Synchrotron radiation as a light source in confocal microscopy

Oord, C. J. R. van der; Gerritsen, Hans C.; Levine, Y.K.; Myring, W. J.; Jones, Gareth R.; Munro, Ian

doi:10.1063/1.1142674

Cited by 16 publications

(6 citation statements)

References 1 publication

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“…Illumination at 442 nm was provided by an Omnichrome helium-cadmium laser using a 20-~tm pinhole and a 40 • objective. Fluorescence was selected using a 580-rim dichroic mirror (van der Oord et al 1992). A region 200 gm x 200 ~m was scanned with a vertical resolution of 4.5 lam.…”

Section: Confocal Fluorescence Imagingmentioning

confidence: 99%

Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence

Scholes¹,

Rolfe²

1996

Planta

141

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Abstract. Localised changes in photosynthesis in oat leaves infected with the biotrophic rust fungus Puccinia coronata Corda were examined at different stages of disease development by quantitative imaging of chlorophyll fluorescence. Following inoculation of oat leaves with crown rust the rate of whole-leaf gas exchange declined. However, crown rust formed discrete areas of infection which expanded as the disease progressed and these localised regions of infection gave rise to heterogeneous changes in photosynthesis. To quantify these changes, images of chlorophyll fluorescence were taken 5, 8 and 11 d after inoculation and used to calculate images representing two parameters; ~u, a measure of PSII photochemical efficiency and AFm/Fm', a measure of non-photochemical energy dissipation (qN). Five days after inoculation, disease symptoms appeared as yellow flecks which were correlated with the extent of the fungal mycelium within the leaf. At this stage, ~l~ was slightly reduced in the infected regions but, in uninfected regions of the leaf, values of ~n were similar to those of healthy leaves. In contrast, qN (AFm/Fm') was greatly reduced throughout the infected leaf in comparison to healthy leaves. We suggest that the low value of qN in an infected leaf reflects a high demand for ATP within these leaves. At sporulation, 8 d after inoculation, Ou was reduced throughout the infected leaf although the reduction was most marked in areas invaded by fungal mycelium. In the infected leaf the pattern of non-photochemical quenching was complex; qN was low within invaded regions, perhaps reflecting high metabolic activity, but was now much higher in uninfected regions of the infected leaf, in comparison to healthy leaves. Eleven days after inoculation "green islands" formed in regions of the leaf associated with the Abbreviations: qP~ = photochemical efficiency of photosystem II; qN = non-photochemical energy quenching; AFm/Fm' = a measure of qN; dai = days after inoculation; IRGA = infra-red gas analyser Correspondence to: J.D. Scholes; FAX: 44 (114)276 0159; E-mail: j.scholes@sheffield.ac.uk fungal mycelium. At this stage, photosynthesis was severely inhibited over the entire leaf; however, heterogeneity was still apparent. In the region not invaded by the fungal mycelium, ~n and qN were very low and these regions of the leaf were highly fluorescent, indicating that the photosynthetic apparatus was severely damaged. In the greenisland tissue, (1)if was lOW but detectable, indicating that some photosynthetic processes were still occurring. Moreover, qN was high and fluorescence low, indicating that the cells in this region were not dead and were capable of significant quenching of chlorophyll fluorescence.

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Section: Confocal Fluorescence Imagingmentioning

confidence: 99%

Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence

Scholes¹,

Rolfe²

1996

Planta

141

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“…A tentative plan to install a DUV microscope coupled to synchrotron radiation was done at the Daresbury Laboratory in the middle of the 1990s~Van der Oord et al, 1992Oord et al, , 1995. However, despite one conceptual paper and one first paper about results in fluorescence decay, the system was subsequently only used with lasers.…”

Section: Introductionmentioning

confidence: 99%

Synchrotron UV Fluorescence Microscopy Uncovers New Probes in Cells and Tissues

et al. 2010

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Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

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“…3a and b show panchromatic CL images of an InGaN epilayer and an InGaN quantum well. 3 The epilayer shows a granular, bubble-like appearance and the emission of light is clearly associated with micrometric structures. High CL yield coincides with the centre of the crystallites.…”

Section: Resultsmentioning

confidence: 99%

“…Confocal microscopy uses a diffractionlimited laser spot to address the sample in a raster scan. The SYCLOPS facility at Daresbury Laboratories employs synchrotron radiation or laser sources for high-resolution imaging and fluorescence lifetime spectroscopy [3]. Sample temperatures are variable in the range from 77 K to room temperature and above.…”

Section: Samples and Experimental Detailsmentioning

confidence: 99%