomarkers for diseases such as cancer, atherosclerosis, and diabetes. Two-photon fluorescence imaging, renowned for its deep penetration and minimal background fluorescence, is an ideal tool for probing these parameters. However, existing two-photon probes lack the ability to simultaneously detect changes in intracellular viscosity, pH, and LD dynamics within a single platform. To address this limitation, we report CyB, a multifunctional single-molecule probe engineered by conjugating a hydroxyethyl-substituted hemicyanine unit to a boron dipyrromethene (BODIPY) core through a flexible double bond. The CyB probe features unique functionalities, including two-photon fluorescence imaging capabilities, dual responsiveness to pH and viscosity for precise microenvironment sensing, ratiometric imaging with distinct absorption/emission shifts through pH-dependent intramolecular cyclization, and specific targeting of LDs. This innovative design of CyB enables simultaneous, multi-parameter detection of pH, viscosity, and LDs behavior in complex disease models (tumor, atherosclerosis, and diabetic cells) using confocal laser scanning microscopy (CLSM) and under two-photon excitation (λ_ex = 940 nm). Furthermore, the dual-response characteristics of CyB have been effectively validated in zebrafish model in vivo, demonstrating its potential for multidimensional analysis of pathological microenvironments.