The Microstructure of Cellulose Nanocrystal Aerogels as Revealed by

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The Microstructure of Cellulose Nanocrystal Aerogels as Revealed by Transmission Electron Microscope Tomography Christian Buesch, Sean Weston Smith, Peter Eschbach, John F. Conley, and John Simonsen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00764 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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The Microstructure of Cellulose Nanocrystal Aerogels as Revealed by Transmission Electron Microscope Tomography Christian Buesch 1, Sean W. Smith2, Peter Eschbach3, John F. Conley, Jr.2, John Simonsen1* 1

Oregon State University, Wood Science and Engineering, 119 Richardson Hall, Corvallis,

Oregon 97331. 2

Oregon State University, School of Electrical Engineering and Computer Science, 1148 Kelley

Engineering Center, Corvallis, Oregon 97331. 3

Oregon State University, Electron Microscopy Facility, 145 Linus Pauling Science Center,

Corvallis, Oregon 97331. *Corresponding Author: [email protected], Phone: +1541-737-4217

KEYWORDS. carboxylation, supercritical drying, atomic layer deposition, FIB, Avizo.

ABSTRACT. The microstructure of highly porous cellulose nanocrystal (CNC) aerogels is investigated via transmission electron microscope (TEM) tomography. The aerogels were fabricated by first supercritically drying a carboxylated CNC organogel and then coating via 1

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atomic layer deposition with a thin conformal layer of Al2O3 to protect the CNCs against prolonged electron beam exposure. A series of images was then acquired, reconstructed, and segmented in order to generate a three dimensional (3D) model of the aerogel. The model agrees well with theory and macroscopic measurements, indicating that a thin conformal inorganic coating enables TEM tomography as an analysis tool for microstructure characterization of CNC aerogels. The 3D model also reveals that the aerogels consist of randomly orientated CNCs that attach to one another primarily in three ways: end to end contact, "T" contact, and "X" contact.

1. Introduction Cellulose nanocrystals (CNCs) have outstanding properties with diameters between 5-10 nm and length in the range of 50-200 nm.1 CNCs are abundant in nature, biocompatible, biodegradable, have exceptional mechanical properties, and chemical modification is straightforward.2 Additionally they are a promising material for a variety of applications as sensors/actuators,3 as barrier films,4 and as substrates for use in solar cells,5 catalysts,6 biomedical applications7 and tablet displays.8 In composites CNCs can be used as additives for coatings,9 plastic composites for cell phones and laptops,2 and thin films with advanced functionality for food packaging.10 Herein we focus on CNC aerogels. CNC aerogels are lightweight and can have low densities (< 0.005 g/cm3) and high specific surface areas (200-500 m2/g).11 Utilizing these properties, CNC aerogels can be used for a variety of applications including thermal insulation; templates for energy storage; sensors; and composites in wind power, construction, automotive, and marine industries.3 Currently, a lack of understanding of the micromechanical properties of CNC aerogels limits progress in developing many of these current and future applications. Proper interpretation of 2


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experimental results requires knowledge of the microstructure. Due to the small size of CNCs, past research efforts have found this to be a major challenge because many traditional analysis and imaging methods applied for materials research at the micrometer scale, such as light microscopy, neutron imaging, and x-ray imaging, cannot be used. Transmission electron microscopy (TEM) is one of the best known techniques for materials analysis at high magnification. TEM imaging of CNCs has been challenging, as reported by Kvien et al.,12 owing to their high sensitivity to the electron beam. In addition, TEM traditionally has been used for imaging in two dimensions (2D) only. Previous work guides the way to possibly solve these issues. It has been shown recently that CNCs become more beam resistant when coated with a thin conformal layer of alumina via atomic layer deposition (ALD).13 Also, in recent years electron tomography (ET) has evolved, emerging as a popular technique to analyze materials in three dimensions (3D) with nanoscale14,15 and even atomic resolution.16,17 Other recent work in the field of ET has resulted in advances in image reconstruction,18 sample preparation with a focused ion beam (FIB),19 imaging of thick samples,15,

