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

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.The majority of land plants possess an extracellular, waxy cuticle that covers the surface of their aerial parts and protects them against desiccation, external physical and chemical stresses, and a variety of biological agents (Grncarevic and Radler, 1967; Barthlott and Neinhuis, 1997; Krauss et al., 1997; Ristic and Jenks, 2002; Yeats and Rose, 2013). The cuticle is a composite layer composed mainly of cutin and overlaid by cuticular waxes. Cutin is a macromolecular structure consisting primarily of hexadecanoic (palmitic) and octadecenoic (vaccenic) acids that are covalently linked by ester bonds to generate a rigid, three-dimensional network that is embedded with polysaccharides. Cuticular waxes are composed of long-chain (C₂₀–C₄₀) aliphatic molecules derived from fatty acids (Samuels et al., 2008), and studies over the last several decades have identified structural and regulatory constituents of the biosynthetic pathways of cuticular components (Kolattukudy, 1981; Beisson et al., 2012). In addition to the physiochemical properties conferred by its lipid components, the architecture of the cuticle plays an essential role in physiological function. For example, through understanding the properties of the cuticular structure, the extraordinary superhydrophobicity of the Lotus spp. leaf has been mimicked in micro- and nanotechnology to generate self-cleaning surfaces (Bhushan and Jung, 2006; Bhushan et al., 2009; Koch et al., 2009).As may be expected, given the diversity of plants, the habitats they inhabit, and individual life histories, the morphology and composition of plant cuticle varies extensively between and within species and includes plate-, needle-, and pillar-shaped wax crystals (Barthlott et al., 2008). In some species, cuticular wax composition is known to vary with depth, giving rise to chemically distinguishable layers (Yeats and Rose, 2013). Finally, the cuticle is increasingly shown to be important in development (Koornneef et al., 1989; Yeats and Rose, 2013) and pathogenesis (Lee and Dean, 1994; Gilbert et al., 1996; Bessire et al., 2007; Delventhal et al., 2014). It is therefore unsurprising that interest in cuticle composition, structure, and physiology is increasing (Buschhaus et al., 2014; Hen-Avivi et al., 2014; Heredia-Guerrero et al., 2014; Xu et al., 2014). Moreover, a greater understanding of the relationship between structure and chemical composition of cuticle waxes is vital for enhancing agriculture yields, as it will further our knowledge of how plants regulate water balance and inform the application of nutrition (foliar feeds) and pesticides, leading to improved formulation strategies for agrochemicals.The chemical composition and topological architecture of cuticular waxes are both critical for optimal physiological function. Analyses of these essential properties have typically been performed separately. Cuticle wax composition is normally determined using gas chromatography; cuticle ultrastructure may be analyzed using destructive imaging techniques such as scanning electron microscopy (SEM; Baker and Holloway, 1971; Jetter et al., 2000; Barthlott et al., 2008) and laser desorption ionizing mass spectroscopy (Jun et al., 2010) or, in vivo, using nondestructive real-time techniques, including white-light scanning interferometry (Kim et al., 2011), atomic force microscopy (Koch et al., 2004), confocal microscopy in reflectance mode (Veraverbeke et al., 2001), fluorescence microscopy of chemical stains (Pighin et al., 2004), coherent anti-Stokes Raman scattering (CARS) microscopy (Yu et al., 2008; Weissflog et al., 2010), and total internal reflection Raman spectroscopy (Greene and Bain, 2005). Despite the advances in our understanding of the cuticle that have been made with these techniques, there is a great need for techniques that combine chemical and structural information to provide in situ high-resolution chemical analysis of epicuticle waxes.Techniques based on vibrational spectroscopy offer in situ chemical analysis derived from the vibrational frequencies of molecular bonds within a sample. However, due to water absorption and the intrinsically low spatial resolution associated with the long infrared (IR) wavelengths required to directly excite molecular vibrations, IR absorption techniques have limited value for bioimaging. Raman scattering, however, provides analysis of vibrational frequencies by examining the inelastic scattering of visible light. Raman scattered light is frequency shifted with respect to the incident light by discrete amounts that correspond to the vibrational frequencies of molecular bonds within the sample. The spectrum of Raman scattered light consists of a series of discrete peaks that each correspond to a molecular bond and can be regarded as a chemical fingerprint holding a wealth of information regarding chemical composition. Unfortunately, Raman scattering is an extremely weak effect, and typical signals from biological samples are at least six orders of magnitude weaker than those from fluorescent labels. This severely limits the application of Raman for studying living systems because long acquisition times (100 ms–1 s per pixel) and relatively high excitation powers (several hundred milliwatts) are required to image most biomolecules with sufficient sensitivity. Furthermore, the lack of sensitivity is compounded by autofluorescence, which in plant tissues completely overwhelms the Raman signal, prohibiting its application in planta.Far stronger Raman signals can be obtained using coherent Raman scattering (CRS; Min et al., 2011). CRS achieves a Raman signal enhancement by focusing the excitation energy onto a specific molecular vibrational frequency (Fig. 1A). A pump and Stokes beam, with frequencies ω_p and ω_S, respectively, are incident upon the sample, with their frequency difference (ω_p–ω_S) tuned to match the molecular vibrational frequency of interest. Under this resonant condition, the excitation fields efficiently drive bonds to produce a strong nonlinear coherent Raman signal. When applied in microscopy format, the nonlinear nature of the CRS process confines the signal to a submicron focus that can be scanned in space, allowing three-dimensional confocal-like mapping of biomolecules. CRS microscopy has particular advantages for bioimaging: (1) Chemically specific contrast is derived from the vibrational signature of endogenous biomolecules within the sample, negating the need for extraneous labels/stains; (2) Low-energy, near-IR excitation wavelengths can be employed, which reduces photodamage and increases depth penetration into scattering tissues; and (3) The CRS process does not leave sample molecules in an excited state, does not suffer from photobleaching, and can be used for time course studies.Open in a separate windowFigure 1.Schematic representation of the two CRS processes: CARS and SRS. A, Energy level diagrams for the CARS and SRS processes, showing the pump (green), Stokes (red), and anti-Stokes (blue) photon energies. B, Diagrammatic representation of the input and output spectra for CARS and SRS, showing the gain and loss in the pump (red) and Stokes (green) beams, respectively. ΔI_S, Change in Stokes beam intensity; ΔI_p, change in pump beam intensity. C, Diagrammatic representation of the modulation transfer detection scheme used to detect stimulated Raman gain and loss with high sensitivity.CRS microscopy may be achieved by detecting either CARS or stimulated Raman scattering (SRS).