Direct Visualization of Crystalline Domains in Carboxylated Nanocellulose Fibers.

Direct visualization of soft organic molecules like cellulose is extremely challenging under a high-energy electron beam. Herein, we adopt two ionization damage extenuation strategies to visualize the lattice arrangements of the β-(1→4)-d-glucan chains in carboxylated nanocellulose fibers (C-NCFs) having cellulose II crystalline phase using high-resolution transmission electron microscopy. Direct imaging of individual nanocellulose fibrils with high-resolution and least damage under high-energy electron beam is achieved by employing reduced graphene oxide, a conducting material with high electron transmittance and Ag+ ions, with high electron density, eliminating the use of sample-specific, toxic staining agents, or other advanced add-on techniques. Furthermore, the imaging of cellulose lattices in a C-NCF/TiO2 nanohybrid system is accomplished in the presence of Ag+ ions in a medium revealing the mode of association of C-NCFs in the system, which validates the feasibility of the presented strategy. The methods adopted here can provide further understanding of the fine structures of carboxylated nanocellulose fibrils for studying their structure-property relationship for various applications.

Introduction

Owing to its immense potential for a broad range of applications and diverse nature of samples from a variety of biomass resources, a molecular-level understanding of nanocellulose (NC) has a great significance in revealing its structure–property relationship. Previously, the crystal dimension and molecular level arrangements of the elementary units in cellulose have been determined with the help of high-resolution neutron and X-ray scattering,[1−5] magic angle NMR,[1,6] and so forth. However, the lack of direct evidence and the complexity to get results from these techniques have made NC chemistry more challenging. Therefore, molecular level imaging using various microscopic techniques is considered more straightforward in determining microstructures and supramolecular arrangements of elementary cellulose molecules. Scanning probe microscopic techniques like atomic force microscopy (AFM), working on the radiation-less principle, have already been successfully employed to image NC lattices, revealing the crystalline orientations of cellulose molecules in cellulose microfibrils.[7,8] Apart from AFM, high-resolution transmission electron microscopy (HR-TEM) is another alternative key imaging technique to provide direct evidence of structural morphology and molecular arrangements of its components with subnanometer lateral resolution.[6,9−15] However, being the most beam-sensitive material, NC entails special care in order to reduce the detrimental effects caused by high-energy electron radiation during TEM imaging.[16] This is because cellulose exhibits little variations in electron density because of the low-Z (atomic number) constituent elements and, therefore, results in poor contrast in transmission electron images. Despite these challenges, researchers have made attempts to visualize the fundamental building blocks of cellulosic materials, cellulose nanocrystals (CNCs), and NC fibers (NCFs) using a transmission electron beam with the advent of advanced add-on technologies like cryo-TEM,[8,17,18] high-angle annular dark-field scanning TEM,[19] aberration-corrected TEM,[11] and so forth. However, complex auxiliary setups/software settings are required for these high-end techniques. Low-dose/low-voltage HR-TEM is another choice for imaging beam-sensitive samples.[20,21] Luo et al. for the first time utilized a conventional TEM to reveal the solution phase morphology and dissolution process of CNCs by visualizing a specimen at the molecular level in ionic liquids.[22] This was made possible because of negligible vapor pressure of ionic liquids. Recently, Zhu et al. have made similar observations with acid-hydrolyzed CNCs with the help of a zero loss energy-filtered bright field TEM.[23]

Thus, on account of the increasing demand of direct supramolecular level imaging of NCFs, retaining their semicrystalline nature and hierarchical arrangements, herein, we realized the molecular level imaging of crystalline domains of carboxylated NCFs (C-NCFs) using HR-TEM technique exploiting the supporting role of conducting reduced graphene oxide (rGO), and high-Z Ag+ ions in a medium. Succeeding to that the feasibility of the presented strategy was validated by visualizing C-NCFs in high-resolution using a C-NCF/TiO2 nanohybrid system by introducing Ag+ ions in the medium, revealing the mode of association of NCFs in the nanocomposite.

