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Running Head: Label-free in vivo SRS analysis of plant cuticle.
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Corresponding author (Stimulated Raman Scattering Microscopy and Submission):
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Julian Moger, Physics and Astronomy, Physics Building, College of Engineering, Mathematics
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and Physical Sciences, University of Exeter, Stocker Road, Exeter, U.K.
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EX4 4QL.
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+44 (0) 1392 724181
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J.Moger@exeter.ac.uk
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Corresponding author (Plant Cuticle): John Love, Biosciences, Geoffrey Pope Building,
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College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, U.K.
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EX4 4QD.
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+44 (0) 1392 725169
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J.Love@exeter.ac.uk
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Research Area: Breakthrough Technologies.
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IN VIVO CHEMICAL AND STRUCTURAL ANALYSIS OF PLANT CUTICULAR WAXES USING STIMULATED
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RAMAN SCATTERING (SRS) MICROSCOPY
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George R. Littlejohn1*, Jessica C. Mansfield2*, David Parker3, Rob Lind4, Sarah Perfect4, Mark
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Seymour4, Nicholas Smirnoff1, John Love1 and Julian Moger2
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Exeter, Stocker Road, Exeter, Devon, EX4 4QD. U.K.
Biosciences, Geoffrey Pope Building, College of Life and Environmental Sciences, University of
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Sciences, Stocker Road, University of Exeter, Exeter, Devon, EX4 4QL. U.K.
Physics and Astronomy, Physics Building, College of Engineering, Mathematics and Physical
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Westhollow Technology Center, 3333 Highway 6 South, Houston, TX 77082-3101. U.S.A.
Biodomain
Technology Group,
Shell
International
Exploration
and
Production
Inc.,
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Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG42 6EY U.K.
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*These authors contributed equally to the research.
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One-Sentence Summary of the Research: Stimulated Raman Microscopy is a new in vivo
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imaging technique that enables simultaneous chemical and structural analysis of plant cuticle.
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Footnotes: This work was supported by grants from the Biotechnology and Biological Sciences
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Research Council (J.L, J.M), Shell Research Ltd. (J.L), and Syngenta (J.M).
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Abstract
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The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that
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fulfils a number of essential physiological roles, including regulating evapotranspiration, light
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reflection and heat tolerance, control of development, and providing an essential barrier
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between the organism and environmental agents such as chemicals or some pathogens. The
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structure and composition of the cuticle are closely associated, but are typically investigated
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separately using a combination of structural imaging and biochemical analysis of extracted
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waxes.
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including Fourier transform infrared spectroscopy (FTIR) microscopy and coherent anti-Stokes
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Raman spectroscopy (CARS) microscopy, have been used to investigate the cuticle, but the
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detection sensitivity is severely limited by the background signals from plant pigments.
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We present a new method for label-free, in vivo structural and biochemical analysis of plant
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cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof-of-principle, we
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used SRS microscopy to analyse the cuticles from a variety of plants, at different times in
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development. We demonstrate the SRS virtually eliminates the background interference
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compared to CARS imaging, and results in label-free chemically specific, confocal images of
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cuticle architecture with simultaneous characterisation of cuticle composition. This innovative
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use of the SRS spectroscopy may find applications in agrochemical R&D or in studies of wax
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deposition during leaf development and, as such, represents an important step in the study of
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the higher plant cuticle.
Recently, techniques that combine stain-free imaging and biochemical analysis,
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Introduction
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The Plant Cuticle
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The majority of land plants possess an extracellular, waxy cuticle that covers the surface of their
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aerial parts and protects them against desiccation, external physical and chemical stresses and
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a variety of biological agents (Grncarevic and Radler 1967; Ristic and Jenks 2002; Krauss et al.
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1997; Barthlott and Neinhuis, 1997; Yeats and Rose, 2013). The cuticle is a composite layer
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composed mainly of cutin and overlaid by cuticular waxes. Cutin is a macromolecular structure
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consisting primarily of hexadecanoic (palmitic) and octadecenoic (vaccenic) acids that are
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covalently linked by ester bonds, to generate a rigid, three-dimensional network that is
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embedded with polysaccharides. Cuticular waxes are composed of long-chain (C20-C40)
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aliphatic molecules derived from fatty acids (Samuels et al. 2008) and studies over the last
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several decades have identified structural and regulatory constituents of the biosynthetic
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pathways of cuticular components (Kolattukudy 1981; Beisson et al. 2012). In addition to the
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physio-chemical properties conferred by its lipid components, the architecture of the cuticle
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plays an essential role in physiological function.
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properties of the cuticular structure, the extraordinary super-hydrophobicity of the Lotus leaf has
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been mimicked in micro and nano-technology to generate self-cleaning surfaces (Jung and
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Bhushan 2006; Koch et al., 2009; Bhushan et al. 2009).
