Thiol-polarity dual cascade-triggered two-photon imaging of protein unfolding for early atherosclerosis diagnosis

Protein unfolding has emerged as a critical factor in the initiation and progression of atherosclerosis. Direct visu-alization of unfolded proteins remains technically challenging yet critical for elucidating atherosclerosis patho-genesis and developing early diagnostic tools. Here, we report a dual-reporter unlocking two-photon fluorescence probe for dynamic tracking of unfolded proteins in early atherosclerotic plaques. The probe achieves synergistic fluorescence activation through covalent binding with exposed thiols and subsequent stabilization within the local hydrophobic microenvironment of unfolded proteins. This dual-activation property allows for precise differentiation between folded and unfolded proteins in foam cells, facilitating spatiotemporal mapping of protein disorder at various stages of atherosclerosis. Notably, the probe detects early-stage unknown or tiny plaques within 4 to 8 weeks by identifying protein unfolding events before conventional Oil Red O staining and clinical ultrasonography, demonstrating unprecedented sensitivity for subclinical plaque prediction. Our findings establish a paradigm for investigating proteostasis disorder in atherogenesis and introduce a powerful molecular tool for early atherosclerosis diagnosis.