CRC 1449/2: Development, characterization and application of nanoprobes for advanced FLIM studies of synthetic and cellular hydrogels (SP A04)

In the first funding period, we analyzed the fluorescence lifetime signatures of fluorescent molecular rotor (FMR) dyes on mucus and mucus-mimicking hydrogels. In this fubding period, we will develop viscosity-sensing probes with high cell surface affinity (Kd ≤ 1 nM), to achieve high contrast in the selective imaging of mucus/glycocalyx and enable a wash-free analysis of delicate cells, preserving the mucus. Towards this end, we will systematically vary the boronic acid units, their number and distance, and explore the influence of charge and flexibility of the peptide scaffold. Attention will also be paid to the role of linkers, which have to provide the glycan-independent viscosity sensing by separating the FMR from the glycan recognition module. We aim to investigate synthetic hydrogels for dissecting the impact of hydrogel rheology and mesh size as well as of small molecule/particle diffusion (e.g., proteins) on the photodynamics of FMRs. Based on this investigation, we will apply FLIM to FMR-stained mucus at increasing levels of complexity (ranging from ex situ studies of patient-derived sputum sample up to native mucus grown on live cells) and determine mucus viscosity and its spatial heterogeneity in health, disease and treatment. In addition to using the advanced FLIM methods, we will investigate whether viscosity changes can also be visualized by fluorescence intensity ratio imaging, which would be feasible with standard fluorescence microscopes