Development and application of nanoviscosity probes in advanced FLIM studies on synthetic and native hydrogels in vitro

An abnormally high viscosity of the hydrogel layer on the airway epithelium is the characteristic hallmark of many respiratory tract diseases. Existing knowledge relies on measurement of bulk samples. However, little is known about the regional viscosity of mucus on living cells and organs, even less is known about its spatial distribution. We are aiming for the development of a method that provides information about the viscosity of hydrogels (mucus and glycocalyx) on specific areas/sites/depths of living cells or organs. To achieve this goal, we will develop a platform to apply newly developed molecular probes of hydrogel viscosity in advanced fluorescence lifetime imaging microscopy (FLIM) studies, in-vitro and in living cells (primarily 2D and 3D cultures). We will perform spatial viscosity mapping by means of our recently developed Cluster-FLIM methodology. The viscosity probes are based on fluorescent molecular rotor (FMR) dyes. The fluorescence properties (i.e., fluorescence lifetime) of the small organic dyes are strongly correlated with (nano)viscosity. FMR dyes will be appended to sugar-binding boronic acids. The conjugates will enrich in the glycan-rich mucus layer and FLIM will provide superior sensitivity to obtain spatially resolved information about viscosity, polarity or pH. The behavior of FMR dyes in highly complex hydrogels system will be systematically characterized in mucin-mimetics, synthetic and native mucins. To enable FLIM measurements of local viscosity determined by select mucins, membrane-standing proteins such as MUC1, and syndecan-1 of the alveolar glycocalx will be labeled with FMR dyes on living cells; FMR-labeled glyco-engineered mucins will be used as well. Cluster-FLIM and FMR probes will distinguish the contributions of small and macromolecule induced friction to viscosity and provide a sensitive read-out for mucin crosslinking. The method will show the minute changes in viscosity at and near cell surfaces that occur upon variation of ion concentration, pH, and bacterial challenge. We further will use fluorescence lifetime and anisotropy measurements to investigate nanodynamics, and determine diffusivities in synthetic and native mucins. We envision that our methodologies will enable cell biological studies to unravel how biological perturbations, such as abnormal mucin expression, ion transport, aberrant glycosylation, or bacterial challenge affect mucus viscosity at the site of action.