Cellulose-Infused Carbon Nanotube Tape Eases Array Tomography Imaging

A novel tape combining natural cellulose fibrils, carbon nanotubes, and silver nanowires has been developed to enhance serial-section electron microscopy. This composite tape, detailed in Applied Microscopy, eliminates the need for glow discharge or carbon coating, which are typically required with conventional tapes. The innovation aims to resolve persistent challenges in array tomography, a technique that reconstructs large volumes of brain tissue at nanometer resolution using scanning electron microscopy.

Array tomography with scanning electron microscopy allows for the detailed reconstruction of neural circuits, resolving synaptic vesicles and other ultrastructural features. When destructive methods like serial block face SEM or focused ion beam SEM are not employed, preparing hundreds or thousands of serial thin sections becomes technically demanding and heavily reliant on operator skill. Sections collected on fragile formvar film-coated single-slot EM grids are prone to breaking, disrupting serial imaging. Automatic tape-collecting ultramicrotomy (ATUM) addresses this by automatically collecting thousands of serial ultrathin sections onto tape, and when combined with mosaic acquisition software, it enables millimeter-scale volumes to be imaged serially.

The tape is a crucial component of this workflow. Currently, Kapton polyimide tape is the most widely used commercial option, known for its stable supply and mechanical robustness, but it lacks sufficient electrical conductivity and surface hydrophilicity. A carbon nanotube-coated polyethylene terephthalate (CNT-PET) tape reported in 2018 improved surface conductivity and imaging stability, but glow-discharged CNT-PET tape is not commercially available. The new study aimed to achieve stable hydrophilicity and sufficient conductivity in one material by depositing a composite of natural cellulose fibrils, carbon nanotubes, and silver nanowires onto a PET substrate.

The fabrication process was simple and chemical-free, involving high-power shaking of an aqueous mixture of cellulose fibrils and carbon nanotubes with zirconia beads. This collision process deagglomerates the materials and promotes the self-alignment of carbon nanotubes within the cellulose network, resulting in a homogeneous viscous dope. Silver nanowires were then added for the AgNW-integrated version. The team evaluated four tape types: Kapton, CNT tape, CNF-CNT, and CNF-CNT-AgNW, measuring water contact angle, electrical conductivity by four-point probe, and surface roughness at two magnifications.

Water contact angle measurements indicated that cellulose incorporation reduced the intrinsic hydrophobicity of CNT-PET tapes without plasma treatment. CNT-PET tape had the highest contact angle at 86.68 ± 2.29 degrees, while adding cellulose fibrils lowered it to 69.26 ± 4.27 degrees, comparable to Kapton at 71.72 ± 3.57 degrees but without glow discharge. The CNF-CNT-AgNW tape maintained moderate hydrophilicity at 74.56 ± 3.22 degrees, showing that silver nanowire incorporation did not significantly affect wettability. The authors suggest a contact angle below 75 degrees may suffice for stable section adhesion without plasma treatment.

Conductivity saw a dramatic improvement. The surface sheet resistance of Kapton exceeded the instrument's measurement limit, while the values for CNT, CNF-CNT, and CNF-CNT-AgNW tapes were 4,419 ± 1,589, 58.68 ± 3.61, and 4.54 ± 0.97 ohms per square, respectively. This enhancement is attributed to the zirconia bead collision process, which separates bundled carbon nanotubes into individual nanotubes and aligns them along cellulose fibril chains, forming highly conductive networks. This conductivity improvement correlates with the suppression of charging artifacts during SEM imaging.

Surface roughness measurements revealed that cellulose-containing tapes are more textured than Kapton or CNT tape. At 200-times magnification, roughness values were 0.34, 0.16, 3.39, and 1.13 for Kapton, CNT, CNF-CNT, and CNF-CNT-AgNW respectively, and at 500-times magnification, the values were 0.78, 0.76, 2.12, and 2.05. The authors note these surface features may aid section adhesion and imaging behavior, but the observed charging suppression is more directly linked to improved conductivity. Profilometry maps showed that the cellulose-containing tapes have a more heterogeneous, fibrous surface consistent with the formation of a cellulose-based network.

To test performance with real specimens, the team embedded mouse brain tissue in epoxy resin using an osmium-tetroxide-thiocarbohydrazide staining procedure and cut 50-nanometer-thick serial sections with an automatic tape ultramicrotome. Brain slices from two male mice, prepared from hippocampal CA1 stratum radiatum, were fixed, heavy-metal stained, dehydrated, and resin-infiltrated before sectioning. When sections on CNF-CNT and CNF-CNT-AgNW tapes were imaged on a Gemini 300 SEM with a backscattered electron detector at 5-kilovolt beam voltage, without plasma treatment or carbon coating, organelles including synaptic vesicles, mitochondria, and endoplasmic reticulum were visible without wrinkles or charging artifacts.

The silver nanowire version did present a drawback. In some areas of images from the CNF-CNT-AgNW tape, fiber-like signals appeared against the sample background, attributed to silver nanowire aggregates. The authors emphasize the need for uniform and well-dispersed nanowires within the material to prevent aggregates from appearing in images and interfering with high-resolution imaging quality. The cellulose-only CNF-CNT tape did not exhibit this artifact, making it the cleaner option for maximum image fidelity, while the AgNW version offers substantially lower sheet resistance.

The researchers argue that the intrinsic, stable hydrophilicity of cellulose is a significant practical advantage over plasma-treated surfaces, which gradually lose hydrophilicity over time and require re-treatment. Conventional Kapton-based ATUM workflows also require carbon coating to suppress charging, a step that introduces variability in coating thickness and consumes time in serial electron microscopy. By integrating hydrophilic nanofibers and conductive nanowires through a chemical-free collision process, the new tapes simplify the workflow and, according to the authors, enhance ultrastructural imaging fidelity for large-scale serial SEM imaging, offering an alternative to conventional Kapton-based ATUM tape.