Results and Discussion

2,2,6,6-Tetramethylpiperidin-1-oxyl (TEMPO)-oxidized NCFs prepared from banana pseudo-stem fibers (BPSFs), a natural soft wood source, were chosen as the key material throughout the analysis. During the preparation of the NC samples, ionizable −COOH groups were introduced (with a calculated average carboxylic content of 1.27 mmol/g) that electrostatically improve the stability of cellulosic fibers in an aqueous suspension. Detailed characterization of the carboxyl-functionalized NCFs has been discussed in our previous study.[24] It should be taken into account that the mercerization using concentrated aqueous NaOH (15%) solution to remove the non-cellulosic contents from raw fibers from the plant origin often ends up in a change in the crystalline order from the native cellulose I to cellulose II phase.[25,26] The supramolecular structural difference between cellulose I and II phases is illustrated in Figure a. The wide-angle X-ray scattering (WAXS) pattern of C-NCF derived from BPSF is given in Figure b, consistent with the cellulose II phase. The peaks at 12.1° and 20.0° are assigned to the (1–10) and (110) planes, respectively, which are characteristic features of the cellulose II allomorph.[27,28] Furthermore, the existence of the cellulose II phase was also justified by the characteristic features, that is, weak yet noticeable vibrational peaks at 3440 and 3480 cm–1, present in the Fourier transform infrared (FTIR) spectrum of C-NCFs (see Figure c).[25]

Figure 1

(a) Schematic showing supramolecular structural difference between the cellulose I and II phases upon mercerization. (b) WAXS pattern and (c) FTIR spectrum showing characteristic peaks of cellulose II.

Transmission Electron Imaging of Crystalline Domains of C-NCFs

Though the cellulose phase and hence the orientation are predicted apparently using the X-ray diffraction and FTIR methods, the direct imaging of the crystalline phase is still necessary for the direct determination of the number of elementary cellulose chains present in the NCFs. Therefore, herein we adopted two simple approaches to visualize the lattice arrangement of β-glucan chains in C-NCFs using HR-TEM in the presence of either (i) rGO, a conducting material with high electron transmittance or (ii) high-Z metallic Ag+ added to the C-NCF suspension. Figure consolidates the HR-TEM images of C-NCFs obtained in the presence of rGO (Figure a) and Ag+ ions (Figure b). Remarkably, in both cases the resolution was sufficient enough to visualize the separation between individual β-(1→4)-d-glucan chains, which enables direct interpretation of supramolecular arrangements of elementary fibrils. The average distance between two parallel chains was calculated from the fast Fourier transform (FFT) patterns (shown in Figure a,b, insets) taken from the labelled areas (red boxes) of the respective images to be 0.40 nm in both cases, corresponding to the crystal lattice parameters of cellulose II (020) plane.[29,30] The average width of superfine fibrils in Figure a,b is estimated to be ≈0.30 nm, respectively. The HR-TEM pixel profiles (yellow lines i and ii) in Figure a,b are presented in the bottom panel. Both these values are in consistent with the reported dimension of a single cellulose chain.[22]Figure c is the schematic representation of lateral arrangements of 10 linear β-(1→4)-d-glucan chains. It is estimated from the above observation that each 3.98 ± 0.06 nm lateral width of C-NCFs consists of an average of 10 β-(1→4)-d-glucan chains.

Figure 2

HR-TEM images of C-NCFs taken in the presence of (a) rGO and (b) Ag+ ions. Insets show the FFT patterns taken from the labelled areas (red box) of the respective images. Corresponding pixel profile given for yellow lines, (i) of (a) and (ii) of (b). (c) The schematic of lateral arrangement of 10 glucan chains in cellulose II. Blue and red colors represent alternate 1-4-linked β-D-anhydroglucopyranose units.

Initially, we used AFM and TEM techniques to image the morphology of the C-NCFs and the details are consolidated in Figure S1, Supporting Information. An average fiber width of 13 ± 6 was calculated from the AFM height image of C-NCFs (see Figure S1a, Supporting Information). Figure S1b,c (Supporting Information) shows the TEM images of C-NCFs with different magnifications taken under unstained conditions and under a 300 kV electron beam. The contrast is obviously lost upon magnifying the image to the next level. It should be taken into account that the size measurements using AFM are vulnerable to errors associated with the tip-convolution artifacts, whereas TEM fails to provide exact molecular details of soft organic materials like NCs that scatter electrons to a smaller extent. This is because the contrast in the electron microscope depends on the atomic number of elements present in the specimen.[31] Therefore, any attempts to achieve higher magnification may cause rapid deterioration of the sample. Therefore, it is often recommended that transmission electron imaging studies of polymers and biomaterials be preferably done at a low voltage in order to suppress knock-on damage because of the elastic collision of electrons. However, a decrease in the acceleration voltage causes inelastic scattering, which in turn can lead to ionization damage and sample heating.[20]

