Individual Cellulose Nanocrystals Research Articles

Study on the interactions and interfacial behaviors of cellulose nanocrystal/β-lactoglobulin complexes for pickering emulsions

Pickering emulsions stabilized by polysaccharides and protein complexes have attracted increasing attention. Exploring their interfacial activities and adsorption behaviors at the oil–water interface is of great significance for deepening our understanding of the emulsification mechanism and improving the properties of polysaccharide/protein-stabilized emulsions. Here, cellulose nanocrystals (CNCs) with rod-like structures and spherical β-lactoglobulin (β-LG) proteins were selected as representative stabilizers. We initially investigated the interaction between the 2 at pH 2.0–7.0 through a multiscale approach. Then, oil–water interfacial tension tests were used to assess the interfacial activities of CNCs and β-LG, revealing that β-LG exerted primary influence over the interfacial behavior in their complexes. Meanwhile, quartz crystal microbalance with dissipation was used to monitor the interfacial adsorption behaviors of CNCs, β-LG, and CNC/β-LG complexes, demonstrating that the interfacial layer formed by the CNC/β-LG complexes on the oil film (∼696.7 ng/cm2) was thicker and more stable compared with that formed by individual CNCs (∼48.4 ng/cm2) or β-LG (∼87.1 ng/cm2). Finally, we applied the CNC/β-LG complexes as stabilizers in Pickering emulsions and the emulsions prepared with the complexes at concentrations above 0.8 wt% exhibited storage stability for up to 28 d which confirmed their exceptional emulsifying ability and practical value. This study provides profound insights into the interfacial behavior of polysaccharide–protein complexes in Pickering emulsions and is expected to facilitate the design of novel food-grade emulsifiers.

Supramolecular Materials Chemistry in the MacLachlan Group

AbstractThis invited Team Profile was created by the MacLachlan group at the University of British Columbia. They recently published an article on the uniform growth of a metal–organic framework, ZIF‐8, on the surface of individual cellulose nanocrystals (CNCs). After the ZIF‐8/CNC fibers had been coated with a microporous organic polymer, the ZIF‐8 was removed to leave a microporous polymer with CNCs encapsulated in a ship‐in‐a‐bottle architecture. This material proved to be effective for CO2 fixation. “Uniform Growth of Nanocrystalline ZIF‐8 on Cellulose Nanocrystals: Useful Template for Microporous Organic Polymers”, K. Cho, L. J. Andrew, M. J. MacLachlan, Angew. Chem. Int. Ed. 2023, e202300960.

Supramolecular Materials Chemistry in the MacLachlan Group.

This invited Team Profile was created by the MacLachlan group at the University of British Columbia. They recently published an article on the uniform growth of a metal-organic framework, ZIF-8, on the surface of individual cellulose nanocrystals (CNCs). After the ZIF-8/CNC fibers had been coated with a microporous organic polymer, the ZIF-8 was removed to leave a microporous polymer with CNCs encapsulated in a ship-in-a-bottle architecture. This material proved to be effective for CO2 fixation. "Uniform Growth of Nanocrystalline ZIF-8 on Cellulose Nanocrystals: Useful Template for Microporous Organic Polymers", K. Cho, L. J. Andrew, M. J. MacLachlan, Angew. Chem. Int. Ed. 2023, e202300960.

Preparation and characterization of cellulose nanocrystals from paper mulberry fibers

The applicability of paper mulberry fiber (PM-FB), which is bast fiber, for manufacturing cellulose nanocrystals (CNCs) with high-yield was investigated. The PM-FB and hardwood bleached kraft pulp (Hw-BKP) were hydrolyzed under different sulfuric acid concentrations, reaction times, and reaction temperatures. The dependence of the CNC yield on the hydrolysis conditions and crystallinity of the raw materials was analyzed. The functional groups of the CNCs and their zeta potentials were determined. The fiber length and width of the individual CNCs were determined by transmission electron microscopy image analysis. The PM-FB showed a higher crystallinity of 86.8% compared to that of Hw-BKP and exhibited a high CNC yield of 55% under strong hydrolysis conditions. The sulfate group was introduced into the CNCs, which increased their negative charge. The fiber width of PM-FB-based CNCs (PM-CNCs) was larger than that of Hw-BKP-based CNCs (Hw-CNCs), and the aspect ratio ranged from 29.0 to 12.2 depending on the hydrolysis conditions. The yield of PM-CNCs was higher than that of Hw-CNCs under the same hydrolysis conditions. In addition, both CNCs exhibited similar quality. Therefore, PM-FB is a promising raw material for efficient CNC manufacturing.