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As may be expected given the diversity of plants, the habitats they inhabit and individual life-
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histories, the morphology and composition of plant cuticle varies extensively between and within
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species and includes plate, needle and pillar shaped wax crystals (Barthlott et al., 2008). In
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some species, cuticular wax composition is known to vary with depth giving rise to chemically
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distinguishable layers (Yeats and Rose, 2013). Finally, the cuticle is increasingly shown to be
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important in development (Koornneef et al., 1989; Yeats and Rose, 2013) and pathogenesis
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(Lee and Dean, 1994; Gilbert et al. 1996; Bessire et al., 2007; Delventhal et al., 2014). It is
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therefore unsurprising that interest in cuticle composition, structure and physiology is increasing
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(Heredia-Guerrero et al., 2014; Buschhaus et al., 2014; Xu et al., 2014; Hen-Aviv et al., 2014).
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Moreover, a greater understanding of the relationship between structure and chemical
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composition of cuticle waxes is vital for enhancing agriculture yields as it will further our
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knowledge of how plants regulate water balance, and inform the application of nutrition (foliar
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feeds) and pesticides, leading to improved formulation strategies for agrochemicals.
For example, through understanding the
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The chemical composition and topological architecture of cuticular waxes are both critical for
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optimal physiological function. Analyses of these essential properties have typically been
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performed separately.
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chromatography; cuticle ultrastructure may be analysed using destructive imaging techniques
Cuticle wax composition is normally determined using gas-
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such as scanning electron microscopy (Baker and Holloway 1971; Jetter et al., 2000; Barthlott
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et al. 2008) and laser desorption ionising mass spectroscopy (Jun et al., 2010) or, in vivo, using
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non-destructive real-time techniques including white light scanning interferometry (Kim et al.,
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2011), atomic force microscopy (Koch et al., 2004), confocal microscopy in reflectance mode
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(Veraverbeke et al., 2001), fluorescence microscopy of chemical stains (Pighin, et al., 2004),
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CARS microscopy (Yu et al., 2008; Weissflog et al., 2010) and total internal reflection Raman
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spectroscopy (Greene and Bain, 2005). Despite the advances in our understanding of the
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cuticle that have been made with these techniques, there is a great need for techniques that
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combine chemical and structural information to provide in-situ high-resolution chemical analysis
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of epicuticle waxes.
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Techniques based on vibrational spectroscopy offer in-situ chemical analysis derived from the
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vibrational frequencies of molecular bonds within a sample. However, due to water absorption
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and the intrinsically low spatial resolution associated with the long infrared (IR) wavelengths
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required to directly excite molecular vibrations, IR absorption techniques have limited value for
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bio-imaging. Raman scattering however, provides analysis of vibrational frequencies by
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examining the inelastic scattering of visible light. Raman scattered light is frequency-shifted with
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respect to the incident light by discrete amounts that correspond to the vibrational frequencies
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of molecular bonds within the sample. The spectrum of Raman scattered light consists of a
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series of discrete peaks that each correspond to a molecular bond and can be regarded as a
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chemical fingerprint holding a wealth of information regarding chemical composition.
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Unfortunately, Raman scattering is an extremely weak effect and typical signals from biological
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samples are at least six orders of magnitude weaker than those from fluorescent labels. This
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severely limits the application of Raman for studying living systems since long acquisition times
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(100 ms to 1 s per pixel) and relatively high excitation powers (several hundred mW) are
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required to image most biomolecules with sufficient sensitivity. Furthermore, the lack of
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sensitivity is compounded by autofluorescence, which in plant tissues completely overwhelms
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the Raman signal, prohibiting its application in-planta.
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Far stronger Raman signals can be obtained using coherent Raman scattering (CRS) (Min et al,
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2011). CRS achieves a Raman signal enhancement by focusing the excitation energy onto a
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specific molecular vibrational frequency (see Fig 1 (a)). A pump and Stokes beam, with
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frequencies ωp and ωS respectively, are incident upon the sample with their frequency
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difference (ωp – ωS) tuned to match the molecular vibrational frequency of interest (Ω). Under
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this resonant condition the excitation fields efficiently drive bonds to produce a strong non-linear
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coherent Raman signal. When applied in microscopy format the non-linear nature of the CRS
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process confines the signal to a sub-micron focus that can be scanned in space, allowing three-
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dimensional ‘confocal-like’ mapping of biomolecules. CRS microscopy has particular
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advantages for bio-imaging:
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(1) Chemically specific contrast is derived from the vibrational signature of endogenous
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biomolecules with in the sample, negating the need for extraneous labels/stains.
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(2) Low energy near-IR excitation wavelengths can be employed which reduce photodamage
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and increase depth penetration into scattering tissues.
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(3) The CRS process does not leave sample molecules in an excited state it does not suffer
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from photobleaching and can be used for time-course studies.
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Coherent Raman scattering microscopy may be achieved by detecting either coherent anti-
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Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS).
