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Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals[para][dagger]

Photochemistry and Photobiology, May 2003 by Gilmore, Adam M, Larkum, Anthony W D, Salih, Anya, Itoh, Shigeru, Et al

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MATERIALS AND METHODS

Sample collection, identification, storage and preparation. Samples of Plesiastrea versipora (blue and green color morphs) were collected from 5 to 9 m depth at Port Jackson (latitude 33[degrees]52'S), New South Wales, Australia, and were subsequently maintained in flow-through seawater aquaria in Sydney, New South Wales, Australia. Small samples (2-4 cm diameter) were shipped in less than 18 h from Sydney to either Canberra, Australian Capital Territories, Australia, or Nagoya, Japan, under room temperature and low light in tightly closed 50 mL plastic containers filled with seawater; this mode of transportation was previously found to be well tolerated by this species. Samples of P. versipora transported from Sydney to Canberra were analyzed within 24 h, during which they were stored in aerated natural seawater at room temperature in dim fluorescent room light.

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Coral specimens were collected under a special permit from the Okinawa Prefectural Fisheries issued to R.V.W. in 2001. Small fragments (5-10 cm) of Acropora nasuta and A. digitifera were collected from 1.5 m depth at Cape Maeda (Okinawa, Japan). Each fragment was placed inside a small plastic bag underwater and then the bags were placed inside a 3 L plastic container with a tight-fitting lid. The corals were shipped to Nagoya within several hours after collection using the "dry method" described by Carlson (25). Specimens of A. nasuta and A. digitifera arrived at Nagoya, Japan, by airfreight on the same day from Okinawa, Japan. The specimens were immediately unpacked and placed in a skim-filtered, aerated, room temperature (minimum 20[degrees]C-maximum 25[degrees]C) aquarium filled with 18 L of artificial seawater. The aquarium was supplemented with commercially available purple photosynthetic bacteria cultures to reduce toxic effects of nitrogen (ammonia waste from the corals). The aquarium was illuminated with fluorescent lamps (

Contour mapping of excitation-emission fluorescence spectra. Contour maps of the instrument-corrected emission versus excitation spectra were obtained with an SLM 8100 Spectrofluorimeter (Spectronic Instruments, Rochester, NY) in photon-counting mode (26). Sample specimens were cut with bone-cutters (0.5-1 cm^sup 2^ front-surface area) and submersed in natural seawater at room temperature in a quartz cuvette that was positioned in a front-surface cell-holder in the light-tight sample compartment. Second-order emissions were blocked from the excitation monochromator by a filter (Schott LS-500) that prohibited all light >500 nm from illuminating the sample. Contour maps focusing on the FP emission were constructed by scanning the excitation monochromator (4 mm, 2 nm/mm slit) from 350 to 450 nm in 1.5 nm increments; the emission monochromator (2 mm slit) was scanned from 450 to 700 nm at a rate of 3 nm/s corresponding to an integration rate of 0.33 s/nm. Contour maps focusing on the zooxanthellae emission were constructed by scanning the excitation monochromator (16 mm slit) from 350 to 450 nm in 2 nm increments; the emission monochromator (4 mm slit resolution) was scanned from 650 to 750 nm at an integration rate of 0.33 s/nm. The contour maps were exported using the SLM software in ASCII format and imported into Microsoft Excel 97 wherein they were normalized to the peak emission value. Two integral profiles were constructed as functions of wavelength for each map corresponding to the excitation and emission axes and were normalized to their maximal intensities.

Simultaneous time resolution of the emission spectra of fluorescent proteins and zooxanthellar chlorophyll in reef-building corals[para][dagger]

Photochemistry and Photobiology, May 2003 by Gilmore, Adam M, Larkum, Anthony W D, Salih, Anya, Itoh, Shigeru, Et al

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