Astrauskaite, G. | Search bioRxiv

bioRxiv2026

Fluorescence-lifetime optical electrophysiology in contracting cardiomyocytes

Precise monitoring of cardiac electrophysiology in vitro is crucial to understanding heart function and cardiac disease. However, high-throughput, contact-free methods for directly measuring excitation-contraction coupling remain limited. Here, we introduce a new paradigm for quantitative electrophysiological imaging that combines fluorescence lifetime and intensity information to capture dynamic cardiac signals with high fidelity. We show that lifetime measurements are intrinsically decoupled from motion artifacts and provide calibrated calcium concentration and membrane potential readouts across wide fields of view. Using a gated single-photon avalanche diode camera, we acquire fluorescence lifetime images at up to 200 frames per second with sufficient signal-to-noise ratio such that each frame contains meaningful lifetime information without temporal averaging. This approach yields spatially resolved maps of absolute voltage and calcium values across contracting cardiomyocyte monolayers, revealing heterogeneous cell behaviors within individual assays and uncovering previously unreported dynamics during late-phase repolarization for real-time analysis of excitation-contraction coupling.

bioengineering↗

bioRxiv2025

Age-associated Tissue Organisation Shifts Retinal Pigment Epithelium Actomyosin Plasticity in Phagocytosis

Tissue homeostasis relies on mechanical feedback loops balanced by cell loss and proliferation. However, maintaining this balance becomes particularly challenging in postmitotic tissues, where alternative mechanisms replace compensatory proliferation. In the postmitotic retinal pigment epithelium (RPE), these mechanisms include significant structural adaptations over time. Yet, how these adaptations relate to epithelial mechanical homeostasis and age-associated functional decline remains poorly understood. To establish the relationship between structural changes, mechanical homeostasis and function, we developed an in vitro reductionistic model mimicking age-related reduction in RPE cell density. Inducing large-scale apoptosis in postmitotic stem cell-derived RPE monolayers recapitulates structural hallmarks of aged tissue, such as reduced cell height, shortened microvilli and cytoskeletal reorganisation. This new structure acquires a new mechanical equilibrium, evidenced by tissue stiffening and enhanced junctional contractility. Functionally, the monolayers display impaired vision-supporting phagocytosis of photoreceptor outer segments. Mechanistically, modulation of actin nucleators, Arp2/3 and formins, demonstrates that apicolateral monolayer deformation is critical for phagocytosis and may be compromised in aged RPE. Our findings suggest that a shift in mechanical homeostasis due to cell loss is a major driver of age-related RPE functional decline. Importantly, we show that structural remodelling in ageing alone can compromise tissue function, independent of other stressors.

cell biology↗