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avoiding loss of

information due to the missing wedge effect,21 and the development of the high-angle annular dark field scanning TEM technique.22,23 These advances have enabled a powerful technique for 3D imaging at the nanoscale which has been applied, for example, to the investigation of the morphology of nanoparticles in composite structures.24-26 To our knowledge, the 3D analysis of CNC aerogels has not been performed. In this work, we perform 3D analysis of CNC aerogels produced using supercritical CO2 drying, which has been shown to result in the lowest densities and least amount of CNC agglomeration.36 The aerogel properties are acquired in two ways: First, bulk aerogel properties 3


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are measured macroscopically. Second, for protection of the CNCs during sample preparation and imaging, as well as enhancement of contrast formation, the aerogels are coated with Al2O3, and analyzed microscopically via TEM tomography. The tomography data are then manually simplified into a 3D model that represents the uncoated CNC aerogel. This model is further used to study the detailed arrangement and bonding of the CNCs.

2. Experimental Section i.

CNC and aerogel preparation

While many different aerogels have been created from various materials, only six research groups have reported the creation of pure CNC aerogels.27-32 Even though the aerogel backbones (the CNCs) are similar, the aerogel properties vary significantly. This can be explained by their different manufacturing procedures, which have a major influence on the degree of agglomeration of the CNCs, the density of the produced aerogel, and its microstructure. Thus direct comparisons are difficult. The CNC aerogels used in this work were created from carboxylated CNCs in aqueous solution. CNCs with a high degree of carboxylation are not currently produced commercially. However, we found consistent gel formation with sulfated CNCs was more difficult than with carboxylated CNCs and so chose carboxylated for this study. The CNCs were prepared from pure cotton cellulose by hydrochloric acid hydrolysis followed by TEMPO regioselective oxidation using sodium hypochlorite/sodium bromide using a previously described method.33-34 The crystallinity was ~85% and the surface charge due to carboxylation was ~1.3 mmol/g.

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The aqueous CNC solution (~1%) was placed in a beaker and a second layer of acetone was carefully laid on top of it to allow diffusion of the solvents without disturbing the CNCs. After a time period of approximately one day the top solution was carefully removed with a syringe and a new layer of acetone was introduced. Over a period of two to three weeks several exchanges were conducted until only a small fraction of water remained in the beaker. The water content was monitored using Fourier transform infrared spectroscopy. The CNCs formed a gel in the acetone environment, an organogel. The gel was then removed from the beaker and supercritically dried. The supercritical dryer is custom made and consists of a vessel, a CO2 tank, a pump and several valves. A computer records and monitors process parameters. The vessel and valves have separate heaters equipped with overheat protection and a controlled internal vessel temperature. The gel was introduced into the vessel under ambient conditions. Next, the vessel was filled with liquid CO2, which is miscible with acetone. The vessel pressure was then increased to ~100 atm (~1500 psi), which is above the supercritical pressure of CO2 (Psc = 72.9 atm). However, at this point the temperature was kept below the supercritical temperature (Tsc = 31.1 °C) so the CO2 remained liquid. After 30 min of CO2-acetone diffusion, the vessel was partially flushed to replace a portion of the CO2-acetone mixture with new CO2 while the pressure was maintained well above Psc. Several flushes were performed until the majority of acetone was removed from the vessel. Then the system was maintained under pressure for one day to allow complete diffusion of acetone into the CO2. Next the vessel was heated above Tsc while the pressure was maintained above Psc. After one hour the vessel was slowly depressurized while maintaining the temperature 5


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above Tsc. The dried aerogel was removed and stored in plastic containers. The supercritical drying procedure is summarized in Table 1 and Fig 1 shows the processing parameters plotted as a function of time. Table 1: Overview of supercritical drying process. Temperature and pressure are used to control the phase of CO2. Phase Description 1 Pressurizing the system

Temperature < Tsc (< supercritical) > -80 ◦C (> solidification)

2

< 31.3 ◦C, outlet valve is Flush to 1200 psi, re-pressure to 1500 psi, repeat 3x, limit dP/dt to heated for flushes 200 psi/min up to 1500 psi ◦ > 800 psi < 31.3 C