The conventional method used to tackle the above issue is selective staining to obtain a high-contrast surface view in an electron microscope. This procedure involves either chemical or physical incorporation of a heavy metal element into the samples composed of low molecular mass components prior to imaging.[32] Figure S1d,e (Supporting Information) shows the TEM images of C-NCFs generated, taking advantage of staining using 1% phosphotungstic acid. Though the resultant images show an improved resolution, we could see the granularity of the dry stain stuck on the C-NCF surfaces that limits the resolution of the size of the smallest particles in the stain. It should be noted that staining can also partly promote the local flocculation of C-NCFs on the supporting grid. Besides, most of the other chemicals used as stains so far are toxic or hazardous (e.g., osmium tetroxide, uranyl acetate, etc.).[33]

C-NCFs in the Presence of rGO

All the above-mentioned complications have raised questions about the conventional staining methods, and in turn highlight the indigence for a better deal with transmission imaging of soft samples like NCFs. Therefore, here we used an alternative procedure over the existing staining methods enabling direct visualization of the crystalline phases of C-NCFs using nontoxic materials. In this context, it is remarkable to consider graphene and its derivatives, which have been recognized as excellent supports as liquid cells for hydrated biological samples for dynamic imaging using microscopy TEM.[34,35] It has also been used for filming mechanistic pathways in organic reactions with a sub-Angstrom resolution.[36] Thus, this conducting material, having a high electron transparency, can be used as a possible material to suppress the sensitivity of soft materials like C-NCFs toward radiation damage to a great extent. When compared to laborious sample-specific staining methods, our method involves simple mixing of samples with rGO by sonication before drop-casting on a carbon-coated Cu TEM grid. TEM images of control rGO flakes are shown in Figure S2a,b, Supporting Information. Figure a provides a representative TEM image of C-NCFs obtained after mixing with rGO. Remarkably, we could see the clear lattice arrangements of the elementary chains without affecting specimen stability for a sufficient time available for examination. The sheet-like appearance of C-NCFs (red arrow mark in Figure a) could possibly be misinterpreted as two-dimensional (2D) graphene sheets whereas the EDX spectra failed to distinguish between these two materials because the same peaks were expected for both. This incongruity could be avoided by a closer assessment (see Figure b), which revealed crystalline arrangement of closely stacked cellulose chains with a lattice spacing of 0.40 nm specific for the (020) plane of cellulose. It is apparent that being a low-Z material, unlike the common staining agents that are mostly from a heavy metal family, the addition of rGO neither increases the electron density nor gets incorporated chemically with the sample. However, the combination of exceptional properties of graphene such as (a) high electron-transparency: high-energy electrons can transmit through thin graphene sheets, (b) high electrical conductivity: reduces the electrostatic charge build-up because of the presence of mobile π-electrons, and (c) high thermal conductivity: dissipates the heat generated, makes it an ideal nanomaterial for HR-TEM imaging.[35,37] Therefore, it sounds reasonable to say that the role of rGO is more or less like a protective conducting layer, which reduces the vulnerability of cellulose toward electron beam damage. However, prolonged exposure causes ionization damage to the specimen and is demonstrated by focusing a particular crystalline area of C-NCFs (see Figure c). Further magnifying, the wavy nature of individual super fine cellulose fibrils can be seen in Figure d most likely originating either from the inherent semicrystallinity or perturbation from the well-stacked arrangements in C-NCFs. On the other hand, the incident high-energy electron can cause inelastic scattering, which generates molecular excitations and ionization, resulting in local heating of the low-electron density cellulose sample leading to the breakage of weak inter-chain hydrogen bonds followed by strong covalent bonds. The FFT pattern (inset, Figure c) of the magnified portion (white marked area) shows obscured spots corresponding to cellulose (020) plane, which implied the unorganized nature of individual fibrils. The red arrow in Figure d points to the gradual loosening of individual fibrils from the parent microfibrils leading to the deterioration of specimen morphology.