3D Printing-Assisted Self-Assembly to Bio-Inspired Bouligand Nanostructures.

Architected materials with nano/microscale orders can provide superior mechanical properties; however, reproducing such levels of ordering in complex structures has remained challenging. Inspired by Bouligand structures in nature, here, 3D printing of complex geometries with guided long-order radially twisted chiral hierarchy, using cellulose nanocrystals (CNC)-based inks is presented. Detailed rheological measurements, in situ flow analysis, polarized optical microscopy (POM), and director field analysis are employed to evaluate the chiral assembly over the printing process. It is demonstrated that shear flow forces inside the 3D printer's nozzle orient individual CNC particles forming a pseudo-nematic phase that relaxes to uniformly aligned concentric chiral nematic structures after the flow cessation. Acrylamide, a photo-curable monomer, is incorporated to arrest the concentric chiral arrangements within the printed filaments. The time series POM snapshots show that adding the photo-curable monomer at the optimized concentrations does not interfere with chiral self-assemblies and instead increases the chiral relaxation rate. Due to the liquid-like nature of the as-printed inks, optimized Carbopol microgels are used to support printed filaments before photo-polymerization. By paving the path towards developing bio-inspired materials with nanoscale hierarchies in larger-scale printed constructs, this biomimetic approach expands 3D printing materials beyond what has been realized so far.

Elucidating the fine-scale structural morphology of nanocellulose by nano infrared spectroscopy

Nanoscale infrared (IR) spectroscopy and microscopy, enabling the acquisition of IR spectra and images with a lateral resolution of 20 nm, is employed to chemically characterize individual cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) to elucidate if the CNCs and CNFs consist of alternating crystalline and amorphous domains along the CNF/CNC. The high lateral resolution enables studies of the nanoscale morphology at different domains of the CNFs/CNCs: flat segments, kinks, twisted areas, and end points. The types of nanocellulose investigated are CNFs from tunicate, CNCs from cotton, and anionic and cationic wood-derived CNFs. All nano-FTIR spectra acquired from the different samples and different domains of the individual nanocellulose particles resemble a spectrum of crystalline cellulose, suggesting that the non-crystalline cellulose signal observed in macroscopic measurements of nanocellulose most likely originate from cellulose chains present at the surface of the nanocellulose particles.

Molecular insights on the crystalline cellulose-water interfaces via three-dimensional atomic force microscopy.

Cellulose, a renewable structural biopolymer, is ubiquitous in nature and is the basic reinforcement component of the natural hierarchical structures of living plants, bacteria, and tunicates. However, a detailed picture of the crystalline cellulose surface at the molecular level is still unavailable. Here, using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we revealed the molecular details of the cellulose chain arrangements on the surfaces of individual cellulose nanocrystals (CNCs) in water. Furthermore, we visualized the three-dimensional (3D) local arrangement of water molecules near the CNC surface using 3D AFM. AFM experiments and MD simulations showed anisotropic water structuring, as determined by the surface topologies and exposed chemical moieties. These findings provide important insights into our understanding of the interfacial interactions between CNCs and water at the molecular level. This may allow the establishment of the structure-property relationship of CNCs extracted from various biomass sources.

Capillary Flow Characterizations of Chiral Nematic Cellulose Nanocrystal Suspensions.

Studying the flow-induced alignment of anisotropic liquid crystalline materials is of major importance in the 3D printing of advanced architectures. However, in situ characterization and quantitative measurements of local orientations during the 3D printing process are challenging. Here, we report a microfluidic strategy integrated with polarized optical microscopy (POM) to perform the in situ characterization of the alignment of cellulose nanocrystals (CNCs) under the shear-flow condition of the 3D printer's nozzle in the direct ink writing process. To quantify the alignment, we exploited birefringence measurements under white and monochromatic light. We show that the flow-induced birefringence patterns are significantly influenced by the initial structure of the aqueous CNC suspensions. Depending on the CNC concentration and sonication treatment, various structures can form in the CNC suspensions, such as isotropic, chiral nematic (cholesteric), and nematic (gel-like) structures. In the chiral nematic phase, in particular, the shear flow in the microfluidic capillary has a distinct effect on the alignment of the CNC particles. Our experimental results, complemented by hydrodynamic simulations, reveal that at high flow rates (Er ≈ 1000), individual CNC particles align with the flow exhibiting a weak chiral structure. In contrast, at lower flow rates (Er ≈ 241), they display the double-twisted cylinder structure. Understanding the flow effect on the alignment of the chiral liquid crystal can pave the way to designing 3D printed architectures with internal chirality for advanced mechanical and smart photonic applications.

