Near-infrared–Fourier-transform–Raman microspectroscopic imaging of flax stems

Abstract

Near-infrared–Fourier-transform–Raman (NIR–FT–Raman) microscopy was employed to image and subsequently produce in situ maps of the distribution of chemical components in flax ( Linum usitatissimum L.) stem tissue. Thick (∼80 μm) cross-sections of flax were cryotomed, thawed, equilibrated at 85% RH for >3 h and sealed under cover glasses on gold-mirrored microscope slides. Spectra for each image pixel were collected from selected tissue sections over the Raman shifted region 3600–300 cm −1 at 16 cm −1 with 256–512 scans and employing ∼185 mW of focused laser power at the sample. A computer controlled automated microscope stage was used to collect area maps of 50–150 μm in rectangular dimensions in 6–10 μm steps and register them to the visible images that were captured using a CCD camera. Data collection required 8–10 h per image. Chemical profiles were produced from area integrations of specific spectral regions. The chemical profiles showed the location of all of the major components of flax by anatomical cell type. A sharp shoulder at 2850 cm −1 in the CH stretch region provided evidence of waxes in the cuticular/epidermal tissue. The bands occurring around 1600 cm −1, due to aromatic ring stretching vibrations, gave evidence of lignin in core tissue and pigments in epidermal tissue. Bands occurring between 1175–1050 cm −1, due to COC heavy atom mixed mode vibrations, showed the greatest concentration of carbohydrate in fiber cells with lesser (but significant) amounts indicated in core tissue. In addition, bands occurring between 870–800, 515–476 and 400–360 cm −1 gave specific evidence for the presence of pectins, other non-cellulosic polysaccharides and cellulose, respectively, in parenchyma tissue.