Plant dry matter or lignocellulosic biomass is a feedstock of bioenergy, biofuel production and novel biomaterials. Understanding the cell wall structure is important for effective utilization of the lignocellulosic biomass.

Structure, chemistry and mechanics of the polymer assembly of the secondary cell walls have been studied for decades on the macrolevel. However, characterization of the chemical components of the cell walls on the nanolevel are still unknown due to the limitation in spatial resolution of spectral techniques such as FTIR and Raman, which are used  for chemical analysis.  

Most secondary cell walls of xylem cells are made up of three dominating polymers: cellulose, lignin and hemicelluloses. Cellulose fibrils with diameter 3-4 nm are arranged in larger agglomerates with size of 20-25 nm and are embedded in a matrix consisting of lignin and hemicelluloses. Different theoretical models of special arrangement of the polymers in the cell walls have been suggested. Most of these models are based on the SEM or AFM study but not on the chemical information.

In this work, a Nanonics MultiView 2000 with Near-field Scanning Optical Microscopy (NSOM) was used  for the first time to study the photo-optical and thus chemical properties of cell walls at the nanoscale. This technique enables scientists to overcome the optical diffraction limit and to reach the spatial resolution of 50nm (limited to size of the NSOM aperture)

The cell walls of three different plants-beeches (hard-wood), spruce (soft wood) and bamboo (grass) were studied with NSOM.


Since the samples are semi-transparent, the NSOM measurements were conducted in reflection mode. In this mode the light coming out from the probe aperture interacts with the sample, is reflected and then collected from the top with an optical objective of an upright microscope. The NSOM measurements were performed with a Nanonics MultiView 2000 and Nanonics cantilevered NSOM probes. The MV-2000 scanning head has a completely open optical axis from the top and from the bottom and can be easily integrated with almost any kind of optical microscopes. MV-2000 together with cantilevered NSOM probes, which have extended tip and special geometry, enable true reflection NSOM measurements.

Three images were acquired simultaneously: height, phase and NSOM. The obtained NSOM images showed curls- like structures with dimensions about 125 nm and more, which are not seen in the height and phase images. It is most likely that lignin contributes a stronger NSOM signal than cellulose and hemicelluloses due to its interaction with light (autofluorescence and resonance effect). NSOM results obtained on the three different plants species (hard wood, soft wood and grasses) point to the universal principle of the special cellulose and lignin assembly in secondary cell walls. For the first time, NSOM images provide sub diffraction limited chemical information about the spatial distribution of the secondary cell wall components. This contributes to the understanding of the cell wall structure and its enzymatic degradation for energy conversion from ligninocellulosic raw material in general.  

Published:  Keplinger et al. Plant Methods 2014, 10:1 

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