3

4

5

Exchanging CO2 3x, Waiting 30 min in between Waiting 24 h for CO2 to fully diffuse into aerogel Heating vessel pressure and temperature increase Venting

Pressure Limit dP/dt to 200 psi/min up to 1500 psi (safety limit)

Increase > Tsc and stay ≥ Psc at all times, CO2 must be above at all times released to keep pressure below 1500 psi Reduce from 1500 to 0 psi with > 31.3 ◦ C 5 psi/min, faster (200 psi/min) once pressure is < 1050 psi

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Figure 1: Values of process parameters during the supercritical drying process of CNC aerogels. ii.

Surface area characterization

BET surface area analysis was performed using nitrogen gas adsorption. Surface areas were calculated using the Brunauer-Emmet-Tell (BET) model based on the aerogel mass. Measurements were performed on a Micromeritics ASAP 2020 instrument and isotherms generated using the onboard software. iii.

Bulk density

Bulk dimensions and mass were measured manually using carefully handled calipers and an analytical balance, respectively. iv.

Aerogel characterization

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The main aerogel properties of interest for structural analysis are porosity, density, surface area, and CNC arrangement. The porosity is related to the pore volume divided by the total volume (CNCs + pores) as: = 1 −

Where P = porosity, Vcnc = CNC volume, and VT = total volume. P can take values from almost zero to one and strongly depends on the manufacturing procedure of the aerogel. Density is related to porosity as:

= (1 − ) Where da = density of the aerogel and dc = density of CNCs, assumed to be 1.6 g/cm3.33, 34 The surface area is the sum of the surface of each individual CNC minus the bonding area (which for low density aerogels amounts to only a small amount of the total). The specific surface area takes the density of the CNC or aerogel into account as well and is defined as the area divided by the mass. We can estimate CNC aerogel properties by assuming the CNCs are cylindrical in shape with constant length and diameter, of uniform distribution, and with a constant density.32, 35 The ends of the cylinder are ignored for the surface area calculation due to the high aspect ratio of the CNCs. For a CNC with an 8 nm diameter and 200 nm length and no agglomeration, the surface area is calculated to be ~5,000 nm2. The volume is ~10,000 nm3 and with a density of 1.6 g/cm3 the CNC mass is 1.6*10-17 g. Then the specific surface area is 313 m2/g. When a conformal coating is applied to the surface of the CNCs the cylinders gain length and diameter but the bulk size is unchanged and thus the number of CNCs per unit volume remains constant. For an aerogel with coated CNCs with a diameter of 50 nm the surface area calculates to be 50 m2/g. 8


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v.

TEM imaging and modeling

While the process parameters varied slightly between samples, here a typical procedure is described. The dried aerogels were conformally coated with approximately 20 nm Al2O3 via ALD. The procedure involves prebaking the aerogel at 150 ◦C for 24 h followed by 50 cycles of ALD at 80 ◦C. Each ALD cycle consisted of (i) a pulse of trimethylaluminum for 1 sec, (ii) 120 sec exposure, (iii) 120 sec N2 purge, (iv) a pulse of water vapor for 0.5 sec, (v) 120 sec exposure, and (vi) another 120 sec N2 purge. The alumina coated aerogels were then used to prepare TEM samples. Small pieces of the aerogel were removed with an ordinary razor blade and mounted on a SEM sample holder with the use of Ag paste. The samples were then coated with Au/Pd to improve stability when exposed to an electron beam during the lift-out procedure, and to prevent charging. The samples were then loaded into a FEI QUANTA 3D dual beam SEM/FIB electron microscope operated under high vacuum. A standard FIB lift out technique was used to remove samples from the bulk. Care was taken for the sensitive cellulose material when exposed to large currents, especially by the ion beam. Then the sample was thinned to approximately 500 nm. For imaging, a FEI TITAN 80-200 TEM/STEM with ChemiSTEM Technology was used in 200 kV and HAADF mode. The samples were loaded into a single tilt tomography holder. The magnification was 57 kx. The acquisition of the tilt series was performed semiautomatically with manual focusing for each image. The rotation step was chosen to be 1.5 degrees with maximum angles of ± 70 degrees. The tilt series was then reconstructed using FEI Inspect3D in basic mode. First an intensity threshold was used to isolate the material of interest and remove noise. The segmentation was performed using FEI Avizo Fire 8.1. Due to the imaging difficulty no 9


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algorithm could be found for the automated segmentation of the individual objects (CNCs + alumina coating). Instead the CNCs were segmented and labeled manually using the intensity gradient at the edges of the CNCs as guiding aid. CNCs were manually isolated from their coatings assuming a diameter of 8 nm and uniform coating. vi.

