Quantification using rheological blending-law analysis and verification with 3D confocal laser scanning microscopy of the phase behaviour in agarose-gelatin co-gels

Abstract

The efficacy of commercially available image analysis software was evaluated in the quantification of 3D images captured with confocal laser scanning microscopy (CLSM). The focus was on the phase behaviour of a mixed system of agarose and gelatin that was allowed to gel by controlled cooling to refrigeration temperature. Microstructural characteristics of the binary system were first probed with FTIR and microDSC to document the development of phase separated networks. Rheological experiments in the form of small-deformation dynamic oscillation in-shear were then employed to monitor the formation of a gelatin continuous network throughout the range of experimental concentrations tested. Agarose, on the other hand, formed discontinuous inclusions at low concentrations that inverted to another continuous phase, alongside that of gelatin, at the high end of polysaccharide addition in the mixture. Blending law analysis was able to model the phase behaviour of these preparations in relation to the mechanical strength of each polymer phase and its water holding capacity. Depending on the principle of operation, image analysis software sampled phase volumes in the co-gel that were either a close match to the predictions of blending laws or yielded statistically different values. Imaging software outcomes are discussed with a view to developing an alternative tool for estimating phase morphology in composite biopolymer gels. • Blending law can follow the phase behaviour of agarose-gelatin composite gels. • 3D CLSM imaging paired with image analysis generates a Z-stack of the composite. • Volumetric quantification of agarose-gelatin phase volumes matched the predictions of blending law. • A rapid microscopy protocol can replace blending law in determining composite morphology.