Alginate microbeads are extensively used in tissue engineering as microcarriers and cell encapsulation vessels. In this study, we used atomic force microscopy (AFM) based indentation using 20 μm colloidal probes to assess the local reduced elastic modulus (E*) using a novel method to detect the contact point based on the principle of virtual work, to measure microbead mechanical stability under cell culture conditions for 2 weeks. The bead diameter and swelling were assessed in parallel. Alginate beads swelled up to 150 % of their original diameter following addition of cell culture media. The diameter eventually stabilized from day 2 onwards. This behaviour was mirrored in E* where a significant decrease was observed at the start of the culture period before stabilization was observed at ~ 2.1 kPa. Furthermore, the mechanical properties of freeze dried alginate beads after re-swelling them in culture media were measured. These beads displayed vastly different structural and mechanical properties compared those that did not go through the freeze drying process, with around 125 % swelling and a significantly higher E* at values over 3 kPa.

The Arabidopsis sensitive-to-freezing8 (sfr8) mutant exhibits reduced cell-wall (CW) fucose levels and compromised freezing tolerance. To examine whether CW fucosylation affects the response to desiccation also, we tested the effect of leaf excision in sfr8 and the allelic mutant mur1-1. Leaf water loss was strikingly higher than wild type in these, but not other, fucosylation mutants. We hypothesised that reduced fucosylation in guard cell (GC) walls might limit stomatal closure through altering mechanical properties. Multifrequency atomic force microscopy (AFM) measurements revealed a reduced elastic modulus (E’), representing reduced stiffness, in sfr8 GC walls. Interestingly, however, we discovered a compensatory mechanism whereby a concomitant reduction in the storage modulus (E’’) maintained a wild type viscoelastic time response (tau) in sfr8. Stomata in intact leaf discs of sfr8 responded normally to a closure stimulus, ABA, suggesting the time response may relate more to closure properties than stiffness does. sfr8 stomatal pore complexes were larger than wild type and GCs lacked a fully developed cuticular ledge, both potential contributors to the greater leaf water loss in sfr8. We present data that indicate fucosylation-dependent dimerisation of the CW pectic domain rhamnogalacturonan-II may be essential for normal cuticular ledge development and leaf water retention.

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Cell lipid membranes are the primary site of irreversible injury during freezing/thawing and cryopreservation of cells, but the underlying causes remain unknown. Here, we probe the effect of cooling from 20 °C to 0 °C on the structure and mechanical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using atomic force microscopy (AFM) imaging and AFM-based nanoindentation in a liquid environment. The Young’s modulus of elasticity (E) at each temperature for DPPC was obtained at different ionic strengths. Both at 20 mM and 150 mM NaCl, E of DPPC bilayers increases exponentially –as expected–as the temperature is lowered between 20 °C and 5 °C, but at 0 °C E drops from the values measured at 5 °C. Our results support the hypothesis that mechanical weakening of the bilayer at 0 °C  is produced by  structural changes at the lipid-fluid interface.

AbstractThe shapes of living organisms are formed and maintained by precise control in time and space of growth, which is achieved by dynamically fine-tuning the mechanical (viscous and elastic) properties of their hierarchically built structures from the nanometer up. Most organisms on Earth including plants grow by yield (under pressure) of cell walls (bio-polymeric matrices equivalent to extracellular matrix in animal tissues) whose underlying nanoscale viscoelastic properties remain unknown. Multifrequency atomic force microscopy (AFM) techniques exist that are able to map properties to a small subgroup of linear viscoelastic materials (those obeying the Kelvin-Voigt model), but are not applicable to growing materials, and hence are of limited interest to most biological situations. Here, we extend existing dynamic AFM methods to image linear viscoelastic behavior in general, and relaxation times of cells of multicellular organisms in vivo with nanoscale resolution, featuring a simple method to test the validity of the mechanical model used to interpret the data. We use this technique to image cells at the surface of living Arabidopsis thaliana hypocotyls to obtain topographical maps of storage E’ = 120 − 200 MPa and loss E’’= 46 − 111 MPa moduli as well as relaxation times τ = 2.2 − 2.7 µs of their cell walls. Our results demonstrate that cell walls, despite their complex molecular composition, display a striking continuity of simple, linear, viscoelastic behavior across scales–following almost perfectly the standard linear solid model–with characteristic nanometer scale patterns of relaxation times, elasticity and viscosity, whose values correlate linearly with the speed of macroscopic growth. We show that the time-scales probed by dynamic AFM experiments (milliseconds) are key to understand macroscopic scale dynamics (e.g. growth) as predicted by physics of polymer dynamics.