Characterization of the hierarchical architecture and micromechanical properties of walnut shell (Juglans regia L.).

In the present work a comprehensive characterization of the hierarchical architecture of the walnut shell (Juglans regia L.) was carried out using scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). Furthermore, micromechanical properties (hardness, HIT and elastic modulus, EIT) of plant tissues were evaluated at cell wall level by applying the instrumented indentation technique (IIT). The complex architecture of the material was described in terms of four hierarchical levels (HL): endocarp (H1), plant tissues (H2), plant cells (H3) and cell wall (H4). Our findings revealed that the walnut shell consists of a multilayer structure (sclerenchyma tissue, ST; interface tissue, IT; porous tissue, PT; and flattened parenchyma tissue, FPT), where differences in the microstructure and composition of plant tissues generate parallel gradients along the cross-section. The indentation tests showed a functional gradient with a sandwich-like configuration, i.e., a lightweight and soft layer (PT, HIT = 0.04 GPa) is located between two dense and hard layers (ST, HIT = 0.33 GPa; FPT, HIT = 0.28 GPa); where additionally there is an interface between ST and PT (IT, HIT = 0.16 GPa). This configuration is a successful strategy designed by nature to improve the protection of the kernel by increasing the strength of the shell. Therefore, the walnut shell can be considered as a functionally graded material (FGM), which can be used as bioinspiration for the design of new functional synthetic materials. In addition, we proposed some structure-property-function relationships in the whole walnut shell and in each of the plant tissues.

Effect of pepper extracts on the viability kinetics, topography and Quantitative NanoMechanics (QNM) of Campylobacter jejuni evaluated with AFM.

Campylobacter jejuni is a pathogen bacterium that causes foodborne gastroenteritis in humans. However, phenolic compounds extracted from natural sources such as capsicum pepper plant (Capsicum annuum L. var. aviculare) could inhibit the growth of C. jejuni. Therefore, different extracts were prepared using ultrasonic extraction (USE), conventional extraction (CE) and thermosonic extraction (TSE). C. jejuni was then exposed to chili extracts to examine the antimicrobial effect and their growth/death bacterial kinetics were studied using different mathematical models. Atomic force microscopy was applied to investigate the microstructural and nanomechanical changes in the bacteria. Extracts obtained by TSE had the highest phenolic content (4.59 ± 0.03 mg/g of chili fresh weight [FW]) in comparison to USE (4.12 ± 0.05 mg/g of chili FW) and CE (4.28 ± 0.07 mg/g of chili FW). The inactivation of C. jejuni was more efficient when thermosonic extract was used. The Gompertz model was the most suitable mathematical model to describe the inactivation kinetics of C. jejuni. Roughness and nanomechanical analysis performed by atomic force microscopy (AFM) provided evidence that the chili extracts had significant effects on morphology, surface, and the reduced Young's modulus of C. jejuni. The novelty of this work was integrating growth/death bacterial kinetics of C. jejuni using different mathematical models and chili extracts, and its relationship with the morphological, topographic and nanomechanical changes estimated by AFM.

Effect of calcium oxalate crystals on the micromechanical properties of sclerenchyma tissue from the pecan nutshell (Carya illinoinensis).

The objective of this study was to evaluate the effect of the presence of calcium oxalate (CaOx) crystals on the micromechanical properties of sclerenchyma tissue from the pecan nutshell (Carya illinoinensis). The microstructure of the cross-section nutshell was examined using light microscopy (LM) and atomic force microscopy (AFM). Using an instrumented indentation system, indentation tests with maximum loads of 500 mN were made on the biological material where the variables studied were the number of crystals present in the evaluated area and the size of individual crystals. Microscopic analysis revealed that the pecan nutshell consists of sclerenchyma tissue with multiple CaOx crystals randomly distributed throughout the material, exhibiting prismatic shapes and various sizes. The results of the indentation tests showed that the examined areas where there were crystals (1, 2 or up to 3) presented values of hardness and elastic modulus significantly higher (P < 0.05) compared to the sclerenchyma (without crystals). Likewise, there were no significant differences (P > 0.05) between the values of the micromechanical properties of the areas evaluated as a function of the number of crystals. On the other hand, it was observed that the size of the crystals did not show a direct correlation with the mechanical properties evaluated as expected. In conclusion, the biomineralization phenomenon is a successful strategy designed by nature to improve the rigidity of the pecan nutshell, where the CaOx crystals strengthen the structure by increasing the micromechanical properties.

Evaluation of Nanomechanical Properties of Tomato Root by Atomic Force Microscopy.

