title: 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.
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: George R
authors.identifier: http://orcid.org/0000-0002-8768-2598
authors.initials: GR
authors.lastname: Littlejohn
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Mansfield
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: David
authors.identifier: http://orcid.org/0000-0003-2191-6416
authors.initials: D
authors.lastname: Parker
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: Rob
authors.identifier: http://orcid.org/0000-0002-1410-4033
authors.initials: R
authors.lastname: Lind
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: Sarah
authors.initials: S
authors.lastname: Perfect
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: Mark
authors.initials: M
authors.lastname: Seymour
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.).
authors.forename: Nicholas
authors.initials: N
authors.lastname: Smirnoff
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.) j.moger@exeter.ac.uk j.love@exeter.ac.uk.
authors.forename: John
authors.initials: J
authors.lastname: Love
authors.affiliation: School of Biosciences, College of Life and Environmental Sciences (G.R.L., N.S., J.L.), and Department of Physics and Astronomy, College of Engineering, Mathematics, and Physical Sciences (J.C.M., J.M.), University of Exeter, Exeter, Devon EX4 4QD, United Kingdom;Biodomain Technology Group, Shell International Exploration and Production, Inc., Westhollow Technology Center, Houston, Texas 77082 (D.P.); andSyngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom (R.L., S.P., M.S.) j.moger@exeter.ac.uk j.love@exeter.ac.uk.
authors.forename: Julian
authors.initials: J
authors.lastname: Moger
mesh_terms: D008853:Microscopy / Q000379:methods*; D019441:Plant Epidermis / Q000737:chemistry* / Q000648:ultrastructure; D018515:Plant Leaves / Q000737:chemistry; D010944:Plants / Q000737:chemistry*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014885:Waxes / Q000737:chemistry*
chemical_list: D014885:Waxes