Eclipse t1 inverted optical microscope

Whole alginate gels were mounted on glass slides and hydrated, then nano-indented with a spherically tipped cantilever (nominal radius 5 µm, spring constant 1 Nm⁻¹, Windsor Scientific Ltd, Slough, UK) fitted to a Bioscope Catalyst AFM (Bruker, Coventry, UK) mounted on an Eclipse T1 inverted optical microscope (Nikon, Kingston, UK). Gels were indented 25 times over a 50 µm×50 µm area, with contact points evenly distributed across the area. Each gel was indented in 3 regions, and 3 gels were used per group. Force curves were analysed using Nanoscope Analysis v1.40 (Bruker). Curves were fit with a baseline correction before a force fit was applied to a Herzian (spherical) model with a maximum force fit of 70%. Contact-based values for reduced moduli were analysed using a Mann–Whitney U-test.

Samples were rehydrated in distilled water and left to adhere to a charged glass slide overnight at room temperature. They were then rehydrated in distilled water for micromechanical assessment by atomic force microscopy (AFM) indentation using a Bioscope Catalyst AFM (Bruker, Billerica, MA, USA) mounted onto an Eclipse T1 inverted optical microscope (Nikon, Tokyo, Japan) fitted with a spherically tipped cantilever (nominal radius and spring constant of 1 μm and 3 N/m, respectively; Windsor Scientific, Slough, United Kingdom) running Nanoscope Software 8.15 (Bruker). The local reduced modulus was determined for each of 25 points in a 50-µm × 50-µm region, indented at a frequency of 1 Hz with lateral spacing of 2 µm. The approach force curve was used to calculate the reduced modulus using the Hertzian contact mechanics model and a 70% force fit.