Thixotropy of cellulose nanocrystal suspensions

The thixotropy of cellulose nanocrystal (CNC) water suspensions is intrinsically dependent on the hierarchical structure of the suspension. The diverse hierarchies that comprise individual CNC nanoparticles and mesophase liquid crystalline domains, chiral nematic and nematic structures, contribute selectively to the rheological material response. Here, we combine rheology with polarized light imaging (PLI) to elucidate the thixotropic behavior of CNCs suspended in water. The simultaneous monitoring of PLI and rheological tests enables the observation of mesogens and their orientation dynamics. Creep, dynamic time sweep, ramped hysteresis loop, and thixotropic recovery tests combined with PLI aim to differentiate the contribution of the different hierarchical levels of CNC suspensions to their thixotropy. The range of concentrations investigated comprised biphasic (4 and 5 wt. %) and liquid crystalline phase suspensions (6, 7, and 8 wt. %). The CNC suspensions exhibited complex thixotropy behavior, such as viscosity bifurcations in creep tests and overshoot in ramped hysteresis loop tests. The restructuring and destructuring appeared to correspond to different levels of their hierarchical structure, depending mainly on the phase, in agreement with previous studies. Restructuring was attributed to re-organizations of an individual CNC, e.g., in the isotropic fraction of biphasic suspensions and at the mesogen interfaces in liquid crystalline phase suspensions. However, by increasing liquid crystalline fraction in the biphasic concentrations, restructuring could also involve mesogens, as indicated in the creep tests. For flow conditions above the yield stress, as evidenced by the ramped hysteresis and thixotropy recovery tests, destructuring was dominated by orientation in the flow direction, a process that is readily observable in the form of PLI “Maltese-cross” patterns. Finally, we show that a simple thixotropy model, while unable to capture the finer details of the suspension’s thixotropic behavior, could be employed to predict general features thereof.

Viscoelastic properties of poly (vinyl alcohol) hydrogels with cellulose nanocrystals fabricated through sodium chloride addition: Rheological evidence of double network formation

This report investigates viscoelastic and mechanical behavior of hybrid polyvinyl alcohol (PVA)-cellulose nanocrystals (CNC) hydrogel aggregated with sodium chloride (NaCl) salt. In almost all samples, wide distribution of pores with roughly equal average pore size was observed, denoting that the average porosity is independent from CNC concentration. To assess the dispersion quality of CNCs in CNC-PVA/salt hybrid samples, transmission electron microscopy (TEM) was employed. Small and large amplitude oscillatory shear (SAOS and LAOS) measurements were also performed on CNC-loaded PVA-salt hydrogels to further characterize their microstructure. At CNC loadings near the percolation threshold (15 g/L), a sudden jump in storage modulus was observed, attributing to hybrid polymer-CNC 3D network formation. The appearance of a more dramatic jump in the value of the rheological parameters at higher CNC concentrations (30 g/L) indicates the establishment of a secondary network structure upon formation of direct contacts between individual CNC particles and/or clusters. The viscoelastic behavior of the hydrogels was further investigated via LAOS approach based on Lissajous-Bowditch plots and sequence of physical processes method. In addition, through a wide range of rheological characterizations, we demonstrated successful fabrication of a healable hydrogel, able to regain original strength after disruption of the structure at sufficiently large deformations. Afterward, to explore the interactions between CNC-CNC and PVA-CNC, the Foglar-Tucker model, designed to account for modeling the orientation of particles within a matrix, was fitted on stress-overshoot (start-up) experiments. The present study on PVA-CNC hydrogels opens avenues for further developing advanced materials with tunable microstructures for tissue engineering and regenerative medicine applications.

Particle Size Distributions for Cellulose Nanocrystals Measured by Transmission Electron Microscopy: An Interlaboratory Comparison.