Figure 3

HR-TEM images of (a) C-NCFs taken in the presence of rGO with scale bar: 10 nm, the red arrow points to the sheet-like appearance of C-NCFs, (b) cellulose crystal lattice showing (020) planes, scale bar: 1 nm, (c) crystalline region showing the wavy nature of the crystal lattice with scale bar: 5 nm, the inset shows the FFT pattern of the white marked area, (d) magnified image showing the initial stage of radiation damage of loosening of elementary fibrils under prolonged exposure to the electron beam, scale bar: 2 nm.

C-NCFs in the Presence of Ag+ Ions

Though staining textile cellulose fibers with high-Z elements has been established since the mid-1950s, most of the chemicals used to date further demand precautionary steps during handling because of their hazardous nature, limiting their applicability in routine studies.[33] Therefore, our second attempt was to use a relatively nontoxic staining protocol. Considering the presence of a large number of carboxyl groups on the surface of TEMPO-oxidized NCFs, which could act as effective anchoring sites for Ag+ ions,[24] AgNO3, a relatively less-hazardous metallic salt, was selected as a staining agent for C-NCFs. The visibility under a TEM was checked and a series of images were taken with different magnifications to examine the crystalline arrangements of β-glucan chains in C-NCFs. The 2D sheet-like appearance of C-NCFs shown in Figure a resembles the observation made in the previous case. Furthermore, HR-TEM studies disclose the hierarchical arrangements of cellulose elementary fibrils to form 2D structures (Figure b). On close examination, it is possible to observe the segregation and/or reintegration of individual fibrils to form bundles, which in turn form 2D sheets (Figure c). It is noteworthy that C-NCFs seem to be stabilized under a high-energy electron beam in the presence of Ag+ ions, which enables further magnification of the field view with enhanced resolution. A likely explanation for this observation is the possible replacement of Na+ by Ag+ ions at surface nucleophilic COO– ends by the addition AgNO3, providing an additional contrast for images by increasing the electron density at the probed area. The EDX spectrum (Figure a, inset) verifies the presence of Ag. It should be noted that both the inherent amorphous nature as well as the beam-generated sample damage can cause deviation from original crystallinity as observed in the first case (see Figure d).

Figure 4

Bright-field TEM images taken in the presence of Ag+ ions showing, (a) 2D sheet-like appearance of C-NCFs along with the EDX spectrum, (b) the corresponding magnified image, scale bar: 10 nm; (c) HR-TEM showing the crystal lattice of cellulose with a red arrow pointing to the segregation/reintegration of nanofibers, scale bar: 5 nm, the inset shows the respective low-magnification image with scale bar: 20 nm, (d) an area of C-NCFs subjected for a long-time exposure under a high-energy electron beam with a red arrow pointing to the amorphous part generated by radiation damage, scale bar: 5 nm, the inset shows the corresponding FFT pattern of the white-marked area.

