title: An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.
abstract: Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.
authors.affiliation: Division of Plant and Microbial Sciences, School of Biosciences, University of Exeter Exeter, UK.
authors.forename: George R
authors.initials: GR
authors.lastname: Littlejohn
authors.affiliation: School of Physics, University of Exeter Exeter, UK.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Mansfield
authors.affiliation: Computer Science, University of Exeter Exeter, UK.
authors.forename: Jacqueline T
authors.initials: JT
authors.lastname: Christmas
authors.affiliation: Division of Plant and Microbial Sciences, School of Biosciences, University of Exeter Exeter, UK.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Witterick
authors.affiliation: Department of Plant Sciences, University of Oxford Oxford, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Fricker
authors.affiliation: Division of Plant and Microbial Sciences, School of Biosciences, University of Exeter Exeter, UK.
authors.forename: Murray R
authors.initials: MR
authors.lastname: Grant
authors.affiliation: Division of Plant and Microbial Sciences, School of Biosciences, University of Exeter Exeter, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Smirnoff
authors.affiliation: Computer Science, University of Exeter Exeter, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Everson
authors.affiliation: School of Physics, University of Exeter Exeter, UK.
authors.forename: Julian
authors.initials: J
authors.lastname: Moger
authors.affiliation: Division of Plant and Microbial Sciences, School of Biosciences, University of Exeter Exeter, UK.
authors.forename: John
authors.initials: J
authors.lastname: Love
keywords: Arabidopsis; confocal; imaging; microscopy; multi-photon; perfluorocarbon; perfluoroperhydrophenanthrene