Two-Photon FLIM Imaging of Mitochondrial Microenvironment in Apoptosis, Ferroptosis, and Fatty Liver Disease Models Using a Carbazole-Pyridinium Probe

Mitochondria are dynamic organelles whose microenvironmental state is tightly linked to cell death pathways and metabolic disease progression. However, directly visualizing mitochondrial microenvironment dynamics (e.g., viscosity changes) in living systems remains challenging. Here, we report an innovative two-photon fluorescent probe with a donor-π-acceptor architecture - featuring a hexyl-carbazole donor and a pyridinium acceptor - that exhibits bright near-infrared two-photon fluorescence. The probe's design enables robust mitochondrial targeting and high-performance two-photon excitation in the NIR region. By employing two-photon fluorescence lifetime imaging microscopy (TP-FLIM), we achieve quantitative, real-time and high-resolution mapping of mitochondrial functional status in live cells and tissues. Using this TP-FLIM approach, the probe sensitively tracks dynamic mitochondrial alterations under stress. In cultured cells undergoing apoptosis or ferroptosis, it reports distinct microenvironmental changes associated with mitochondrial stress and remodeling - for instance, revealing increased mitochondrial viscosity during apoptotic condensation and compaction of the organelle during ferroptotic cell death. In a nonalcoholic fatty liver disease (NAFLD) mouse model, longitudinal imaging with the probe visualizes progressive mitochondrial dysfunction and remodeling across different disease stages, reflecting the mounting stress on hepatic mitochondria as NAFLD advances. Overall, this D-π-A based two-photon FLIM probe provides a powerful biosensing tool for functional imaging of mitochondria, highlighting dynamic mitochondrial remodeling and microenvironment changes in cell death and disease contexts with high spatiotemporal resolution.