CNC contact types

A contact between two CNCs can generally be characterized as end contact (head to head, head to tail, tail to tail), X contact (two CNCs cross in their midsections), T contact (head to midsection, tail to midsection), or side contact (CNCs agglomerated side to side). A contact involving more than two CNCs can be described by a combination of X, T, and end contacts. 3. Results and Discussion i.

Macroscopic surface area and porosity

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Figure 2: Gas adsorption isotherm for uncoated and ALD-coated aerogels. Open symbols show desorption, closed symbols show adsorption.

BET analysis resulted in surface areas of 429 m2/g and 43 m2/g for representative uncoated and coated aerogels, respectively (Fig 2). The measurements are in reasonable agreement with our estimates (313 m2/g for uncoated and 50 m2/g for coated) implying the aerogel is nearly ideal in terms of having minimal agglomeration. Also, the results for uncoated aerogels fall in between 250 m2/g, measured by Xuan et al.,32 and the range reported by Heath et al. (78 - 605 m²/g).29 The assumption of CNCs being ideal cylinders may not be exactly correct and CNC surface roughness, leading to increased adsorption area, is a possible reason for the higher than predicted 11


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BET surface area value for the uncoated aerogel. Another possible reason is the lack of CNC crystallinity causing nitrogen molecules to potentially penetrate into the cellulose. The macroscopic density of the aerogel was calculated from the measured volume and weight of a representative sample to be 0.0057 g/cm3 and a porosity of 0.996 was calculated. For coated CNC aerogels, values for density and porosity were derived from the uncoated aerogel with a volume gain due to the increase of the CNC diameter from 8 nm to 50 nm to result in 0.22 g/cm3 for density and 0.861 for porosity. ii.

TEM analysis of aerogels

A representative sample with a volume of 1000 nm * 1000 nm * 500 nm or 0.5 µm3 was selected from the TEM data. After imaging, reconstruction, and initial manual thresholding the raw data of the coated aerogel can be visualized (Fig 3) (original images are in Supporting Information 1). It was observed that the intensities of the CNCs and the coating are not uniform indicating the possibility to distinguish between the two so as to analyze the CNCs directly. However, only the coating can be accurately and reliably measured. From the raw data the diameter of the coated CNCs was measured to be 47.72 ± 5.17 nm (Fig 3). It also indicates that the sample preparation works adequately and that the coated CNCs can withstand the electron beam. The data was idealized to a 3D model and compared with experimental data (Table 2, additional data in Supporting Information 2).

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Figure 3: 3D view of segmented tomography data. The aerogel is virtually cut using the Avizo clipping plane module. Table 2: Summary of results from theory, experiments, and 3D modeling

Uncoated

BET Gas Bulk macro Absorption data measurements Porosity 0.9964a Specific surface area [m2/g] 429 Density [g/cm3] 0.0057

3D TEM reconstruction 0.9962 493a 0.0061a 13


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Coated

Porosity Specific surface area [m2/g] 43 Density [g/cm3] Number CNCs Number Contacts a assuming density of CNCs to be 1.6 g/cm3

0.861 0.22a

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0.88 97a 0.19a 120 122b

b

while most CNCs had at least two points of contact, we did not double count, so each contact point was assigned to an individual CNC.

