3D Confocal Laser Scanning Microscopy for Quantification of the Phase Behaviour in Agarose-MCC co-gels in Comparison to
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ORIGINAL ARTICLE
3D Confocal Laser Scanning Microscopy for Quantification of the Phase Behaviour in Agarose-MCC co-gels in Comparison to the Rheological Blending-law Analysis Pranita Mhaske 1 & Asgar Farahnaky 1 & Stefan Kasapis 1 Received: 3 September 2020 / Accepted: 5 October 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract The need for a rapid and direct alternative to the rheology-based blending laws in quantifying phase behaviour in biopolymer composite gels is explored in this study. In doing so, the efficacy of confocal laser scanning microscopy (CLSM) paired with image analysis software – FIJI and Imaris - in quantifying phase volume was studied. That was carried out in a model system of agarose with varying concentrations of microcrystalline cellulose (MCC) in comparison to the rheological blending laws. Structural studies performed using SEM, FTIR, differential scanning calorimetry and dynamic oscillation in-shear unveiled a continuous, weak agarose network supporting the hard, rod-shaped MCC inclusions where the composite gel strength increased with higher ‘filler’ concentration. The phase volumes of MCC, estimated with the microscopic protocol, matched the predictions obtained from computerized modelling using the Lewis-Nielsen blending laws. Results highlight the suitability of the microscopic protocol in estimating the water partition and effective phase volumes in the agarose-MCC composite gel. Keywords Phase behaviour . Blending law analysis . 3D imaging . Confocal laser scanning microscopy . Image analysis
Introduction The recent years have seen a drastic increase in the use of proteins and polysaccharides in food product formulations to improve/control processability, shelf life, texture/mouthfeel and the kinetics of bioactive compound release [1]. For the most part, such techno-functionality is governed by the solvent partitioning amongst the macromolecules in the phaseseparated mixture [2]. Fundamental understanding of solvent distribution and molecular interactions between constituents depends on their concentration, water addition, ionic strength, pH and processing, for example, thermal treatment or applied shear. Despite the range and depth of research in the literature, a thorough understanding of phase behaviour and interactions in complex biomaterial systems remains of great interest. These systems generally comprise highly viscous and gelled phases, whose phase behaviour cannot be accurately predicted
* Stefan Kasapis [email protected] 1
School of Science, RMIT University, Bundoora West Campus, Plenty Road, Melbourne, VIC 3083, Australia
using the classical methods of centrifugal separation or determination of osmotic pressure from composite solutions [3, 4]. Other attempts to probe phase behaviour of composite systems employed turbidimetric [5], spectrometric [6] and rheological protocols. The latter, in particular, paired with blending law analysis estimated accurately the phase behaviour of synthetic and natural polymer mixtures [7, 8].
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