Perfluorodecalin enhances <i>in vivo</i> confocal microscopy resolution of <i>Arabidopsis thaliana</i> mesophyll

Littlejohn, George R.; Gouveia, João D.; Edner, Christoph; Smirnoff, Nicholas; Love, John

doi:10.1111/j.1469-8137.2010.03244.x

Cited by 81 publications

(70 citation statements)

References 35 publications

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“…Arabidopsis cotyledons and N. benthamiana leaves were prepared as described 65 and a Zeiss LSM710 laser scanning confocal microscope (Zeiss, Germany) was used to visualize GFP fluorescence (excitation: 488 nm; emission: 505-530 nm), RFP fluorescence (excitation: 561 nm; emission: 570 to 620 nm) and chlorophyll autofluorescence (excitation: 633; emission: 650-750 nm). Images were taken using a LD C-Apochromat 40x/1.1 W Korr objective.…”

Section: Methodsmentioning

confidence: 99%

Arabidopsis AZI1 family proteins mediate signal mobilization for systemic defence priming

et al. 2015

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Priming is a major mechanism behind the immunological 'memory' observed during two key plant systemic defences: systemic acquired resistance (SAR) and induced systemic resistance (ISR). Lipid-derived azelaic acid (AZA) is a mobile priming signal. Here, we show that the lipid transfer protein (LTP)-like AZI1 and its closest paralog EARLI1 are necessary for SAR, ISR and the systemic movement and uptake of AZA in Arabidopsis. Imaging and fractionation studies indicate that AZI1 and EARLI1 localize to expected places for lipid exchange/movement to occur. These are the ER/plasmodesmata, chloroplast outer envelopes and membrane contact sites between them. Furthermore, these LTP-like proteins form complexes and act at the site of SAR establishment. The plastid targeting of AZI1 and AZI1 paralogs occurs through a mechanism that may enable/facilitate their roles in signal mobilization.

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Section: Methodsmentioning

confidence: 99%

Arabidopsis AZI1 family proteins mediate signal mobilization for systemic defence priming

et al. 2015

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“…During stratification and germination time courses, embryos were carefully removed from the testa and endosperm by the application of gentle pressure to seeds in water between microscope slide and cover slip. All tissue was mounted in perfluorodecalin (octadecafluorodecahydronaphtalene; Fluka) (Littlejohn et al, 2010). For all experiments, each image stack corresponds to a different embryo.…”

Section: Live-cell Imagingmentioning

confidence: 99%

Arabidopsis Seed Mitochondria Are Bioenergetically Active Immediately upon Imbibition and Specialize via Biogenesis in Preparation for Autotrophic Growth

et al. 2017

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Seed germination is a vital developmental transition for production of progeny by sexual reproduction in spermatophytes. Quiescent cells in nondormant dry embryos are reawakened first by imbibition and then by perception of germination triggers. Reanimated tissues enter into a germination program requiring energy for expansion growth. However, germination requires that embryonic tissues develop to support the more energy-demanding processes of cell division and organogenesis of the new seedling. Reactivation of mitochondria to supply the required energy is thus a key process underpinning germination and seedling survival. Using live imaging, we investigated reactivation of mitochondrial bioenergetics and dynamics using Arabidopsis thaliana as a model. Bioenergetic reactivation, visualized by presence of a membrane potential, is immediate upon rehydration. However, reactivation of mitochondrial dynamics only occurs after transfer to germination conditions. Reactivation of mitochondrial bioenergetics is followed by dramatic reorganization of the chondriome (all mitochondrial in a cell, collectively) involving massive fusion and membrane biogenesis to form a perinuclear tubuloreticular structure enabling mixing of previously discrete mitochondrial DNA nucleoids. The end of germination coincides with fragmentation of the chondriome, doubling of mitochondrial number, and heterogeneous redistribution of nucleoids among the mitochondria, generating a population of mitochondria tailored to seedling growth.

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“…The leaves were treated 24hrs prior to imaging for azoxystrobin and chlorothalonil and 30minutes prior to imaging for deuterated glyphosate. For imaging, the leaves were mounted between 2 coverslips using perfluorocarbon as a mounting medium 17 .…”

Section: Sample Preparationmentioning

confidence: 99%

Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy

Mansfield

Littlejohn²,

Seymour³

et al. 2013

Anal. Chem.

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The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas.There is currently a lack of analytical tools that provide non-invasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide labelfree chemically specific image contrast based on vibrational spectroscopy. Over the past decade these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis; it does not suffer from photobleaching; and allows near realtime imaging in 3D with submicron spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll) the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored.In this paper we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally we demonstrate the potential of SRS for a range of in planta applications by presenting in-situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

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Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll

Cited by 81 publications

References 35 publications

Arabidopsis AZI1 family proteins mediate signal mobilization for systemic defence priming

Arabidopsis AZI1 family proteins mediate signal mobilization for systemic defence priming

Arabidopsis Seed Mitochondria Are Bioenergetically Active Immediately upon Imbibition and Specialize via Biogenesis in Preparation for Autotrophic Growth

Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy

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