Fig 1.
Scanning electron micrographs of the preparation steps of phytolith lamellas.
(a) bilobate phytoliths at the surface of the sugarcane stalk. (b-c) Platinum cover strip deposited along the longitudinal axis of a phytolith. (d-e) Trenches carved by the focused ion beam to form the phytolith lamella. (f-g) Fixation of the micromanipulator to the platinum strip and removal of the lamella from the plant tissue. (h-i) Fixation of the lamella to a copper lift-out grid (j). The arrow points to the lamella in (j). Detail of the phytolith lamella fixed to the lift-out grid before (k) and after (l) thinning (<500 nm) the central region of the lamella under the focused ion beam.
Fig 2.
Panoramic images of the phytolith lamellas.
The images were obtained by Scanning Electron Microscopy (SEM, secondary electrons, top) and Scanning Transmission X-ray Microspectroscopy (STXM, photon energy 285.0 eV, bottom) from the four mounted phytolith lamellas (L1–L4). The rectangular regions selected for spectral analysis are approximately indicated in the STXM images.
Fig 3.
Principal component analysis of the image stacks in the 1840–1880 eV energy range.
The scree plot indicates the significance is limited to PC1 for each one of the four phytolith lamellas (L1–L4). The inset shows the corresponding PC1 eigenspectra, which are consistent with opaline silica.
Fig 4.
Spectral weight maps obtained for the phytolith lamellas L1–L4.
(left) Maps of the silicon K edge PC1 eigenspectrum in the 1840–1880 eV stacks. Maps of the linear (center) and carbon K edge (right) components in the 280–310 eV stacks. In the carbon K edge maps, carbon is concentrated near the lamella borders (white arrows), as gray islands (yellow arrows), and as areas of carbon dispersed in the silica matrix (ellipses).
Fig 5.
X-ray absorption spectra in the carbon K edge.
(Colored) Target spectra were obtained from the linear combination of the eigenspectra of the image stacks measured in the phytolith lamellas (L1–L4) of the present study. (Gray) Reference spectra of protein reported by Zubavichus et al. [48] and of plant cell wall biopolymers lignin, cellulose, and polygalacturonic acid (PGA) reported by Karunakaran et al. [47] after shifting the energy axis (–0.2 eV). Approximate positions of peaks from aromatic (aryl), carboxyl (O = COH), and protein carbonyl (O = CN) groups are marked.
Fig 6.
Selected regions of interest (ROIs) and their measured spectra.
At the top, selected ROIs are presented for the silicon K edge energy range (1840–1880 eV), having L1 as an example of phytolith lamella. Selected ROIs are also presented for the four phytolith lamellas (L1–L4) in the carbon K edge energy range (280–310 eV).
Fig 7.
Analysis of the spectral weight maps for the L1–L4 phytolith lamellas.
(left column) Scatter plots of weights obtained from the linear component in the 280–310 eV stacks versus the silicon K edge in the 1840–1880 eV stacks, evidencing proportionality. (center column) Scatter plots of weights obtained from the carbon K edge and the linear components in the 280–310 eV stacks, evidencing negative correlations. In the center top graph, the datapoints are distributed across diagonal streak lines, whose interpretation is illustrated by the arrows. Weights in the x and y axes tend to increase with local lamella thickness. The complementarity of carbon and silicon tends to disperse the datapoints across streaks of negative slope that scale with carbon density, which is, for example, higher for lignin than protein. (right column) Histograms of carbon signals are calculated by dividing the carbon K edge weights (pixel value in the y-axis in the center column graphs) by the mean linear weight of each lamella where the carbon K edge weight is approximately zero (x value of the stars in the center column graphs). The reference of zero silicon is marked as a vertical line (right column) and a gray circle (top graph of the central column).