C-NCFs in the C-NCF/TiO2 Nanohybrid System

Finally, we moved forward to examine the feasibility of the above-adopted methods in real samples by visualizing the C-NCFs in the C-NCF/TiO2 hybrid system, which can be developed to fabricate photocatalytic membranes.[38] TEM images were collected after 5 min treatment of the colloidal hybrid system with the AgNO3 solution (0.25 mM) under sonication and drop-cast on a carbon-coated copper grid. Figure a represents a low-magnification TEM image of C-NCF/TiO2 with the corresponding EDX spectrum, revealing the coexistence of TiO2 and Ag. The red-marked arrows point to the entangled thin cellulose microfibrils observed on the periphery and the surface of the TiO2 sheets (see Figure a, inset). On close examination, it is possible to observe crystal lattice structures in Figure b,c that clearly outline the NC chains stacked together to form microfibrils. Both amorphous and short-range ordered crystalline patterns were identified. In the crystalline region, the observed lattice spacing of 0.40 nm calculated from the FFT pattern (Figure c, inset) for the white-dotted area is in accordance with the interlayer distance of the (020) plane of cellulose II. The HR-TEM imaging also provides an insight to the curly appearance (yellow-dotted curves in Figure d) and the exceptionally narrow width (∼0.8–4.7 nm) of C-NCFs in the C-NCF/TiO2 system, different from that of the initial dimension (13 ± 6 nm). We could also see that the knock-on damage was significantly slowed down (but not completely suppressed) when Ag+ ions were introduced in the system. The FTIR spectra of the lyophilized samples of the C-NCFs and the C-NCF/TiO2 hybrid composite are provided in Figure S3, Supporting Information. The results suggest the preservation of the cellulose II phase, even after the lateral dimension of C-NCFs was reduced considerably by the possible loosening of the elementary fibrils from the stacked bundles upon introducing TiO2 into the system. Thus, this sample preparation method provides direct evidence for how C-NCFs interact and undergo changes in different environments in hybrid systems. To further prove that the observed lattices are not from TiO2, the TEM images of TiO2 alone nanoparticles are provided in Figure S4, Supporting Information. We could see that unlike C-NCF lattices, the inorganic TiO2 lattice planes are highly stable under a 300 kV transmission electron beam. The d spacings observed for TiO2 in the C-NCF/TiO2 composites were 0.36 and 0.24 nm, which correspond to the (100) and (004) planes of anatase TiO2, respectively.[39]

Figure 5

(a) TEM image of the C-NCFs on TiO2 sheets with the EDX spectrum, the inset shows curly C-NCFs appearing at the periphery of TiO2, scale bar: 20 nm; (b) HR-TEM images showing super lattices of C-NCFs with scale bar: 10 nm and (c) scale bar: 5 nm, the inset shows the FFT pattern of the white-dotted area showing a d spacing of 0.40 nm corresponding to the (020) plane of cellulose II; (d) HR-TEM showing radiation-induced damage of the curly C-NCFs (yellow-dotted curve).

Additionally, the X-ray diffraction analysis of C-NCFs was performed (see Figure S5, Supporting Information) after introducing rGO (Figure S5, pattern a) and Ag+ ions (Figure S5, pattern b) and after lyophilizing the respective aqueous suspensions. The WAXS patterns confirm that the initial cellulose II phase of the C-NCFs remain unaffected in the presence of these additional entities introduced for getting a better resolution under transmission electron imaging. A similar trend was observed for C-NCF/TiO2 also (see Figure S5, pattern c). In the light of the above discussions, the additional stability and hence the contrast acquired by C-NCFs are attributed to the presence of either light, non-metallic, and conductive material like rGO or electron dense, metallic Ag+ ions. These additional entities protect the C-NCFs from high-energy electron beam damage and thereby enabling high-resolution imaging. The strategies adopted above may help in finding out different types of defects (point, line, and area defects) existing in NC[40] similar to those of the inorganic clusters and nanoparticles.[41] However, more detailed modelling studies are still needed to check the versatility of the adopted methods for variety of applications. Besides, the structural dynamics is yet to be completely explored as the current methods provide limited information about the real-time changes, which point to the directions for future research.

Conclusions

In conclusion, direct imaging of C-NCFs (having cellulose II crystalline phase) with a resolution down to the molecular level was made possible using standard HR-TEM, revealing crystalline arrangements of the β-(1→4)-d-glucan chains. Low-Z-conducting rGO and/or high-Z Ag+ ions were employed as a charge neutralization strategy, which significantly slowed down knock-on damage and permit the direct visualization of crystalline domains with an exceptional resolution. Finally, the mode of association of C-NCFs in the C-NCF/TiO2 hybrid system was visualized using a HR-TEM with least damage to NC by introducing Ag+ in the medium, which validated the versatility of the introduced strategy. Thus, our procedures promise easy and direct sample preparation methods for low-density C-NCFs prior to TEM analysis without the use of conventional toxic staining reagents, specific tools, or complicated sample preparation methods. The methods adopted here are facile and the results can provide input for modelling studies and promote the theoretical understanding of the carboxylated NC hybrid systems.