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Coherent anti-Stokes Raman scattering (CARS) microscopy
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CARS microscopy relies on detection of the anti-Stokes signal generated at frequency ωas =
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2ωp – ωS, which, by using filters, is spectrally isolated from the pump and Stokes beams, and its
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intensity used to map the location of biomolecules of interest (Zumbusch et al, 1999). The
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CARS signal is blue-shifted with respect to the pump and Stokes wavelengths (see Fig. 1(b))
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making CARS more resilient to sample autofluorescence than spontaneous Raman. However,
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in highly autofluorescent samples such as plant tissues, the usually weak two-photon
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fluorescence (also blue-shifted with respect to the excitation wavelengths) overwhelms the
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CARS signal. Consequently CARS imaging in planta has only been applied to samples with
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reduced chlorophyll autofluorescence including dried tissues (Zeng et al., 2010, Ding et al.,
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2012), roots (Ly et al., 2007) and cuticle waxes after they have been stripped away from the leaf
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(Weissflog et al., 2010).
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Stimulated Raman Scattering (SRS) microscopy
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SRS relies on detecting subtle changes in the intensities of the excitation fields that occur by
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virtue of stimulated excitation (Freudiger et al, 2008). When the difference frequency, ωp – ωS,
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matches the frequency of a molecular vibration the Stokes beam intensity (IS) experiences a
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gain (ΔIS) while the intensity of the pump beam (Ip) experiences a loss (ΔIp), shown in Fig. 1 (c).
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This transfer of intensity between the excitation beams only occurs when both beams are
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incident simultaneously on the sample and is measured by detecting the modulation that is
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transferred to the pump beam after it has passed through the sample when the intensity of the
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Stokes beam is modulated. The amplitude of the transferred intensity modulation is directly
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proportional to the concentration of target molecules and by modulating at frequencies above
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laser noise (>1 MHz) can be detected with a lock-in amplifier with great sensitivity (Ye et al,
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2009).
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Since SRS is detected at the same wavelength as the excitation fields it is not affected by
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fluorescent emission and as recently demonstrated by Mansfield (Mansfield et al., 2013), may
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also be used in the presence of highly pigmented samples such as plant tissues.
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We have recently shown that it is possible use phase-sensitive detection to separate the
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vibrational SRS signal from the electronic absorption processes (Mansfield et al., 2013). In the
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current investigation, we used SRS imaging to investigate both the structure and chemical
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composition of plant cuticular waxes, simultaneously and in-vivo. We compared SRS images of
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cuticle wax structures from variety of plant species that display characteristic wax morphologies
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to SEM and CARS images of the leaf surface. We demonstrated that spectroscopic information
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can be obtained to provide chemically-specific, in-situ analysis of waxes on living leaves with
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submicron spatial resolution. Using SRS microscopy we compared the cuticle structure and
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composition between wild type Arabidopsis thaliana and an eciferum-1 (cer1) mutant with
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impaired cuticle deposition and have also shown that SRS imaging is sufficiently sensitive to
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track temperature-induced changes in cuticle formation in the salt-cress, Thellungellia
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salsuginea. We conclude that, with the increasing availability of commercial Raman-based
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microscopes, SRS imaging has the potential to radically advance our understanding of plant
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cuticular structure and composition, and their effects on physiology and development.
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Results
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Characterisation of the Raman Spectra of Dissolved Cuticle Waxes.
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Spontaneous Raman spectra of hexane-extracted wax samples over the CH vibrational region
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from 2700-3200cm-1 were acquired as a baseline reference for each plant species (Fig. 2).
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Gaussian peaks were fitted underneath each of the spectra to visualise the relative contribution
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of different molecular vibrations, based on peaks previously identified in the literature (Greene
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and Bain, 2005, Snyder et al., 1978, Ho and Pemberton, 1998). The wax extracts from
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Arabidopsis thaliana, Thellungiella parvula (Saltwater Cress), Musa acuminata (Banana) and
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Monstera delicosa (Cheeseplant) had similar spectral profiles that matched those reported for
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other waxes and alkanes reported in the literature (Snyder et al., 1978, Greene and Bain, 2005)
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and deconvolved into 7 identifiable peaks (Table 1). The peaks, corresponding to the
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symmetrical and anti-symmetrical CH2 stretch, are prominent on all spectra. The ratio anti-
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symmetric to symmetric peak height is known to give a strong indication of the alkyl chain
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conformational order with a ratio 1.6-2 indicating a highly crystalline structure and a ratio of 0.6-
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0.9 indicating a liquid structure (Ho and Pemberton, 1998, Greene and Bain, 2005, Snyder et
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al., 1978). For the wax samples analysed here, the ratios were as follows: A. thaliana 1.13, T.
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parvula 1.58, M. acuminata 1.34 and M. delicosa 1.54, indicating structures intermediate
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between a highly crystalline structure and a liquid. The curve fits to the spectra also included
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two Fermi resonances of the CH2 bond one at 2930 cm-1 and another very broad Fermi
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resonance at approximately 2870 cm-1 (Snyder et al., 1978). The contributions due to the CH3
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symmetric and anti-symmetric stretches were small as proportionally there are much less of
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these groups due to the long chain lengths. The fitting parameters for each wax along with the
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goodness of fit are included in the supplementary information.
