Visualizing cellulose chains with cryo scanning transmission electron microscopy

Abstract text (incl. figure legends and references)

Cellulose is the most abundant biopolymer on earth and is found in the cell walls of most plants and some algae, but also occurs in bacteria, fungi and even some sea animals. Plant cellulose, in particular, has a wide range of applications as a renewable, biodegradable and non-toxic material in papers, textiles, packaging and medical products, to name a few. Cellulose in plants is organized in supramolecular structures where the basic structural element the cellulose microfibril (CMF). The supramolecular cellulose comprises of ordered periodic crystalline regions that can be liberated by acid hydrolysis into nanoparticles, namely the cellulose nanocrystals (CNC), with dimensions of 100-200 nm in length and a few nanometers in width. Despite its abundance in nature and its technological relevance, the structural details of cellulose still remain elusive and various structural models have been proposed in recent years [1].

Transmission electron microscopy (TEM) under cryogenic conditions has proven to be a highly valuable technique for the structural analysis of such biomolecules. However, imaging cellulose at sufficiently high resolution has been challenging due to its very high susceptibility to electron beam damage, combined with the low contrast provided by its light constituents. These problems have been addressed in the past by applying contrast agents, staining with uranyl acetate or low voltage imaging [2]. While, by this, great progress has been made regarding the visualization of nanoscale cellulose features, atomic scale visualization still remains problematic [3].

Here, we report on the visualization of sulfated cellulose chains by low-dose cryo high-resolution scanning TEM (STEM). To this end we exploit the high contrast provided by indvidual heavy ions in annular dark field (ADF) imaging for visualization of cellulose chains.

For imaging a FEI Titan G2 STEM, operated at 300 kV, has been used. Samples were prepared by drop casting the CNCs, dispersed in water, on a TEM grid, which is covered by a 2-3 nm thick amorphous carbon film. During imaging the sample is kept at liquid nitrogen temperature.

In the obtained ADF images (Figure 1) the individual atoms providing contrast can clearly be discerned and exhibit a regular, linear arrangement along the long axis of the CNCs. By comparing the micrographs with multislice simulations based on atomistic structural models, we obtain information about possible arrangements of the sulfate groups, linked to the position of carbon 6 sites in the glucose unit within single CNC chains. Exemplary, a possible structural configuration on the amorphous carbon substrate is depicted in Figure 1c with the corresponding ADF multislice simulation shown in (f).

References

[1] Habibi, Youssef et al.; Chemical reviews 110 (6), pp. 3479–3500. DOI: 10.1021/cr900339w.

[2] Sugiyama, J. et al.; International Journal of Biological Macromolecules 9 (3), pp. 122–130. DOI: 10.1016/0141-8130(87)90039-0.

[2] Ogawa, Yu et al.; Cellulose 26 (1), pp. 5–15. DOI: 10.1007/s10570-018-2076-9.

Acknowledgements

The authors acknowledge financial support by the European Union"s Horizon 2020 research and innovation program under Grant 823717-ESTEEM3.