Experimental Section

Materials

Bioextracted BPSFs were used for NCF extraction. Silver nitrate (AgNO3), TEMPO (C9H18NO), sodium chlorite (NaClO2), sodium bromide (NaBr), 15% solution of sodium hypochlorite (NaClO2), sodium hydroxide (NaOH), graphite (<150 μm), and phosphotungstic acid (H3[P(W3O10)4]·xH2O) were procured from Sigma-Aldrich. Potassium permanganate (KMnO4) was purchased from SD Fine-Chemicals Limited.

Preparation of C-NCFs

Raw lignocellulosic fibers were obtained from natural soft wood source BPSF, following the TEMPO oxidation method discussed in our previous report.[24] The first step of C-NCF preparation was pretreatments for the removal of noncellulosic contents by mercerization using 15% NaOH and bleaching using acidified NaClO2. Further C6-carboxyl functionalization was carried out by TEMPO-mediated oxidation. This carboxyl-functionalized micron-sized cellulose fibers were then defibrillated under high intensity ultra-sonication to obtain C-NCFs. The synthesized C-NCFs were washed, freeze dried, and desiccated for further use.

Preparation of rGO

The rGO was prepared using Hummers’ method and the detailed procedure is given elsewhere.[42,43] The oxidation of graphite was carried out using acidified KMnO4 maintaining the temperature below 10 °C. The synthesized rGO was further purified by filtration, washing, and drying at 60 °C.

Preparation of the C-NCF/TiO2 Composite

Mesocrystal TiO2 sheets were prepared adopting a low-temperature microwave-assisted sonochemical method explained in our previous report.[39] For the preparation of the C-NCF/TiO2 nanocomposite, 1 mL dispersion of 0.1 wt % mesocrystal TiO2 sheets and 1 mL dispersion of 0.25 wt % C-NCF were mixed well in a sonication bath for 10 min. The mixture was then heated to boiling for 1 h. The as-prepared C-NCF/TiO2 colloid was then cooled at room temperature.

Characterization

AFM imaging was performed under dry conditions at a temperature of 22 ± 2 °C using a MultiMode 8 atomic force microscope equipped with a NanoScope controller (Bruker, Santa Barbara, CA). The carboxyl content of the C-NCF sample was determined using the conductometric titration method.[21,44] TEM images and the corresponding EDX patterns were obtained on a FEI Tecnai 30 G2S-TWIN transmission electron microscope operating at an accelerating voltage of 300 kV at a temperature of 22 ± 2 °C. The images were processed with the help of Gatan Inc. Version 2.31.734.0 software. The FTIR spectra were obtained using a PerkinElmer Series Spectrum Two FTIR spectrometer over the 4000–400 cm–1 wavenumber range. The sample was directly mixed and pelletized with KBr. The C-NCFs were analyzed with small-angle/wide-angle X-ray scattering on a XEUSS, WAXS system with Cu Kα radiation (λ = 1.54 Å) using a Genisxmicro source from Xenocs operated at 50 kV.

TEM Sample Preparation of C-NCFs

With 1% phosphotungstic acid as a staining agent: a small drop of the C-NCF aqueous sample was placed onto a carbon coated copper TEM grid. Staining was carried out by adding a single drop of 1% phosphotungstic acid on a semi-dried C-NCF sample on a Cu grid allowing a homogeneous distribution throughout the grid. The excess of liquid was gently blotted out. The sample was then allowed to dry at room temperature.

In the Presence of rGO

rGO was used as the conducting supporting material for C-NCFs. Freeze-dried C-NCFs were dispersed in deionized water and mixed with rGO in a 1:1 ratio, and sonicated for 30 min in a high-intensity ultrasonication bath. A small amount of the above mixture was drop-casted on the TEM grid and dried at ambient temperature.

In Presence of Ag+ Ions

A well-dispersed 0.25 wt % suspension of C-NCFs was mixed with 0.25 mM AgNO3 solution and sonicated for 10 min. A small drop of the mixture was drop-casted and dried on a carbon-coated copper grid at room temperature.

Incorporation of Ag+ Ions into the C-NCF/TiO2 System

The prepared C-NCF/TiO2 colloidal suspension was mixed with 0.25 mM of the AgNO3 solution and sonicated for 10 min. A single drop of the mixture was drop-casted on a copper grid and allowed to dry at room temperature.