In situ plant materials hyperspectral imaging by multimodal scattering near-field optical microscopy

Revealing plant cell walls physical and chemical properties by s-SNOM. In this work, we combine nanotechnology and engineering for plant morphogenesis. We report for the first time the local optical properties of the cell walls regions correlated to their chemistry.

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3000 billion trees on Earth

With 3000 billion individuals, trees are one of the most widespread materials on Earth, i.e. wood, but also one of the most essential for human survival. Their great diversity is expressed in a wide variety of physical and bio-chemical properties giving them different functionalities involved in our daily life, ranging from simple functions such as our breathing, our nutrition or our well-being, to exploitation functions for building, paper or energy... Despite this diversity, wood has a particularity. Its great paradox is that this diversity is obtained only from three polymers, namely cellulose, hemicellulose and lignin, constituting nearly 90% of it.

 The diversity then comes from the arrangement and proportions of these polymers. Trees have indeed a hierarchical structure, meaning that their macroscopic properties and appearances inevitably come from their structures at micro and nanometric scales. This leads to the question of the relationship between chemical distribution, structural organization and mechanical, thermal and optical properties at the nanoscale. How would nanometric variations influence the global response of the tree? And conversely, how can an external action modify the molecular arrangement and the associated properties? These questions constitute a major societal challenge and to tackle them, it is necessary to access properties at the nanoscale. Thanks to various interdisciplinary research initiatives, plant biologists and physicists have opened up a new avenue of research combining advanced technologies, engineering and wood biology.

Fig. 1 : Multimodal s-SNOM was used to measure, in situ, and correlate the local mechanical, chemical and optical properties of poplar cell walls at the nanoscale.

Unlocking the nanoworld of plant cell walls

In our research consortium, Institut Fresnel, CINaM, ORNL, we are physicist expert in local probe microscopy techniques applied to biomaterials. Our first studies on wood in 2014, using atomic force microscopy (AFM) nanoindentation, allowed us to extract nanomechanical properties of the cell wall. To go further in the understanding of wood morphogenesis, and thus correlate the physical properties to the chemical distribution, it seemed important to us to go towards a technique combining AFM and optical spectroscopy. We then turned to the company Neaspec gmbh (Attocube) known for its scattering optical near-field system (s-SNOM) in the infrared, spectral range specific to cellulose, hemicellulose and lignin. Our results gathered in this paper show the potential of this technique to access and correlate mechanical properties, optical constants and in situ chemical distribution of the wood cell wall. The study here was performed on poplar samples in their first year of growth.

Fig.2: A) Hyperspectral intensity data-cube, I(x,y,v) of the absorption signal obtained in situ on a 1x1 µm² region of a young poplar stem. The measurement was performed in the spectral range 974-1986 cm-1, where main wood polymers (cellulose, lignin and hemicellulose) absorption bands are present. B) Mappings of the local n (a,b) and k (c,d) dielectric optical constants were extracted from the reflectance and absorption mappings at 1034 cm-1 and 1590 cm-1, two wavenumbers that are specific to cellulose and lignin respectively. The reddest regions in the k mappings correlate with a high amount of cellulose in (c) and of lignin in (d), showing the associated chemical distribution and co-localization of the different polymers.

These properties were determined, in-situ, and correlated for the first time on plant cell walls, by measuring the near-field interaction between the sample and an oscillating nanometric tip used in s-SNOM obtaining a spatial resolution of 20nm. The use of hyperspectral imaging in absorption and reflection allows obtaining simultaneous spatial and spectral mappings; thus tracing the chemical distribution of the different wood constituents and their optical dielectric constants (n and k) at the nanometer scale. This AFM-based technique also allows tracing back some local mechanical properties such as viscoelasticity.

 Using a combination of all these AFM modes we followed a delignification process that allowed us to establish factors preventing the complete removal of lignin, an important process for a rational wood industry. We also correlated chemistry and optics and demonstrated how environmental chemical variation is imprinted in the variability of the n and k at the nanoscale.

Read the full paper here:

Charrier, A.M., Normand, A.C., Passian, A. et al. In situ plant materials hyperspectral imaging by multimodal scattering near-field optical microscopy. Commun Mater 2, 59 (2021)


Researcher, Institut Fresnel, CNRS