386 doi:10.1017/S1431927618014216 Microsc. Microanal. 24 (Suppl 2), 2018 © Microscopy Society of America 2018 Lignin Deposition In Arabidopsis thaliana Cell Walls Unveiled By Ptychographic X-Ray Computed Tomography (PXCT). Carla Polo 1 , Luciano Pereira 2 , Denisele Flores 2 , Manuel Guizar-Sicairos 3 , Mirko Holler 3 , Paulo Mazzafera 2 , Juliana Mayer 2 , Harry Westfahl 1 , Florian Meneau 1 1. Brazilian Light Synchrotron Laboratory (LNLS)/ Brazilian Center for Research in Energy and Materials (CNPEM), Campinas- Brazil. 2. Vegetal Biology Department of Biology Institute (IB)/ University of Campinas (UNICAMP), Campinas- Brazil. 3. Swiss Light Source (SLS)/ Paul Scherrer Institut (PSI), Villigen- Switzerland. * Corresponding author, carla.polo@lnls.br During plant development, lignin is deposited along with cellulose and hemicellulose in the secondary cell walls of structural fibers and water-conducting cells, being essential for their strength and rigidity [1]. Attempts to reduce the biomass recalcitrance through genetic manipulation of lignin deposition have met with some success, but the stunted growth of many of the resulting plants and the associated yield penalty have made the use of similar genetic modifications in commercial biomass crops problematic [2]. Although extensive biochemical characterization has already been performed showing chemical cell wall differences among wild-type and lignin-deficient mutants, information regarding the 3D structural changes introduced in the lignocellulosic layer is still missing. Ptychographic X-ray computed tomography (PXCT) is a coherent X-ray based technique, which, combined with tomography, enables to obtain a 3D density map of several tens of micrometers thick specimens with few tens of nanometers resolution [3]. Therefore, we present the use of PXCT to study the three-dimensional organization and interaction of the complex cross-linked molecules within the plant cell at the nanometer scale. Wild type and C4H Arabidopsis thaliana petiole fragments were selected for these studies and manually cut down to volumes of around 0.5 x 0.5 x 1 mm. These fragments were chemically fixed and infiltrated with LR white acrylic resin. The resin block containing the small samples volume (Fig. 1A and 1B) were reduced to 50 x 50 μm (d x h) pillar (Fig. 1C) by Ga FIB-SEM (FEI Helios 600i) from the Scientific Center for Optical and Electron Microscopy (ScopeM-ETH Zurich). The pillars were transferred onto a flat Au coated pin and glued with platinum deposition (Fig. 1D). The PXCT measurements were done at cSAXS beamline of the Swiss Light Source (PSI- Villigen, Switzerland). The pin was introduced into the OMNY chamber enabling sample cryocooling [4] in order to reduce X-ray radiation damage. The sample was placed 3 mm downstream the Fresnel zone plate (220 μm diameter with outermost width of 60 nm, with a focal distance of 66 mm) resulting in a 10 x 10 μm 2 coherent X-ray beam, at 6.2 keV, with 10 8 photons/s. The Eiger 500 K detector, with 75×75 μm 2 pixel size was placed 7.3 m from the sample position. Ptychographic scans were performed with a circular pattern [3] with a step size of 2.5 μm over a field of view of 60 x 25 μm 2 (w x h) with a total number of 238 points/projection. Each point was exposed for 0.1 s and total angular coverage was 0ᵒ to180ᵒ with an angular step of 0.48 ᵒ resulting in 750 projections. https://doi.org/10.1017/S1431927618014216 Downloaded from https://www.cambridge.org/core. IP address: 54.162.69.248, on 01 Jun 2020 at 05:32:01, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms.