Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging

Figure S1. Schematic illustration of the experimental set-up for NIR and visible (VIS) laser excitations. Within 1 microspectroscopic setup, spontaneous Raman scattering, SHG and 2PF spectra can be observed. NIR excitation for SGH and of 2PF: 150 fs, 73 MHz laser pulses at 850 nm with 6 × 10¹¹ W/cm² peak intensity and 7 ps pulses at 1064 nm, 75 MHz, 2.4 × 10¹⁰ W/cm². NIR excitation of Raman scattering: intensity of 2.2 × 10⁶ W/cm² at 785 nm (cw); VIS excitation of Raman scattering: intensity of 1.6 × 10⁶ W/cm² at 532 nm (cw). Step size for each mapping measurement was 1 μm.

Figure S2. (A, B) SHG images of sorghum root cross-sections corresponding to the Figures 1A and D, respectively. (A) SHG signal excited at 1064 nm: 0 to 2100 counts (white to red), (B) polarization-resolved SHG signal excited at 850 nm: 0 to 800 counts (white to red), detection was perpendicular to the excitation field. Scale bars in are 10 μm, step size 1 μm. Excitation was horizontally.

Figure S3. Chemical Raman images of sorghum root sections. (A, B) Images correspond to Figure 1A-C and (C, D) images correspond to Figures 2 and Figure 3A-D. (A, C) Chemical images based on the linear baseline corrected (1160-1185 cm⁻¹) intensity of the ferula and p-coumaric acid Raman signal at 1171 cm⁻¹, representing mostly the xylem cell wall regions. (B, D) Chemical image based on the baseline corrected (1115-1160 cm⁻¹) intensity of the suberin Raman signal at 1138 cm⁻¹, representing mostly the endodermis regions. Image scales are (A) 0 to 920 cps, (B) 0 to 155 cps, (C) 0 to 1660 cps and (D) 0 to 380 cps. Scale bars are 10 μm and step size is 1 μm.

Figure S4. Selected chemical images based on the intensity of the cellulose Raman signal at 1093 cm⁻¹. The cellulose maps (A, B and C) correspond to the lignin chemical images shown in Figure 3C, G and K, respectively. The maps show the baseline corrected (1106-1079 cm⁻¹) maximum signal with color scales (from black to red to white) of (A) 70 to 180 cps, (B) 100 to 360 cps and (C) 120 to 530 cps. The polarization of the excitation light is indicated by an arrow in each map. Scale bars are 10 μm and step size is 1 μm.

Figure S5. (A-C) Overlay of SHG images of data shown in Figure 3A and B, E and F, and I and J, respectively. (D) Overlay of SHG images of data shown in Figure 4A and E. Color scales are the same as in corresponding Figures in Figure 3 and Figure 4. (A) Normalized to 3800 counts; (B) normalized to 7800 counts; (C) normalized to 7200 counts and (D) normalized to 1700 counts. The excitation polarization colored blue was along the vertical, and colored red was along the horizontal axis in the plane of each figure. The detection direction was vertical in all cases. Scale bars are 10 μm and step size is 1 μm.

Figure S6. (A) Overlay of Phenyl ring stretching mode (green, intensity at 1602 cm⁻¹) and cellulose (red, intensity at 1093 cm⁻¹) Raman map of sorghum root section with bright field image and (B) Phenyl ring stretching mode (green, intensity at 1602 cm⁻¹) and 2-photon autofluorescence (purple, at 530 nm) map of sorghum root section with bright field image. The vibrational band at 1602 cm⁻¹ attributable to lignin and bound or unbound components such as ferulic and p-coumaric acid [3,8]. Scale bars are 10 μm and step size is 1 μm.

Table S1. Band assignments in the Raman spectra of sorghum sections. s, symmetric; as; asymmetric; conj, conjugated; ν, stretching; δ, deformation; bend, bending; endodermis, signal more prominent in endodermis; xylem, signal more prominent in xylem region.