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Two of the species investigated, Dudleya anthonyi (Dudleya) and Xerosicyos danguyi (Silver
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Dollar Plant), showed dramatically different Raman spectra from A. thaliana, T. parvula, M.
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acuminata and M. delicosa, characterised by additional peaks which are indicative of a more
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complex chemical structure, with additional chemical bonds to those described in Table 1. In D.
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anthonyi, the Raman spectrum changed depending on the number of the hexane wash and
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hence, we surmise, on the depth of penetration into the cuticle; the initial, most superficial
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hexane wash showed an unusual Raman spectrum, but subsequent washes that solubilise
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waxes deeper in the cuticle showed spectra which matched more closely with those of a typical
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alkane. These differences are most readily explained by both these species possessing a thick,
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glaucous cuticle compared to the other mesophytes studied, and is an adaptation enabling
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survival in xeric environments.
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In-Vivo Comparison of Coherent anti-Stokes Raman scattering (CARS) and stimulated Raman
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scattering (SRS) images of plant cuticle.
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Having characterised the Raman spectra of waxes extracted from the cuticle of a variety of
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plants, we imaged the cuticles of the same plants in vivo, using both coherent anti-Stokes
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Raman scattering (CARS) microscopy (Weissflog et al, 2010) and stimulated Raman scattering
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(SRS) microscopy (Fig. 3). SRS and CARS images of the epicuticular wax layer of T. parvula
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leaves were acquired simultaneously (Fig. 3) and are presented as the exemplar to compare
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the two techniques.
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The
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autofluorescence from cell walls and chloroplasts (Fig. 3A). This autofluorescence is due to
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inevitable two photon-absorption by the compounds in these organelles, and the resulting
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fluorescence emission at similar wavelengths to the CARS signal. Consequently, in the CARS
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images, only the largest wax crystals can be visualized.
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Conversely, in the SRS image (Fig. 3B) there is no fluorescent background because the signal
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measured is not due to a change in wavelength (i.e. fluorescence relative to excitation), but a
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change in intensity between the excitation lasers (the pump and Stokes beams) following
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stimulated Raman excitation of the samples. The cuticular wax crystals are clearly visible and
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the SRS signal spectrally matches that of the spontaneous Raman spectrum of purified cuticular
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waxes (Fig. 3C). Cell walls are also visible in the SRS images, although with a lesser intensity
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than the wax crystals, which can be explained by the overlap between the Raman spectra of the
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cuticular waxes and cellulose. SRS microscopy therefore provides not only clearer images of
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plant cuticle structure than CARS microscopy, but, due to the low-levels of background noise in
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the images, also has the capacity to yield information on specific compounds that compose the
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imaged structures.
images
acquired
using
CARS
are
dominated
by
high-levels
of
background
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Comparison of stimulated Raman scattering (SRS) and Scanning Electron Microscope Images
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of Plant Cuticle.
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To confirm the capacity of SRS microscopy for accurately resolving cuticular structures, the
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surface of A. thaliana stems were imaged using SRS microscopy and the more conventional
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scanning electron microscopy (SEM; Fig. 4). SRS and SEM images of untreated, wild-type
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stems (Fig. 4A and 4B) show an uneven surface composed of similarly-proportioned globules,
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although the level of detail is higher in the SEM image. We ascertained that these globular
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structures were indeed waxes by washing the stems in hexane prior to imaging (Fig. 4C and
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4B). In this case, both images show that the surface of the stem is smooth, with clearly visible
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cells and stomata. Finally, stems from A. thaliana defective for ECERIFERUM1 (CER1) gene
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expression were imaged using either technique (Fig. 4E and 4F). cer1 mutants are impaired in
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cuticle wax biosynthesis (Aarts et al. 1995; Bernard et al. 2012), and the images indeed show
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stems with markedly reduced surface structures than the wild-type (Fig. 4A and 4B).
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Unlike SEM which provides information only on the surface topology of imaged samples, SRS
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microscopy is a technique of multi-photon confocal microscopy that, like conventional confocal
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microscopy, can penetrate materials and provide information on their subsurface structure. To
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illustrate this technical capability, we imaged the cuticles of M. acuminate (Banana) and X.
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danguyi (Silver Dollar Plant; Fig. 5). M. acuminate was chosen as it is a monocot, with a visibly
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waxy cuticle and regular files of elongated, epidermal cells. X. danguyi was chosen because of
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its thick, glaucous cuticle composed of different waxes with different Raman spectra.
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Three-dimensional reconstructions of confocal SRS images of M. acuminate (Fig. 5A and 5C)
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and X. danguyi (Fig. 5B and 5C) cuticles show very different structures. The M. acuminate
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cuticle is formed of a number of hair-like projections from the leaf surface. In the comparator
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SEM image (Fig. 5E), these projections clump together, most likely due to the process of
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fixation, and show a less even distribution. In contrast, the X. danguyi cuticle has an amorphous
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surface, supported by a reticulate pattern (6 - 10 µm below the surface) of columnar structures
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emerging from the leaf epidermis. The amorphous surface of the X. danguyi cuticle is clear on
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the comparator SEM image (Fig. 5F), but the intriguing cuticle sub-structure is not.