Particle size is a key parameter that must be measured to ensure reproducible production of cellulose nanocrystals (CNCs) and to achieve reliable performance metrics for specific CNC applications. Nevertheless, size measurements for CNCs are challenging due to their broad size distribution, irregular rod-shaped particles, and propensity to aggregate and agglomerate. We report an interlaboratory comparison (ILC) that tests transmission electron microscopy (TEM) protocols for image acquisition and analysis. Samples of CNCs were prepared on TEM grids in a single laboratory, and detailed data acquisition and analysis protocols were provided to participants. CNCs were imaged and the size of individual particles was analyzed in 10 participating laboratories that represent a cross section of academic, industrial, and government laboratories with varying levels of experience with imaging CNCs. The data for each laboratory were fit to a skew normal distribution that accommodates the variability in central location and distribution width and asymmetries for the various datasets. Consensus values were obtained by modeling the variation between laboratories using a skew normal distribution. This approach gave consensus distributions with values for mean, standard deviation, and shape factor of 95.8, 38.2, and 6.3 nm for length and 7.7, 2.2, and 2.9 nm for width, respectively. Comparison of the degree of overlap between distributions for individual laboratories indicates that differences in imaging resolution contribute to the variation in measured widths. We conclude that the selection of individual CNCs for analysis and the variability in CNC agglomeration and staining are the main factors that lead to variations in measured length and width between laboratories.

A reproducible method to characterize the bulk morphology of cellulose nanocrystals and nanofibers by transmission electron microscopy

With the increased use of nanocelluloses as additives in many industrial applications, better characterization methods are needed to ensure their effectiveness. Transmission electron microscopy (TEM) is an appropriate image acquisition system to enable their morphological characterization. The use of TEM has typically been focused on determining the diameter and length of individual cellulose nanocrystals (CNCs) or nanofibers (CNFs), so different dispersion practices, such as sonication or the use of dispersants, are commonly applied to separate the particles. However, this study aims to improve the characterization of the bulk morphology of CNCs and CNFs by TEM, taking steps towards the determination of the aggregation/dispersion degree as well as the fibrillation degree of CNFs. TEM has been investigated with two types of grids (holey and Formvar/carbon), three different fixing methods (Poly-l-Lysine, glow discharge and UV radiation) and the use of negative staining. Fractal dimension and lacunarity were used to quantify the reproducibility of the improved method. With the use of Poly-l-Lysine, the attachment of CNCs and CNFs particles on the TEM grids was ensured, due to the electrostatic interactions between negatively charged nanocelluloses and positively charged and hydrophilic Poly-l-Lysine surfaces. The low value of lacunarity, close to 0.3, shows a very high reproducibility of the methodology proposed. With this new approach, the state that the nanocelluloses have in suspension can be directly characterized by TEM.

Linking Film Structure and Mechanical Properties in Nanocomposite Films Formed from Dispersions of Cellulose Nanocrystals and Acrylic Latexes.

Cellulose nanocrystals (CNCs) are unique, lightweight materials that possess high elastic modulus and tensile strength, making them of great interest in the formation of nanocomposite materials. However, efficient design of the composite material is essential in translating the mechanical properties of the individual CNCs into the nanocomposite film. In this work, we demonstrate the formation of structured CNC/acrylic dispersions by physical blending of the anionic CNCs with charged acrylic latex particles. By blending with large cationic latex particles, the CNCs adsorbed onto the acrylic latex surface while blending with small latex particles led to the inverse structure. Films were cast from these dispersions and the physical properties were compared with the aim of understanding the influence of the initial structure of the hybrid dispersion on the structure of the final film. A significant difference in the mechanical properties was observed based on the position of the CNCs in the initial dispersion. Adsorption of latex particles onto the CNC surface led to a random distribution of nonconnected CNCs, which contributed little to improving the Young's modulus, while adsorption of CNC onto the latex led to a honeycomb CNC network and a large increase in the Young's modulus. This work underlines the importance of particle structure on the structure and mechanical properties of nanostructured films.

Effect of Ionic Surfactants on the Viscoelastic Properties of Chiral Nematic Cellulose Nanocrystal Suspensions.