Here, different tissue surfaces of tomato root were characterized employing atomic force microscopy on day 7 and day 21 of growth through Young's modulus and plasticity index. These parameters provide quantitative information regarding the mechanical behavior of the tomato root under fresh conditions in different locations of the cross-section of root [cell surface of the epidermis, parenchyma (Pa), and vascular bundles (Vb)]. The results show that the mechanical parameters depend on the indented region, tissue type, and growth time. Thereby, the stiffness increases in the cell surface of epidermal tissue with increasing growth time (from 9.19 ± 0.68 to 13.90 ± 1.68 MPa) and the cell surface of Pa tissue displays the opposite behavior (from 1.74 ± 0.49 to 0.48 ± 0.55); the stiffness of cell surfaces of Vb tissue changes from 10.60 ± 0.58 to 6.37 ± 0.53 MPa, all cases showed a statistical difference (p < 0.05). Viscoelastic behavior dominates the mechanical forces in the tomato root. The current study is a contribution to a better understanding of the cell mechanics behavior of different tomato root tissues during growth.

Study of the porosity of calcified chicken eggshell using atomic force microscopy and image processing.

In this work, the porosity of the layers of calcified chicken eggshell (vertical crystal layer VCL, palisade layer PL and mammillary layer ML) was evaluated using atomic force microscopy (AFM) and image processing (IP). AFM topographic images were obtained from different locations for each layer and along the cross-section of calcified eggshell. Roughness parameters, surface area values, pore size and shape, surface porosity, area occupied by pores and pore density were obtained from AFM and IP. It was observed that the thickest layer (PL) exhibited the highest degree of porosity (surface porosity = 2.75 ± 1.68%, pore density = 162 ± 60 pores/µm2) when compared to the other two layers. In general, the pores located in all layers ("bubble pores") had circular shape and similar sizes. Measurements revealed a porosity gradient along the cross-section which varied with position, i.e., increasing surface porosity from the VCL towards the region of the PL closer to the ML, and decreasing surface porosity from this location towards the ML innermost surface. This suggests that the calcified eggshell has a sandwich-like structure where porosity may influence gas exchange and mechanical properties. The combination of AFM with IP presented here provides a simple and precise method to study porosity in calcified chicken eggshell, and this methodology could be used to examine other types of porous biological materials.

Morphological and micromechanical characterization of calcium oxalate (CaOx) crystals embedded in the pecan nutshell (Carya illinoinensis).

The morphology and micromechanical properties of the mineral crystals embedded in the pecan nutshell (Carya illinoinensis) were characterized. Qualitative and quantitative morphological analyses carried out revealed that the crystals were comprised of calcium oxalate (CaOx) and have a wide range of sizes, with prismatic shapes, distributed heterogeneously in the sclerenchyma tissue. From indentation tests, it was found that CaOx crystals are stiffer structures compared to stone cells (sclerenchyma tissue), showing hardness and elastic modulus values of 0.53 ±â€¯0.19 GPa and 9.4 ±â€¯1.80 GPa, respectively. Additionally, the values of fracture toughness (0.08 ±â€¯0.02 MPa m0.5) and the brittleness index (9336 m-0.5) revealed that these types of structures are extremely brittle. The results obtained suggest that the main function of the CaOx crystals is to provide structural support to tissue. The presented methodology demonstrates the potential of the instrumented indentation technique (IIT) for in situ micromechanical characterization of mineral crystals located in plant tissues.

Numerical study of the hydrodynamic drag force in atomic force microscopy measurements undertaken in fluids.

When atomic force microscopy (AFM) is employed for in vivo study of immersed biological samples, the fluid medium presents additional complexities, not least of which is the hydrodynamic drag force due to viscous friction of the cantilever with the liquid. This force should be considered when interpreting experimental results and any calculated material properties. In this paper, a numerical model is presented to study the influence of the drag force on experimental data obtained from AFM measurements using computational fluid dynamics (CFD) simulation. The model provides quantification of the drag force in AFM measurements of soft specimens in fluids. The numerical predictions were compared with experimental data obtained using AFM with a V-shaped cantilever fitted with a pyramidal tip. Tip velocities ranging from 1.05 to 105 µm/s were employed in water, polyethylene glycol and glycerol with the platform approaching from a distance of 6000 nm. The model was also compared with an existing analytical model. Good agreement was observed between numerical results, experiments and analytical predictions. Accurate predictions were obtained without the need for extrapolation of experimental data. In addition, the model can be employed over the range of tip geometries and velocities typically utilized in AFM measurements.

Physical and structural characterisation of zein and chitosan edible films using nanotechnology tools.

The use of composite edible films made from biopolymers has attracted interest as a way to reduce pollution and recycling problems; however, the relation between barrier, mechanical and structural properties of the films have been scarcely studied. The aim of this work was to evaluate composite zein-chitosan edible films by applying common nanotechnology tools and to relate the results to zein concentration and film structural changes. Rougher, more elastic, and less hard film structures with better water vapour barrier properties were obtained using larger zein concentrations. Raman spectroscopy exhibited unexpected interactions, as indicated by the disappearance of the thiol groups of cysteine in the zein films and the appearance of O=S=O and C-O-S groups in the blended materials in conjunction with the disappearance of zein ε-amino and -NH2 functional groups in the zein film samples, thereby confirming changes in the blended film structure. Zein concentration presented linear correlations with water vapour permeability (R=-0.978) and film roughness (R=0.929). The composite films presented better barrier and mechanical properties than single ingredient films. This information shows the benefit of using protein-polysaccharide blends to prepare edible films.