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The comparison between SRS and SEM images therefore demonstrates that sufficient contrast
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can be produced using SRS microscopy to provide credible, structural images of waxes with
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submicron resolution and that correlate well with images from SEM and can provide critical,
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structural information not previously available.
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Simultaneous Ultrastructural Imaging and Chemical Analysis of Cuticle by stimulated Raman
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scattering microscopy.
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As previously noted, SRS imaging relies on the chemically-specific Raman spectrum of the
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cuticle constituents. To verify the potential of the technique to provide structural and chemical
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information, simultaneously and in vivo, we first analysed the cuticle of D. anthonyi (Dudleya;
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Fig. 6). D. anthonyi was selected for this aspect of the investigation because previous work had
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shown stratification of the composition of cuticular wax in the D. anthonyi cuticle (Jetter et al.,
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2000).
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Both the SRS (Fig. 6A) and SEM (Fig. 6B) images of the D. anthonyi cuticle show a complex
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structure; the outer cuticle layer has a structure in which many small wax crystals are amassed
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together with the deeper layers of the cuticle having a smoother, somewhat reticulated or
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wavelet-like appearance (Fig. 6D). Most importantly for this investigation, it is clear that the wax
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composition, as reported by the SRS spectra (Fig. 6C) was different depending on cuticle depth.
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The SRS spectrum acquired from deeper in the cuticle was most similar to an alkane whereas
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the SRS spectrum from the surface components was markedly different, indicating a different
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chemical composition.
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It is well documented that changes in wax composition and deposition during development and
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in response to abiotic stress or pathogen attack are important in the life history of many plant
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species (Bourdenx et al 2011, Raffaele et al. 2009). T. salsuginea is particularly interesting
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because it is a close relative of A. thaliana and T. parvula, and its cuticle composition and
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structure alters following exposure to prolonged cold treatment or vernalisation (Teusink et al.
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2002; Amtmann 2009; Xu et al. 2013).
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To determine whether SRS microscopy could, in principle, document the ultrastructural and
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chemical changes in cuticle that may occur during plant development, we imaged leaves from
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T. salsuginea plants that had been grown either in constant temperature or exposed to a 14-day
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long cold-shock, using SRS microcscopy and SEM (Fig. 7).
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temperature of 20°C show a cuticle containing aggregates of waxes on the adaxial surface of
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the leaf (Fig. 7A and 7B), and relatively little cuticular waxes on the abaxial surface (Fig. 7C and
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7D). However, following incubation at 4˚C for 14 days, a marked alteration in cuticule structure
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can be observed on both the adaxial and abaxial surfaces (Fig. 7C to 7H): A marked increase
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in waxy deposits in the cuticle was observed covering the entire leaf.
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ultrastructural aggregation of the cuticle waxes was smaller and more regular that observed on
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the adaxial surface of leaves grown in a constant temperature of 20˚C (Fig. 7A and 7B). SRS
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microscopy therefore precisely reported the anticipated changes in cuticle formation that occur
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following exposure to cold in T. salsuginea, and, by extension may prove a useful, in vivo tool
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for investigating other dynamic changes in cuticle architecture and composition during
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development and in response to environmental or biotic perturbation.
Plants grown at a constant
Moreover, the
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Discussion
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The cuticle is a composite, hydrophobic layer composed mainly of waxes and cutin secreted
328
from the aerial epidermis of land plants. The cuticle performs a number of physiological roles,
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notably acting as a barrier to water loss which, in higher plants, enables the exquisite control of
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evapotranspiration by stomatal guard cells, increasing light reflection and heat tolerance in
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xerophytes, and providing an essential barrier to the ingress of toxins and pathogens into the
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plant.
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techniques that provide only a fragmented understanding of the complexity of this essential
334
structure.
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In this investigation, we have generated in vivo images of cuticle waxes at sub-micron
336
resolution, using spontaneous Raman scattering (SRS) microscopy. Like other forms of non-
337
linear Raman-based imaging techniques, SRS is label-free and enables the simultaneous
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acquisition of chemically specific data and confocal quality images. Most importantly, SRS
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microscopy relies on a change in signal intensity rather than wavelength, so images can be
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acquired without the strong auto-fluorescent background from chloroplasts and cell walls that
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hampers the resolution and interpretation of coherent anti-Stokes Raman scattering (CARS)
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and spontaneous Raman images (Gierlinger et al., 2010, 2012).
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The structures observed using SRS microscopy compared well to those seen under the SEM,
344
albeit with a lesser degree of resolution due to the inherent limitations of optical microscopy
345
relative to electron microscopy. However, unlike SEM which generates images from the surface
346
of fixed or cryogenically preserved samples, SRS imaging, like other forms of single- or multi-
347
photon laser microscopy, enables information to be acquired in vivo from within the sample and
348
three-dimensional reconstructions of internal structures to be reconstructed. This capacity is
349
particularly advantageous for documenting dynamic changes of topography or composition that
350
may occur and that are otherwise undetectable using fixed samples.