The gelation of cellulose nanocrystal (CNC) suspensions is explored in the presence of two ionic surfactants, namely, sodium dodecyl sulfate (SDS) with a negatively charged head and cetyltrimethylammonium bromide (CTAB) with a positively charged head. The viscosity profile of pure CNC suspensions indicated that at concentrations greater than 5 wt %, they exhibit gel-like behavior, where the viscosity shows a single shear-thinning profile. However, at concentrations lower than 1 wt %, the suspension shows isotropic behavior and experiences a transition to chiral nematic biphasic domains at increasing CNC concentration. In addition, the effect of CTAB, SDS, and NaCl on the surface charge of CNCs is analyzed and coupled with rheological measurements in order to study the relative importance of surfactants and ionic strength on the viscoelastic properties of the CNC suspensions. The mechanism of CNC/surfactants and CNC/CNC interactions leading to the gelation of CNC suspensions is also investigated. It was found that the addition of both ionic surfactants results in the gelation of biphasic chiral nematic CNC suspensions by fostering cross-linking between individual CNCs at smaller concentrations. In the case of CTAB, a gradual and ongoing increase in the viscoelastic moduli was observed with the increase of surfactant concentration, implying the induction of dominant attractive forces between CNCs by surfactant molecules. However, the presence of SDS stimulates both attractive and repulsive forces whose relative dominance controls the viscoelastic properties and gelation.

Characterization of size and aggregation for cellulose nanocrystal dispersions separated by asymmetrical-flow field-flow fractionation

Cellulose nanocrystals (CNCs) derived from various types of cellulose biomass have significant potential for applications that take advantage of their availability from renewable natural resources and their high mechanical strength, biocompatibility and ease of modification. However, their high polydispersity and irregular rod-like shape present challenges for the quantitative dimensional determinations that are required for quality control of CNC production processes. Here we have fractionated a CNC certified reference material using a previously reported asymmetrical-flow field-flow fractionation (AF4) method and characterized selected fractions by atomic force microscopy (AFM) and transmission electron microscopy. This work was aimed at addressing discrepancies in length between fractionated and unfractionated CNC and obtaining less polydisperse samples with fewer aggregates to facilitate microscopy dimensional measurements. The results demonstrate that early fractions obtained from an analytical scale AF4 separation contain predominantly individual CNCs. The number of laterally aggregated "dimers" and clusters containing 3 or more particles increases with increasing fraction number. Size analysis of individual particles by AFM for the early fractions demonstrates that the measured CNC length increases with increasing fraction number, in good agreement with the rod length calculated from the AF4 multi-angle light scattering data. The ability to minimize aggregation and polydispersity for CNC samples has important implications for correlating data from different sizing methods.

Responsive and patterned cellulose nanocrystal films modified by N-methylmorpholine-N-oxide

A type of cellulose solvent, i.e., aqueous N-methylmorpholine- N-O xide (NMMO) solutions, was used to modify cellulose nanocrystal (CNC) photonic films. CNC films can be swollen by NMMO, resulting in red-shifted reflected colors. The swelling effect is supposed to come from NMMO permeation into the crystalline regions of individual CNCs and intercalating in between CNC particles. When NMMO was removed, the reflected colors of CNC films blue shifted because of the reduced helical pitches. NMMO-treated CNC films display reversible responsive colors to humidity changes in several minutes. Increasing NMMO content allows CNC films to enlarge the responsive color range. Aqueous NMMO can be used as an ink to depict responsive photonic patterns on CNC films. This post-treatment approach to producing responsive colors and photonic patterns in CNC films may be applied to the areas of sensor, anti-counterfeiting, and decoration.

Preparation and Characterisation of Cellulose Nanocrystal from Sugarcane Peels by XRD, SEM and CP/MAS 13C NMR

Sugarcane peels are agro-waste resources discarded before taking the sugarcane juice. In the present study, cellulose nanocrystal was isolated from sugarcane peel by sulphuric acid hydrolysis. Two pretreatments, alkaline treatment and bleaching with acidified sodium chlorite, were applied. Sulphuric acid hydrolysis was performed at 450C for 45 min using 64% concentrated sulphuric acid. The resulting cellulose nanocrystal (CNC) of the sugarcane peel was characterised by studying the surface morphology using scanning electron microscope (SEM). X-ray diffraction (XRD) was studied to identify the crystalline nature of the CNC. CP/MAS 13C solid-state NMR was used to evaluate the purity and molecular structure of the CNC. The SEM image of the nanocrystal showed that the bundles of fibre were separated into individual CNC, with the size decreasing to a nanosize indicating an effective removal of the amorphous region. XRD diffraction pattern showed that the CNC possessed the cellulose crystalline configuration with crystallinity index of 99.2% and crystallite particle size dimension of 5.56 nm. The NMR spectra of the CNC revealed that all the signals have six carbon atoms of cellulose and the disappearance of several signals also indicated the disruption of the amorphous region. The results revealed effective synthesis of CNC from sugarcane peel, suggesting the leaching of the amorphous domain, apparent crystallinity and purity of the CNC. The cellulose nanocrystal prepared is considered to be a potent material for various industrial applications.

