In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy

Abstract

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles.

Using Raman spectroscopy to characterize biological materials

“…Fluorescence can occur when electrons are excited to electronic energy levels and return to the ground energy level by emitting a photon of light at a longer wavelength. 32,185,186 Chemical analysis beneath physical obstructions 187,188 23,194,195 Single molecule detection 196,197 Tumour targeting 198 Live cell analysis 199 Pharmaceuticals 200 Cancer diagnosis 62,201,202 Bacteria identification 16,203 Plant materials 204 Table 4. Classification rates (% classification ± standard deviation) of control and endometrial cancer patients from blood serum and plasma samples, using surface enhanced Raman spectroscopy (SERS) with two different diameter gold nanoparticles and spontaneous Raman ('No SERS').…”

Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization

“…S5). Moreover, Raman microspectroscopy has a high resolution that fits with structural characterization of cuticle surfaces (Littlejohn et al, 2015). Therefore, Raman imaging at the specific 1,001-cm 21 band was used to monitor the spatial distribution of free OH groups within the cutin polymer.…”

Compositae Plants Differed in Leaf Cuticular Waxes between High and Low Altitudes

“…The content and composition of cuticular waxes vary among different species and organs , and thus most taxonomic studies have been at the level of interspecific differentiation . Cuticular wax protects plants against desiccation and external environmental stresses . Changes of UV‐B radiation, humidity, temperature, and soil water condition have been shown to result in alterations of the crystal structure and chemical compositions of cuticular wax .…”