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As demonstrated here, the chemical specificity of the SRS signal allows differentiation of
352
different wax components from the main constituents of cell walls, cellulose and pectins. In this
353
study, we showed that the xerophytes D. anthonyi and X. danguyi had different cuticular wax
354
compositions to the other, mesophytic plants investigated. Although this result is unsurprising
355
as these plants were selected for precisely their singularly thick and glaucous cuticles, it does
356
demonstrate the potential of SRS for in vivo characterisation of cuticle constituents.
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Moreover, although we did not attempt in this study to quantify in situ the different compounds
358
for which we acquired SRS spectra, it may be possible to use this technique in a more
359
quantifiable manner to characterise, in vivo, the relative abundance of cuticle or cell wall
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components and the changes that may occur in response to environmental or developmental
361
stimuli. In this investigation, we have shown that SRS imaging is sufficiently sensitive enough
Cuticle topography and composition has mainly been investigated using destructive
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to show developmental changes in wax production in T. salsuginea. Moreover, the technique
363
may be useful for characterising wax biosynthesis mutants, such as the exemplar used here,
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cer1, which has been shown to have a role in both water use and Arabidopsis interaction with
365
fungal and bacterial pathogens (Bourdenx et al. 2011, Raffaele et al. 2009). SRS microscopy
366
may therefore provide new insights into the physiological responses of plants to drought,
367
temperature, chemicals or pathogens, and underpin the use of alternative model systems such
368
as T. salsuginea (Amtmann 2009) to investigate the control of cuticle deposition.
369
However promising, any new technology or application must be simplified to enable widespread
370
use.
371
However, commercial, user-friendly CARS microscopes that can be converted to SRS imaging
372
exist and, like laser confocal microscopy, will become increasingly common and add real-time,
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label-free Raman microscopy to the range of imaging techniques widely available to
374
researchers in plant science.
Currently, SRS microscopy is somewhat niche and requires specialist knowledge.
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Conclusion
377
The chemical composition and structure of the cuticle in mesophyllous plants are crucial
378
elements for survival, physiology and development. We have shown that spontaneous Raman
379
scattering (SRS) microscopy can be used to acquire in-vivo, three-dimensional images of the
380
cuticle in a variety of plant species that enable simultaneous analysis of cuticle structure and
381
chemical composition. In contrast to coherent anti-Stokes Raman (CARS) microscopy, which is
382
more common, SRS images of plant tissues contain very low autofluorescence from
383
chloroplasts or cell walls and are therefore easier to interpret. Moreover, SRS images compare
384
well with those acquired using scanning electron microscopy (SEM), albeit with the lower
385
resolution that is due to the use of laser light rather than an electron beam. Moreover, we have
386
shown that SRS can be used to track and quantify dynamic changes in cuticle structure and
387
composition in response to environmental stimuli, and therefore increase our understanding of
388
this essential and often overlooked structure.
13
389
Materials and Methods
390
391
Plants.
392
Xerosicyos danguyi (Silver Dollar Plant), Dudleya anthonyi
393
(Banana), Monstera delicosa (Cheeseplant) were grown in soil, in the University of Exeter
394
glasshouses and harvested between February and April 2012. The Photoperiod was set to 16 h,
395
from 05.00 to 21.00. Supplemental lighting and shading was provided to ensure irradiance
396
beween 540 and 720 µmol s-1 m-2. The greenhouse temperature was set to 19.5 ˚C with a
397
standard deviation of 3.7 ˚C.
398
Thellungiella salsuginea and Thellungiella parvula (Saltwater Cress) were grown in a 16 h light /
399
8 h dark (16L/8D) photoperiod, at 20 ˚C, in growth rooms controlled for temperature and
400
humidity. 4 weeks after germination, Thellungiella species were transferred to an ambient
401
temperature of 4 ˚C for two weeks and then returned to 20 ˚C for a further 2 weeks. The light
402
regime remained at 16L/8D during all experiments. Leaves were harvested for imaging and
403
biochemical analysis.
(Dudleya), Musa acuminate
Arabidopsis thaliana (Mouse-eared Cress; Arabidopsis),
404
405
Purification of Wax from Plant Cuticle.
406
Following excision from plants, leaf surface areas were measured and logged. Leaf surfaces
407
were washed for 30 s in 15 ml HPLC-grade hexane (Sigma, U.K.). The hexane solvent and
408
dissolved cuticular waxes was decanted into a glass vial that had been washed with acetone
409
and dried. Leaves were washed a second time with 2 ml hexane, which was added to the
410
appropriate sample vial. Hexane was evaporated under a continuous stream of N2 to dryness
411
and the cuticle wax re-dissolved in 250 µl hexane.
412
413
Spontaneous Raman Spectroscopy.
414
Spontaneous Raman spectra of purified wax samples were acquired using a Renishaw RM100
415
Raman microscope (Renishaw plc, U.K.), equipped with a 785nm diode laser and a 1200
416
line/mm spectral grating which gave a spectral resolution of 1 cm -1. Samples were mounted on
417
aluminium-coated microscope slides.