Effects of interface properties on the mechanical properties of bio-inspired cellulose nanocrystal (CNC)-based materials

Cellulose nanocrystals (CNCs) is a new family of cellulose materials with outstanding mechanical and chemical properties that have been successfully demonstrated to have potential for many applications. Understanding the mechanical behavior of individual CNCs and their interaction at nanoscale is very important for improving the manufacturing process and mechanical performance of the resulting materials at macroscale. However, due to time and length scale limitations for both, experimental observations and atomistic modeling, predicting the mechanical performance of a system consisting of many CNCs is still challenging. We present a coarse-grained model of CNCs based on both mechanical properties and crystal-crystal interactions to overcome this limitation. Parametrization of the model is carried out in comparison with all-atom (AA) molecular dynamics and experimental results of some specific mechanical and interfacial tests. Subsequently, verification is done with other independent tests. In particular, we note that the parameters of the model can be calibrated to capture the properties of both twisted and untwisted CNC interface without sacrificing the mechanical properties of the individual particle. Finally, we analyze the effect of interface properties on the mechanical performance of CNC-based materials including, bending test of a crystalline bundle, tensile test and fracture in two representative bioinspired architectures. The results revealed different failure mechanisms depending upon the interfacial configuration. For instance, well-aligned CNCs lead to a more brittle and catastrophic failure mechanism, whereas naturally twisted interfaces promote toughening mechanisms that helps attained optimal mechanical performance.

Characterization challenges for a cellulose nanocrystal reference material: dispersion and particle size distributions

Cellulose nanocrystals (CNCs) have high aspect ratios, polydisperse size distributions, and a strong propensity for aggregation, all of which make them a challenging material for detailed size and morphology characterization. A CNC reference material produced by sulfuric acid hydrolysis of softwood pulp was characterized using a combination of dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy, and X-ray diffraction. As a starting point, a dispersion protocol using ultrasonication was developed to provide CNC suspensions with reproducible size distributions as assessed by DLS. Tests of various methods for AFM sample preparation demonstrated that spin coating on a positively charged substrate maximizes the number of individual particles for size analysis, while minimizing the presence of agglomerates. The effects of sample-to-sample variability, analyst bias, and sonication on size distributions were assessed by AFM. The latter experiment indicated that dispersion of agglomerates by sonication did not significantly change the size distribution of individual CNCs in suspension. Comparison with TEM data demonstrated that the two microscopy methods provide similar results for CNC length (mean ~ 80 nm); however, the particle width as measured by TEM is approximately twice that of the CNC height (mean 3.5 nm) measured by AFM. The individual crystallite size measured by X-ray diffraction is intermediate between the two values, although closer to the AFM height, possibly indicating that laterally agglomerated CNCs contribute to the TEM width. Overall, this study provides detailed information that can be used to assess the factors that must be considered in measuring CNC size distributions, information that will be useful for benchmarking the performance of different industrially sourced materials.

Effective cellulose nanocrystal imaging using transmission electron microscopy

Characterization of cellulose nanocrystals (CNCs) is often complex and tedious. With their increased use for biological materials, polymer reinforcing agents, and other applications, better characterization methods of CNCs are needed to ensure product quality. However, because of their small size, hydrogen bonding, and low electron density, individual CNCs are difficult to image with high resolution and magnification using electron microscopy. Methods to help counter these challenges include staining for increased contrast and techniques to increase dispersion. This work tested several stains, dispersing agents, and sample supports to find a consistent method of individualizing CNCs and providing good contrast for imaging in transmission electron microscopy (TEM). The most consistent method found uses a low concentration of CNCs, bovine serum albumin as a dispersing agent, and Nanovan® as the contrasting stain on a silicon monoxide-coated Formvar TEM grid.