Total internal reflection Raman spectroscopy of barley leaf epicuticular waxes in vivo

The use of total internal reflection (TIR)-Raman spectroscopy to examine cuticular wax layers in vivo at the surface of barley leaves (Hordeum vulgare L. cultivar Pastoral) is demonstrated. The limited penetration depth (40 nm) of the evanescent wave compared to the thickness of the wax layer eliminates problems arising from fluorescence from underlying pigments. Epicuticular wax crystals are observed to be more crystalline than the amorphous wax layer, which is analysed after removal of the wax crystals by cellulose acetate stripping. Carotenoids are detected as trace species in the cuticular waxes.