418
419
Stimulated Raman Scattering Microscopy.
420
A detailed technical explanation of the materials and methods for stimulated Raman scattering
421
(SRS) microscopy is provided to promote the implementation of the technique as widely as
422
possible.
423
Stimulated Raman Gain Microscopy (SRG) was two laser systems were used: The Stokes
424
beam was provided by the signal output from the picosecond (ps) Optical Parametric Oscillator
425
(Levante, Emerald APE) pumped by the frequency doubled output from a Nd:Van ps oscillator
14
426
(picoTrain, HighQ laser). The pump beam was provided by a Ti:Sapphire laser (MIRA 900 D,
427
Coherent) tuned to 770 nm and operating in ps mode, which was electronically synchronised to
428
the Nd:Van laser (Coherent, Synchro-lock AP) thereby ensuring the laser pulses were
429
temporally overlapped.
430
The 770nm pump beam was amplitude modulated at 1.7MHz using an acousto-optical
431
modulator (AOM) (Crystal Technology Inc., USA, model 3080-122) and combined with the
432
signal output from the OPO using an 850 nm short pass dichroic mirror. Imaging was performed
433
using a modified confocal laser scanning unit (Flouview 300, Olympus, U.K.) and Olympus IX71
434
inverted microscope. The laser light was focused onto the sample using a 60x 1.2 / NA water
435
immersion microscope objective (UPlanS Apo, Olympus, UK). The transmitted light from the
436
sample was collected with a 60x 1.0 / NA water dipping condenser (LUMFI, Olympus, U.K.).
437
The transmitted Stokes beam was detected using a Si: photo-diode (Thorlabs, FDS1010) with a
438
70 V reverse bias. The pump beam was blocked from the photo-diode using an 850 nm long
439
pass filter (hq850lp, Chroma Technologies). The output from the photo-diode was passed
440
through a long pass filter (mini circuits, BLP-1.9+) to remove modulations at the 76 MHz laser
441
repetition rate and terminated by a 50 Ω resistor. The filtered beam was then fed into a lock-in
442
amplifier (Zurich instruments, HF2L1 Lock-in amplifier) that separated out the modulated
443
stimulated Raman scattering (SRS) signal at 1.7 MHz. SRS spectra were obtained by tuning
444
wavelength of signal beam in 0.2 nm intervals and acquiring a series of images.
445
Stimulated Raman gain was measured in preference to stimulated Raman loss, as it allowed
446
the chemically specific Raman signal to be separated from any additional signal due to two
447
photon absorption (Ye et al., 2009) or photo-thermal lensing (Moger et al., 2012; Lu et al., 2010)
448
by phase-sensitive lock-in detection (Mansfield et al., 2013).
449
Fresh, excised plant leaves were mounted in perfluorodecalin (Littlejohn et al., 2010) prior to
450
imaging, which is has been shown not to interfere with Raman-based imaging in plants
451
(Littlejohn et al. 2014; Mansfield et al. 2013).
452
453
454
Scanning Electron Microscopy
455
Leaf samples were imaged using cryogenic scanning electron microscopy. Samples were flash-
456
frozen in liquid N2 slush, transferred to a vacuum and coated in gold using the Gatan Alto 2100
457
system. Images were acquired using a JEOL JSM-6390 LV scanning electron microscope
458
operating at 5 kV with a working distance of 10-12 nm.
15
459
Acknowledgements
460
461
We thank Peter Splatt of the Exeter Bioimaging Centre for his technical assistance with the
462
scanning electron microscopy and James Chidlow for plant samples from the University of
463
Exeter glasshouse collection. Thanks also to Dr. Anna Amtmann of the University of Glasgow,
464
who generously provided seeds of Thellungiella species used. We also gratefully acknowledge
465
the support of Biomedical Physics and of the Exeter Imaging Network.
16
466
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Figure and Table Legends
681
682
Figure 1 Title: Schematic representation of the two coherent Raman scattering processes:
683
coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS).
684
22
685
Figure 1 Legend: Panel A: Energy level diagrams for the CARS and SRS processes, showing
686
the pump (green), Stokes (red) and anti-Stokes (blue) photon energies,
687
Panel B: Diagrammatic representation of the input and output spectra for CARS and SRS,
688
showing the gain and loss in the pump (red) and Stokes (green) beams respectively.
689
Panel C: Diagrammatic representation of the modulation transfer detection scheme used to
690
detect stimulated Raman gain and loss with high sensitivity.
691
692
693
694
Figure 2 Title: Raman spectra and curve fits of plant leaf waxes.
695
696
Figure 2 Legend: A: Raman spectra of cuticle waxes purified from Xerosicyos danguyi (Silver
697
Dollar Plant), Dudleya anthonyi
698
Arabidopsis), Musa acuminate (Banana) and Thellungiella parvula (Saltwater Cress). Peak
699
fitting to the Raman spectra of T. parvula (B), D. antonyi (C) and X. danguyi (D).
