Abstract Temperature monitoring is critical for endoscopic laser surgery to prevent tissue damage. Thermal injuries of the urothelium have been reported as a known complication of laser lithotripsy, however, in most cases real time temperature measurement is not available due to the absence of sensors in conventional endoscopes. In this study, we present an all-optical fluorescein-based thermal sensing approach enabling temperature measurement via an optical fiber, independent of the endoscope model, using a biocompatible fluorescent probe. The temperature-dependent fluorescence response originates from the redistribution between the monoanionic and dianionic forms of fluorescein, which is influenced by albumin through different mechanisms depending on the solution environment. A comparative analysis in water and in saline solution, which is commonly used for irrigation during surgery, revealed distinct environmental contributions to the temperature response of fluorescein emission. In water, a stable fluorescein–albumin complex was formed, accompanied by a systematic shift in the monoanion–dianion equilibrium, leading to strong temperature-dependent spectral changes. In saline solution, which is more relevant to intraoperative irrigation, no stable complex was observed, as shown by time-resolved fluorescence techniques; nevertheless, a reproducible temperature response was retained and was attributed to weaker, non-specific microenvironmental effects, including local pH variations near the protein surface. These findings demonstrate a reproducible temperature-dependent fluorescence response under defined solution conditions and provide a proof of principle for further development of optical temperature-monitoring approaches relevant to minimally invasive procedures.
Abstract The combination of stimulated emission depletion (STED) microscopy with fluorescence lifetime imaging microscopy (FLIM) offers a powerful route to functional super-resolution imaging, but remains technically challenging for lifetime-based sensing because of the complex interplay between stimulated emission, photon budget, and accurate lifetime quantification. In this technical note, we evaluate and optimize STED-FLIM for nanoscale mapping of bacterial membrane mechanics using the mechanosensitive probe Flipper-TR. This probe is particularly demanding for STED-FLIM because it exhibits a biexponential fluorescence decay, with only the long lifetime reporting on membrane mechanical properties. Using live Bacillus subtilis cells, we establish an acquisition and analysis workflow that combines optimized imaging conditions with tail fitting to minimize distortions introduced by the depletion pulse. Under these conditions, STED imaging achieved a more than twofold improvement in spatial resolution compared with confocal microscopy, while maintaining sufficient photon counts for robust lifetime estimation. Finally, we discuss the trade-offs between spatial resolution and photon budget and provide practical recommendations for implementing STED-FLIM with environmentally sensitive probes. These results extend the application of Flipper-TR to functional super-resolution microscopy and provide a framework for robust STED-FLIM measurements of membrane nanomechanics and other fluorescence lifetime-based sensing applications.
Abstract Lipid packing in cellular membranes exerts direct effect on membrane tension and microviscosity, and plays a central role in cellular adaptation, homeostasis and disease development. According to conventional mechanical descriptions, viscosity and tension are directly interconnected, with increased tension leading to decreased membrane microviscosity. However, intricate molecular interactions that contribute to provide the structure and function of cellular membranes suggest a more complex relationship between these parameters. In this perspective, we review recent progress in using environmentally sensitive fluorophores to map changes in lipid structure, corresponding to alterations in the bilayer hydration, polarity, microviscosity or tension. We focus particularly on the use of fluorescence lifetime imaging microscopy that enables quantitative concentration-independent measurements of these parameters.
Abstract Understanding mass transfer in aquatic biological systems requires simultaneous measurements of velocity fields and transported scalar fields. Such scalar fields, including spatial distributions of dissolved oxygen concentration, can be measured using luminescent sensor particles that also function as flow tracers, but their performance is affected by particle size and dispersity. Reproducible preparation of monodisperse sensor particles is essential for reliable sensing and velocimetry. Here, we systematically optimized flash nanoprecipitation to prepare oxygen-sensitive nanosensors based on poly(styrene-co-maleic anhydride) (PSMA). Dynamic light scattering identified polymer concentration as the primary determinant of particle size, with additional tunability provided by nonionic surfactants. Below the critical overlap concentration, increasing Reynolds number generally reduced particle size with lower dispersity. By varying preparation conditions, monodisperse nanosensors with tunable hydrodynamic diameters of approximately 120–410 nm (polydispersity index, PDI < 0.1) were reproducibly obtained. In contrast, polymer concentrations above the critical overlap concentration produced polydisperse particles with mean hydrodynamic diameters exceeding 800 nm. Frame-straddling lifetime imaging showed an approximately linear Stern–Volmer response from anoxia to air saturation, with nonlinear behavior occurring only under supersaturated conditions. The nanosensors were successfully applied within the sensPIV framework, enabling simultaneous mapping of oxygen distributions and velocity fields around a living coral. Estimated hydrodynamic relaxation times indicated negligible inertial lag across the investigated particle-size range, whereas estimated oxygen equilibration times of approximately 0.04–4 ms suggested that finite sensor response may limit the resolution of the smallest scalar structures. These results establish flash nanoprecipitation as a reproducible and highly controllable route for preparing monodisperse polymeric nanosensors with tunable particle sizes for simultaneous scalar and velocity measurements.
Protein oligomerization equilibria are central to many biological processes and are often highly sensitive to environmental conditions such as ionic strength, pH, and ligand binding. Quantitative characterization of these equilibria remains experimentally challenging because stable protein complexes frequently dissociate only at concentrations that are difficult to access with conventional biophysical methods. Fluorescence correlation spectroscopy (FCS) is uniquely suited to this problem, as it provides direct access to diffusion coefficients of fluorescently labeled proteins at nanomolar concentrations. However, the quantitative interpretation of FCS data from oligomeric systems requires a rigorous mathematical framework and careful experimental practice that have not previously been described in sufficient detail to guide implementation. Here, we provide a comprehensive description of the experimental workflow and analytical framework for determining dissociation equilibrium constants by FCS, covering instrument calibration, sample preparation, data quality control, after-pulse correction, and nonlinear least-squares fitting. We discuss common sources of error and provide practical guidance on critical experimental considerations including surface passivation, buffer preparation, equilibration time, and the role of labeling efficiency. Using the homotrimeric sliding clamp PCNA as a model system, we demonstrate the complete workflow under a range of KCl concentrations and show that moderate ionic strength stabilizes the PCNA trimer while very high salt partially destabilizes the complex. The approach is general and applicable to any reversible protein self-association reaction accessible by fluorescence detection at low protein concentrations. ### Competing Interest Statement The authors have declared no competing interest.
