Combining Colloidal Probe Atomic Force and Reflection Interference Contrast Microscopy to Study the Compressive Mechanics of Hyaluronan Brushes

Abstract

We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.

Cell Surface Access Is Modulated by Tethered Bottlebrush Proteoglycans

“…Furthermore, for the PCM to expand to such large sizes, particularly on the RCJ-P cells, which we have shown has no measurable cross-linking in the PCM (19), the HA strands must be at least 18 mm in length (~7.5 MDa, 1 disaccharide~1 nm~400 Da (37)) or it requires that the PCM is comprised in part by free HA strands entangled with surface bound strands (38). This is unlikely, however, as several studies of biomimetic HA-aggrecan systems suggest that such uncross-linked multilayers do not occur (39,40). Finally, we infer differences between the MSC and RCJ-P cell PCMs by comparing the difference in exogenous aggrecan concentration to reach maximal PCM.…”

Transient Exposure of Pulmonary Surfactant to Hyaluronan Promotes Structural and Compositional Transformations into a Highly Active State

“…Although ⌸ increases linearly with c below c ol , it increases supralinearly above c ol (with a scaling up to c 9/4 (44,45)). The distinct increase in the sensitivity around c ol , which arises from the encounter of HA strands in the meshwork of interpenetrating polymers, may explain why the beneficial effect of HA sets in at this particular concentration.…”

Films of End-Grafted Hyaluronan Are a Prototype of a Brush of a Strongly Charged, Semiflexible Polyelectrolyte with Intrinsic Excluded Volume

“…Polystyrene microspheres of 25 ± 3 μm diameter (Polysciences, Eppelheim, Germany) were attached to tipless V-shaped Si 3 N 4 cantilevers with a nominal spring constant of 0.06 N m −1 (Veeco Probes, CA, U.S.A.), as described previously. 14 The real cantilever spring constant was determined before attaching the microsphere using the thermal noise method. 23 Preparation of the Liquid Chamber.…”

“…The method, which is described in detail elsewhere, 14 is based on the analysis of radially averaged intensity profiles with a simple optical model, the parallel plate approximation with incident light parallel to the surface normal. 27 The correlation of multiple solutions generated by this model provides The deflection vs piezo displacement curves obtained by AFM were converted into deflection vs relative distance curves using established methods.…”

“…With this setup, interaction forces can be quantified at the same time as the absolute distance between the colloidal probe and the substrate on which the brush was built. 14 The latter is not readily available by AFM alone because the distance at closest approach remains unknown when analyzing polymer films. The combined setup provides the level of control on the absolute distance that is needed for a rigorous comparison of experiment and theory.…”