The simplified coated CNC 3D model consists of near perfect cylinders with ~50 nm diameter (Fig 4) and 6.14*107 nm3 volume, resulting in a porosity of 0.88. A surface area of 9.55*106 nm2 was found which results in a specific surface area of 97 m2/g. While the porosity result correlates well with the experimental results the specific surface area obtained is about twice that of the BET measurements. The inaccuracy of the 3D model may partially explain this result. Each individual step of the process to manually generate the model (imaging, reconstruction, segmentation) introduces error. Additional reasons for the discrepancy could be local variations in the aerogel and undetected agglomerations. Our assumptions of no agglomeration and perfect cylinders may not be valid and the sample preparation may be altering the aerogel structure to some extent. Yet as an estimate and a proof of concept, these values are encouraging.

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Figure 4: 3D view of coated CNC aerogel 3D model after image filtering and segmentation steps. After virtual removal of the coating to reveal a constant uncoated core CNC diameter of 8 nm (Fig 5, Supporting Information 3), the CNC volume was measured as 1.9*106 nm3 resulting in a total porosity of 0.9962, a density of 0.0061 g/cm3 and a surface area of 1.5*106 nm2. The specific surface area is then 493 m2/g.

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Figure 5: 3D view of CNC aerogel 3D model. Avizo can be used to virtually remove the alumina coating. The CNCs are shown in nontransparent green; the coating is represented in blue color with partial transparency.

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As seen in Table 2, the density and porosity are comparable to macroscopic volume measurements. While the specific surface area is higher, the difference is much smaller compared to the coated aerogel. In this sample 120 individual CNCs were identified (Fig 6).

Figure 6: 3D view of CNC aerogel 3D model of individualized CNCs. In Avizo neighboring CNCs were colored differently for improved visualization. iii.

Aerogel morphology and CNC dimensions

Two additional samples were analyzed to further quantify the CNCs and their contacts. Thus the average number of CNCs in 0.5 µm3 was estimated to be approximately 177 using the above assumptions (8 nm diameter, no agglomeration, 1.6 g/cm3) and a CNC length of 200 nm. This 17


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results in an estimate of 344 CNCs/µm3. TEM tomography reveals the number to be 294 ± 110 CNCs/µm3, in reasonable agreement. The number average length of the CNCs was found to be 210 ± 27 nm (including truncated CNCs at the border of the sample image). The length distribution is shown in Fig 7.

Figure 7: Length distribution of CNCs in the CNC aerogel.

The length appears to follow a skewed distribution (the error bars are one standard deviation) centered around 150-200 nm with a tail to the large end. Few short CNCs were measured, the majority fall between 100 and 200 nm, and a large fraction exceed 250 nm. The short CNCs can be explained by truncation at the edges of the sample image. A possible reason for the tail to the large end is that due to the alumina coating, agglomerated CNCs parallel and bound together side to side along an extended length can appear as a single CNC underneath the coating. Also CNCs 18


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combined end to end could appear to be a single nanocrystal (Fig 8) and explain why there are fewer CNCs in the 3D model than expected.

Figure 8: 3D view of a small region of a coated CNC aerogel. a) The long CNC at the top right area appears to be an individual CNC. However, in fact two CNCs are bonded together end to end. b) 3D view of a coated CNC aerogel showing the cross section of two CNCs in end to end contact type. When slicing through the CNC, different intensities in the image can be found with maxima at the surface of the CNC and the surface of the coating. With the application of a specific threshold value the CNC can be rendered hollow and the ends of the CNCs inside the coating can be visualized (Fig 8b).

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Figure 9: SEM micrograph showing an ALD coated CNC aerogel with 160 kx magnification. The sample was thinned with a FIB so that black areas in the image represent empty space. iv.

Bonding types in CNC aerogels

The analysis of the CNC contacts was first performed via scanning electron microscopy (SEM) in 2D (Fig 9). It was observed that the CNCs mostly bond to each other in four different ways: "X", "T", "end to end", and multiple contacts. A differentiation between the head and the tail of a CNC cannot be made and those contacts are summarized as end to end contacts. Detailed characterization of the contact types was performed by manual inspection of the 3D TEM model with results shown in Fig 10.

