Nowadays, a large set of microscopy tools are commonly used to investigate the nanostructure of complex biological or synthetic materials with a multiscaled organisation: epi-fluorescence and laser scanning confocal microscopes (LSCM), scanning and transmission electron microscopes (SEM, TEM), atomic force microscopy (AFM), or Raman imaging. In particular, transmission electron microscopy (TEM) allows observation of the internal structures of natural systems (grains, cells and tissues) with a high spatial resolution. However, such observation requires in parallel dedicated sample preparation steps to prepare the fragile hydrated biological samples with successive steps including chemical or physical fixation, dehydration or vitrification of the water content, resin-inclusion and contrast-enhancement staining. Its well-known these steps may induce artefacts that reduce the image resolution and valuable information, but are often considered as the unique way to access to the ultrastructural information.
We propose here to explore alternative routes for the ultrastructure investigation of the grain tissues, as an example of a very complex biological material, with the main objective to reduce the preparation steps as most as possible while adapting the same preparation to be process with several microscopy tools: TEM, SEM, AFM, Raman imaging, Fluorescence imaging, X-ray microscopy. Depending on the successfulness of this approach, a set of complementary experiments from the same zone of a native sample can alloy to connect the plant ultrastructure with a high spatial resolution to accurate chemical information.
The multimodal and multiscale approach has been fully tested to study wheat grains tissues. We will show how unique features in the wheat outer layers organisation have been revealed due to both the capacity of keeping the native tissue “true contrast”, free from any chemical artefacts, or the native mechanical stress between the components, exempt from any alteration from resin infiltration. Moreover, taking advantage from the capacity of keeping the wheat tissue in its native shape, the mechanical properties of the native wheat outer layers (intenal and external pericarp, aleurone and sub-aleurone layers and starchy endosperm) have been characterised by AFM at the nonometer scale, see Figure 1. Additionally, the chemical composition has been acquired by Raman mapping from the different zones of interest, giving in an unique way the capability to understand the natural assemblies of biopolymers and mineral nutrients at the sub-cellular level in regard to their chemical, physicochemical and structural properties.
C. Karunakaran, C.R. Christensen, C. Gaillard, R. Lahlali, L.M. Blair, V. Perumal, S.S Miller, A.P. HitchcockIntroduction of soft X-ray spectromicroscopy as an advanced technique for plant biopolymers research. PLoS One, 2015 26;10 (3) pages: e0122959.
Berquand A., B. Bouchet and C. Gaillard: Investigating the ultrastructure and mechanical properties of wheat grain tissues using optical microscopy and HarmoniX, Application Note, n°AN122, Veeco Instruments (2008)
To cite this abstract:Cédric Gaillard; A multimodal and multiscale approach to investigate the micronsized organisation of a very complex biological material : the wheat grain.. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/a-multimodal-and-multiscale-approach-to-investigate-the-micronsized-organisation-of-a-very-complex-biological-material-the-wheat-grain/. Accessed: December 4, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/a-multimodal-and-multiscale-approach-to-investigate-the-micronsized-organisation-of-a-very-complex-biological-material-the-wheat-grain/