Super-resolution Thermal Mapping and Viscoelastic Modelling of Lipid Raft Dynamics in Lens Epithelium: A Nanoscale Approach to Thermal Damage Mechanisms

Abstract Background: Understanding how lipid rafts respond to sublethal thermal stress in the lens epithelium remains a significant gap in membrane biophysics, particularly in relation to cataractogenesis. This study introduces a high-resolution thermal mapping approach to explore raft dynamics and their potential functional interactions with stress-responsive proteins. Methodology: We developed a super-resolution imaging system using single-molecule localisation microscopy, combining PAmCherry-tagged cholera toxin subunit B with DiD-PEG phospholipid probes to achieve ~20 nm spatial resolution. A custom microfluidic platform generated precisely controlled thermal gradients (0.1°C/s) within a physiologically relevant range (40°C–45°C), minimising bulk heating artefacts. Raft diffusion and membrane mechanics were modelled using a modified Helfrich–Canham viscoelastic framework, further supported by Martini 3.0 coarse-grained molecular dynamics simulations and interpreted via the Stokes–Einstein–Sutherland relation. Bayesian clustering quantified raft colocalisation with HSP70, while a graph neural network enabled drift correction with sub-5 nm precision to enhance spatiotemporal fidelity. Results: Thermal stress induced raft coalescence, decreased lateral mobility and increased spatial colocalisation with HSP70. These results suggest an active role for lipid rafts in sensing and responding to thermal perturbations, rather than serving purely as structural membrane domains. Conclusion: Our integrated platform combines super-resolution imaging, microfluidic thermal control and biophysical modelling to reveal a dynamic, functional role for lipid rafts in early heat stress responses. The system’s modularity and precision offer broad applicability for probing membrane behaviour in other epithelial tissues and present a promising tool for early-stage cataract biomarker discovery.