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Figure 10: Classification of contacts between CNCs in a CNC aerogel showing the distribution of the four most common types. The most common contact type was found to be the end of one CNC contacting the cylindrical surface of another CNC (a T contact). End to end contacts may be undercounted due to the difficulty of identifying these types of contacts beneath the Al2O3 coating. However, we speculate that T contacts would still be the most common type. Agglomerations were not listed, but are likely also present. These would provide side to side contacts of two or more CNCs. Again, these could be difficult to detect as they appear as oval centers in the coated aerogel. Two or more agglomerated CNCs could also appear as one extended CNC, giving an increased length in the distribution, which was observed in Fig 8. We also found contacts of three or more CNCs. In this case it was impossible to determine the exact combination of T, X, and end to end contacts and therefore these contacts were summarized as multiple contacts. Surprisingly, the aerogel is composed primarily of CNCs bonding at their ends with very few CNCs having zero end contacts. While X contacts are present, they do not predominate. Since 21


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cellulose chain ends contain aldehyde groups at one end we can expect them to be carboxylated. Thus it is likely that the formerly aldehyde ends of the CNCs have a higher density of carboxyl groups than the sides of the CNC. In addition, the ends do not appear to have a curved surface, although they are probably not routinely flat, either. But still may provide for a greater density of bonding sites than the sides. We also speculate that hydrogen bonding and bonding between carboxylate groups and their sodium ion salts is stronger than hydroxyl-hydroxyl or carboxylatehydroxyl bonding. However, end contacts would be statistically less favored due to their smaller available bonding area, arguing against end bonding. The analyzed samples also suggest that while most of the CNCs are connected with one end there is no clear preference for the other end to be connected. However, most CNCs have more than one contact so that few dangling CNCs were observed. This further supports the notion of stronger bonding at the carboxylated ends. Our experience, noted above, is that CNCs produced by the sulfuric acid method form gels in acetone with much more difficulty than carboxylated CNCs. This also suggests stronger bonding between carboxylated CNCs than sulfated CNCs. The higher density of carboxylate groups on the ends of the CNCs may be the reason.

4. Conclusion This work demonstrates that a thin conformal ALD coating of Al2O3 allows analysis of the microstructure of highly porous CNC aerogels to be performed using TEM tomography. It appears to be reasonable to assume cylindrical and individual CNCs building a network to form the aerogel. With this information a 3D TEM model was generated and compared to the 22


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macroscopically measured values for porosity and surface area, the main structural properties, resulting in reasonable agreement, given the difficulty of the experiments. Additionally the use of TEM tomography enabled the detailed arrangement of the CNCs in an aerogel for the first time. We found CNCs to arrange randomly with low interconnectivity. Unexpectedly, the majority of CNCs were connected such that one end was attached to the body of another CNC, a T contact. One reason for this may be the supposed higher density of carboxylate groups on the end of the CNC. The favored end bonding of the CNCs and few agglomerations after supercritical CO2 drying explain the extraordinarily high porosity of CNC aerogels which is desired for a variety of applications such as thermal insulation, templates for energy storage, and catalysts. This study has been the first step along what we hope is a path to a more fundamental understanding of CNCs and one of nature’s most interesting polymers, cellulose.

ACKNOWLEDGEMENTS This work was funded by the U.S. Forest Service, Forest Products lab Project 11-JV-11111129137. J.F. Conley and S. W. Smith acknowledge support of the NSF Center for Sustainable Materials Chemistry, grant number CHE-1102637.

AUTHOR INFORMATION Corresponding Author *Email: [email protected], Phone: +1541-737-4217

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SUPPORTING INFORMATION. Supporting Information 1 is a compressed file folder that contains the data after reconstruction. Uncompressed raw data is available from the corresponding author. Supporting Information 2 is a video that shows the processing of the image data to show the 3D model of the coated CNC aerogel. Supporting Information 3 is a video that shows the virtual removal of the coating of the CNCs via software to identify individual CNCs.

Table of Contents Graphic and Synopsis

The internal structure of a carboxylated cellulose nanocrystal aerogel has been examined by TEM tomography and analyzed obtaining reasonable agreement with bulk measurements. The nanocrystals predominately bond at their ends, an unexpected result.

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The Microstructure of Cellulose Nanocrystal Aerogels as Revealed by

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