Use of Z-stack imaging to quantify the phase behaviour of biomaterial composites in relation to theoretical predictions of blending laws from rheological measurements
Creators: name: MHASKE, Pranita
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300602
subject: Food chemistry and food sensory science

description: The importance of estimating phase separation in protein and polysaccharide gels in order to obtain desired structural properties and textural profile in food product formulations remains paramount. Due to thermodynamic imbalance, biopolymer mixtures segregate into distinct phases, with one phase acting as a continuous matrix in which the second phase remains dispersed as a discontinuous 'filler'. Studies up till now show that theoretical models relate the elastic modulus of the biopolymer phases to the topology of their mixtures and can be successfully employed for an indirect estimation of the solvent partition between the biopolymers.  Theoretical modelling, though robust, is a time consuming and tedious method of estimating phase volume of phase separated polymers. Acknowledging this, a need for developing a rapid, efficient, and accurate method of determining solvent partition between the biopolymers is identified.
Therefore, this PhD study aims at exploiting the accelerated advancement in image processing technology and developing a novel Confocal Laser Scanning Microscopy (CLSM)-based approach using Z-stack imaging and image analysis to quantify phase behaviour of biopolymer composites. This is achieved by employing numerous analytical and physicochemical techniques such as scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, rheology based theoretical modelling and confocal laser scanning microscopy.

The first experimental chapter focused mainly on developing a protocol for obtaining 3D images of a simple model system of agarose and canola oil and quantifying the lipid phase volume using two image analysis software, FIJI and Imaris in parallel. For comparison, rheology based- theoretical blending laws were employed. The phase behaviour of composite gels made of agarose and various concentrations of canola oil were examined using a variety of techniques including SEM, FTIR, DSC, dynamic oscillation, polymer blending laws and CLSM-based Z-stack imaging coupled with image analysis. Microscopic, spectroscopic, and thermomechanical observations recorded continuous agarose networks supporting discontinuous canola oil inclusions with increasing levels of canola oil reinforcing the rigidity of the continuous phase. The outcome of the microscopic protocol is in close agreement with the oil phase volume predictions from the isostrain blending law indicating the suitability of the developed protocol in quantifying the phase behaviour of composite gels. 

The second experimental chapter probed the phase behaviour of a model system comprising agarose and a varying concentration of a hard lipid, ghee. Results obtained from SEM, microDSC, FTIR and dynamic oscillation in-shear revealed discontinuous and hard inclusions of ghee reinforcing the continuous, weaker agarose matrix with increasing concentrations of the former. Phase behaviour of the system was quantified in parallel with a novel method combining 3D CLSM imaging and image analysis software - FIJI and Imaris - in an effort to substantiate the efficacy of the microscopic protocol in quantifying phase behaviour. Phase volumes recorded with the microscopic protocol were in close agreement to those modelled with the isostress blending law using small-deformation dynamic oscillation. However, results indicated that the inner filtering effect or `self-shadowing' observed commonly in CLSM images due to the diffraction of laser as it passes through components with different densities and refractive indices may pose a limitation to the application of this technique, necessitating further development before it can be applied to more complex, industrially relevant systems.

This merited further consideration of the suitability of the developed microscopic protocol in estimating solvent partition in an industrially relevant hydrogel. In doing so, the efficacy of confocal laser scanning microscopy (CLSM) paired with image analysis software - FIJI and Imaris - in quantifying phase volume in a model system of agarose with varying concentrations of microcrystalline cellulose (MCC) in comparison to the rheological blending laws was probed. 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 highlighted the suitability of the microscopic protocol in estimating the water partition and effective phase volumes in the agarose-MCC composite gel. 

These were encouraging outcomes which lead us to estimate phase behaviour in a mixed gelling system of a polysaccharide (agarose) and a protein (gelatin) in comparison with the rheology based blending laws. Structural properties of the composites were probed using FTIR spectroscopy, microDSC and small-deformation dynamic oscillation in shear. Throughout the experimental range of concentrations, gelatin formed a continuous network whereas the agarose phase remained dispersed as either a soft or a hard filler at low agarose concentrations (0.1-0.8%) and formed a continuous network alongside that of gelatin at higher concentrations (1.2-3%). The phase volumes of gelatin, recorded using Imaris, were a close match with those obtained from the blending law predictions, whereas FIJI yielded statistically different estimates. Results suggest that the microscopic protocol using Imaris shows promise and can potentially be used as an alternative to the theoretical blending laws in estimating phase behaviour in biomaterial composites.
Work outlined in this thesis thus lays the groundwork for future research, where it can be used to determine phase behaviour accurately and rapidly in complex binary and tertiary gelling biomaterial composites and perhaps to materials in the nanoscale.