Optical assessment of gel-like mechanical and structural properties of surface layers: single particle tracking and molecular rotors

Abstract

Thin gel-like layers form at many surfaces of natural or artificial origin. Important properties of such layers include thickness, viscosity and density. Here we discuss two optical approaches which allow assessment of these properties with high resolution. The first approach relies on centroid calculation and defocus imaging based 3D tracking of fluorescent tracer particles, which is based on standard fluorescent microscopy and allows a precision of particle detection in the range of 10nm. The size of the particle and its surface charge and polarity will determine the particle invasion into the layer. Thus simultaneous application of different colored beads with different size and properties can reveal the thickness and nature of the layer. Via tracking the thermal vibration of particles invading the layer the bulk viscosity of the layer can be calculated. The second approach uses “molecular rotor” fluorophores (MR). Due to their molecular structure, the MR’s fluorescence quantum yield increases as their internal rotation is hampered by e.g. high viscosity of the embedding medium. The MRs are several orders of magnitude smaller than the structural (macro) molecules of a gel-like layer and therefore the MRs are not necessarily directly sensitive toward the bulk viscosity of the layer. In contrast, the MRs internal rotation will be attenuated by the MRs interaction with the structural elements of the layer or the solvent included in it. Depending on their molecular structure MRs exhibit different sensitivity to the mechanical properties of the large macromolecules or the solvent in a layer. Thus, they may be used to assess the microdomain’s viscosity or density in a surface layer. Using a ratiometric imaging approach, they can be used for continuous measurements in very different experimental settings.