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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
Fluorescence lifetime spectral image acquisition. Time-resolved fluorescence emission spectra were collected with a streak-camera spectrograph equipped with a blue laser diode emitting 50 ps pulses at 405 nm (Hamamatsu 4334, Hamamatsu Photonics Inc., Hamamatsu, Japan). The sample compartment consisted of a positionable x, y, z stage fitted to accept the coral samples in an otherwise light-tight compartment. The coral specimens were immersed in seawater at room temperature in a 1 by 1 cm quartz cuvette (or low-UV fluorescence plastic cuvette) in a front-surface configuration to receive the laser excitation. Diode emission was filtered before the sample by an interference filter (409 nm, 10 nm bandwidths, 90% transmittance). The total spectral region of the sample's fluorescence emission was controlled by adjusting the spectrograph's (Chromex 2501-S) central wavelength while maintaining the emission grating properties (grating = 50 grooves/mm, blaze = 600 nm and slit = 30 [mu].m). Separate images were collected to focus on either the FP emissions (central wavelength, between 500 and 545 nm) or both the FP emission and algal chl emission (central wavelength, 610 nm). In both cases sample emission was filtered with long-pass filters to exclude light below 420 nm, including the laser excitation. Sample data were collected in photon-counting mode with a 5 ns time window until a total of 25 000 or 125 000 excitation shots accumulated (at a rate of approximately 32.4 Hz) for the FP-only emission or FP plus zooxanthellar emission, respectively. The scattered excitation light profile of the laser pulse was collected (10000 shots) with the spectrograph center wavelength at 500 nm after placing both 1% and 2% neutral density filters between the sample and the spectrograph and removing the 420 nm long-pass filter. The 640 (wavelength axis) by 480 (kinetic axis) pixel charged coupled device (CCD) image was exported from the Hamamatsu software in ASCII format and imported into Excel 97. The pixels were binned and integrated at a rate of 5 pixels per time channel and 3 pixels per wavelength channel and subjected to a further three-channel adjacent averaging of the spectral profiles. The chosen spectral region of interest was restricted to 100 time channels ([lambda] = 1.11 nm/ch) and 160 wavelength channels (t = 31.25 ps/ch) for global analysis using the DCI method, as described below.
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Global analysis of the time-resolved emission spectra and FRET using the DCI method. The DCI method (27) constituted the foundation for the spectral kinetic models used to simulate the time-resolved spectra and FRET. The DCI method centers around two assumptions, namely, (1) at any point in time after an excitation event the fluorescence emission spectrum of any compound can be simulated assuming it is composed of a sum of a finite number of Gaussian spectral components; and (2) only the amplitude and not the spectral center or width of the component changes as a function of time. In the present analyses, the decay kinetics for each Gaussian spectral band were simulated using three to four Gaussian kinetic distribution modes, with either positive or negative amplitudes, to represent the integral distribution of the pre-exponential amplitude factor as a function of the fluorescence lifetime (([alpha]([tau]);). The integral of ([alpha]([tau]); was then used to calculate the fluorescence intensity as a function of time,
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Most Popular Articles in Reference
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