Interference-based Investigation of Microscopic Objects Near Surfaces: a View From Below

Phenomena occurring when microscopic objects approach planar surfaces are challenging to probe directly because their dynamics cannot be resolved with a sufficiently high spatial/temporal resolution in a non-invasive way, and suitable techniques/methods involve complex instrumentation/computations of limited accessibility/applicability. Interference-based techniques can overcome these barriers. However, because most set-ups and analysis methods are ideal for planar-like geometries, their accurate application for studying microscopic objects has been difficult. Reflection interference contrast microscopy (RICM) has shown particular promise allowing objects in close proximity to a surface to be observed from below, producing interferograms that inherently embed detailed information about the objects? topography near the substrate. Because precise extraction of this information has been challenging, this study seeks to develop analysis methods applicable to RICM to facilitate its practical implementation for accurate investigation of interfacial phenomena between microscopic objects and surfaces.

The most sophisticated theory of RICM was significantly improved and coupled with a general method to simulate the interference pattern from arbitrary convex geometries. Experimental results revealed that accurate reconstruction of an object?s contour is possible by fitting its interferogram; however, this is computationally intensive and of limited applicability, motivating the formulation of a simplified and accurate RICM model. This facilitated a major breakthrough: an innovative analysis of RICM interferograms provides the inclination angles of the geometry under study and a mathematical procedure allows near-instantaneous reconstruction of the contour with nanometer-scale resolution, applicable to arbitrarily shaped convex objects under different experimental conditions.

A method for extracting nanometer-scale topographic information from RICM interferograms has been proposed; in particular, microspheres can be conveniently analyzed to measure surface roughness based on fringe visibility. Also, precise and accurate measurements of microspheres? size were performed by means of optimized and robust fringe spacing analysis. Finally, RICM?s distinctive ?view-from-below? perspective was applied in simple experiments involving the deposition of microspheres on surfaces, directly revealing the existence of different scenarios depending on deposition media and unique femtoliter-scale capillary condensation dynamics underneath micron-sized glass beads. Results show that RICM has a clear potential for near real-time analysis of ensembles of objects near surfaces so that statistical/probabilistic behavior can be realistically captured.