(Dudleya) Arabidopsis thaliana (Mouse-Eared Cress;
700
701
702
703
Figure 3 Title:
704
images of the Thellungelia parvula leaf surface.
Coherent anti-Stokes Raman scattering and stimulated Raman scattering
705
706
Figure 3 Legend: The surface of a leaf from a Thellungelia parvula was simultaneously imaged
707
using coherent anti-Stokes Raman scattering (CARS) microscopy (panel A) and stimulated
708
Raman scattering (SRS) microscopy (panel B). Both images were acquired at 2845cm -1 CH2
709
symmetric stretch. The CARS image (A) is dominated by autofluorescence, and only the largest
710
wax crystals are visible against the background. Conversely, the SRS image (B) is almost
711
background free, enabling the cell walls and wax crystals to be clearly visualised. Panel C
712
shows the spectral scan of the SRS (red crosses) from the wax crystals in situ overlaid onto the
713
spontaneous Raman spectra of purified T. parvula wax (blue line), purified cellulose (green line)
714
and purified pectin (purple line).
715
716
717
718
Figure 4 Title: Stimulated Raman scattering images and scanning electron micrographs of
719
Arabidopsis thaliana cuticle.
720
23
721
Figure 4 Legend: The surface of Arabidopsis thaliana stems were imaged using stimulated
722
Raman scattering microscopy (panels A, C and D) and scanning electron microscopy (panels,
723
B, D and E).
724
ecotype “Landsberg erecta”, with the structure of cuticle wax crystals clearly visible. Panels C
725
and D show the surface of the wild-type A. thaliana stem following a wash in hexane that
726
dissolved the cuticle waxes. Panels E and F show the surface of the stem from a cer 1 A.
727
thaliana mutant. Images A, C and E are generated from 3D stacks taken of a 64 x 64 µm field of
728
view and are displayed in false colour.
Panels A and B show the surface of the untreated stem of wild-type A thaliana,
729
730
731
732
Figure 5 Title: Stimulated Raman scattering images and scanning electron micrographs of
733
cuticle of Musa acuminate and of Xerosicyos danguyi.
734
735
Figure 5 Legend: The surface of Musa acuminate (banana) and of Xerosicyos danguyi (Silver
736
dollar Plant) leaves were imaged using stimulated Raman scattering (SRS) microscopy (panels
737
A, B, C and D) and scanning electron microscopy (SEM) (panels E and F). Panel A is a SRS
738
image of the adaxial leaf surface of surface M. acuminate constructed from an image stack from
739
a 64 x 64 µm field of view. Panel B is a SRS image of X. danguyi leaf reconstructed from an
740
image stack from a 126 x 126 µm field of view. Panels C and D are orthogonal views projected
741
from the image stacks presented in A and B, respectively, and show the depth profile of
742
cuticles. Panels E and F are SEM images of the cuticles of M. acuminate and X. danguyi
743
leaves, respectively.
744
745
746
747
Figure 6 Title: Stimulated Raman scattering spectral images of Dudleya anthonyi cuticle.
748
749
Figure 6 Legend: Stimulated Raman scattering (SRS) spectral imaging of Dudleya anthonyi
750
(Dudleya) cuticle waxes shows changes in chemical composition with depth. Panels A and B
751
are, respectively, the SRS and SEM images of D. anthonyi leaf showing the crystalline structure
752
of cuticle wax deposits on the adaxial surface. The image in panel A is a 3-D reconstruction of
753
an image stack taken from a 126 x 126 µm field of view at the 2840 cm -1 Raman shift. Panel C
754
shows the SRS spectral scans taken from the more superficial and deeper wax areas, indicated
755
on Panel D. The blue rectangle indicates an area of superficial wax and the red line delimits an
756
area deeper in the cuticle. The panels on the right and lower sides of the main image in Panel
24
757
D are from the orthogonal views through the stack showing the depth profile image of the cuticle
758
wax.
759
760
761
762
Figure 7 Title: Stimulated Raman scattering images and scanning electron micrographs of
763
cuticle of the cuticle of Thellungiella salsuginea following cold-induced wax biogenesis. Musa
764
765
Figure 7 Legend: Thellungiella salsuginea (Saltwater Cress) leaves were imaged using
766
Stimulated Raman scattering (SRS) spectral imaging and scanning electron microscopy (SEM)
767
before and after cold treatment. Panels A, C, E and G are three dimensional reconstructions of
768
image stacks from a 250 x 250 µm field of view, at the 2840cm -1 Raman shift. Panels B, D, F,
769
and H are SEM images; scalebars represent 50 µm. Panels A-B and C-D are, respectively, from
770
the adaxial and abaxial leaf surfaces of plants grown at 20°C for 8 weeks. Panels E-F and G-H
771
are, respectively, from adaxial and abaxial leaf surfaces of plants grown at 20°C for 4 weeks,
772
then at 4˚C for 2 weeks and at 20 ˚C for a further 2 weeks.
773
774
775
776
Table 1 Title: Raman peaks used in curve fitting for cuticular waxes.
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