We acknowledge the authors’ considered response to our editorial commentary on their recent work describing the development of a novel near-infrared (NIR) fluorescent probe for monitoring lipid peroxidation–mediated viscosity changes in ferroptotic hepatocytes. We sincerely thank the authors for their thoughtful and comprehensive correspondence in response to our editorial commentary on their recent study published in Clinical and Molecular Hepatology. We agree that visualizing intracellular viscosity dynamics in live hepatocytes represents a significant methodological advance for elucidating oxidative membrane damage and ferroptosis in the progression of metabolic dysfunction–associated steatotic liver disease (MASLD) [1,2]. Multiple lines of evidence indicate that TTM-4, a newly developed NIR fluorescent probe, enables selective detection of oxidized lipid droplets and facilitates visualization of their intracellular distribution, compared with classical fluorescent lipid peroxidation probes. Furthermore, TTM-4 allowed the observation that intracellular viscosity increases more rapidly during ferroptosis than during apoptosis or pyroptosis and effectively monitored these ferroptosis-associated viscosity changes, thereby facilitating visualization of oxidized lipid dynamics. The application of TTM-4 is particularly noteworthy, as it highlights the importance of evaluating oxidized lipids during ferroptosis in the early stages of MASLD, and suggests that TTM-4 may serve as a useful probe in this context. This interpretation is further supported by previous studies reporting pronounced alterations in hepatic tissue viscosity in patients with chronic liver disease, as well as viscosity changes across different grades of steatosis and stages of fibrosis [3]. Therefore, the development of TTM-4 as a sensitive probe for interrogating lipid peroxidation–associated viscosity changes in ferroptotic hepatocytes represents a remarkable technical and conceptual advance. In contrast to conventional lipid probes, TTM-4 selectively visualizes peroxidized lipids, enabling real-time monitoring of lipid oxidation dynamics during ferroptosis in live cells. The authors further demonstrated that lipid peroxidation is initially detectable within cytosolic lipid droplets and subsequently propagates through the endoplasmic reticulum toward the nucleus using this platform. This spatial propagation is conceptually aligned with prior studies implicating lysosomes as potential initiation sites of lipid peroxidation, given their enrichment in redox-active iron and reactive oxygen species [4]. Moreover, these observations further emphasize the organelle-specific nature of ferroptotic signaling driven by lipid dysregulation and extend current mechanistic understanding of how oxidative stress–induced subcellular perturbations contribute to the progression of MASLD. Collectively, TTM-4 emerges as a valuable platform for visualizing ferroptosis-associated liver injury, with potential implications for early detection and mechanistic stratification of liver diseases [5].

As a conclusion, this correspondence provides a revised conceptual context for the early detection and mechanistic characterization of ferroptosis-driven hepatocellular injury through the application of TTM-4. These findings demonstrate that lipid peroxidation–associated viscosity changes constitute a functional feature of hepatocellular susceptibility to metabolic stress. This perspective encourages a reassessment of the conventional view of MASLD as a disorder driven predominantly by oxidative injury, lipid peroxidation, and inflammation, by highlighting spatiotemporal and organelle-level perturbations as critical determinants of disease progression. By identifying intracellular viscosity dynamics and lipid peroxidation as integrative markers of ferroptotic activity, the application of TTM-4, which is capable of detecting these biophysical alterations, aligns with emerging paradigms that emphasize real-time visualization of subcellular stress responses to facilitate early diagnosis and intervention. We acknowledge the authors’ contribution and agree that continued refinement of probes such as TTM-4 would be instrumental in advancing early detection strategies and targeted therapeutic approaches for ferroptosis-associated liver injury.

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