Fluorescence lifetimes of protochlorophyllide in plants with different proportions of short-wavelength and long-wavelength protochlorophyllide spectral forms[para]

Photochemistry and Photobiology, Aug 2003 by Mysliwa-Kurdziel, Beata, Amirjani, Mohammad R, Strzalka, Kazimierz, Sundqvist, Christer

Phase and modulation data were measured in reference to a standard of carhoxyfluorescein at 10-15 modulation frequencies of the excitation light, ranging from 1 to 200 MHz. The fluorescence lifetime of the standard was determined at room temperature, in relation to a scattering solution of glycogen in water. During a lifetime measurement, both the standard and the leaf sample were placed in the dewars to give optimal symmetry. However, only the sample dewar was filled with liquid nitrogen.

Fluorescence lifetimes were measured for whole leaves that were inserted into open-ended glass rods suited for the dewars. Care was taken to cover the area of the light beam with leaf tissue and to load the leaves in a similar way each time. For some samples, leaves were crushed in liquid nitrogen and mixed with glycerol at below 273 K. The resulting homogenate was transferred to the glass rods and used for measurements. The spectral properties were unchanged, but the reproducibility between samples was only slightly better; therefore, this method was not used routinely. For measuring lifetime values of Pchlide in organic solvents, Pchlide was isolated from dark-grown wheat leaves and partly purified as described by Lindsten et al. (25). The pigment was stored in diethyl ether at 255 K.

The evaluated average random errors of or -0.5[degrees] and or -0.01 for phase angle and modulation, respectively, were then used in fluorescence lifetime analysis.

Fluorescence lifetime calculations: analysis of phase and modulation results. A single measurement was repealed several times using a fresh sample. The phase shift and modulation data were analyzed for each measurement using the nonlinear least-square analysis program provided by Spex Industries (GRAMS program, Galactic Industries Corporation, Salem, NH) or the ISS (Urbana, IL) Company. A multiexponential model of fluorescence decay (26,27) was applied for the analysis. Fluorescence lifetime values and corresponding fractional intensities (fractions) for the different spectral components were calculated as results of the analysis. Decay-associated spectra (DAS). DAS were calculated as I^sub i^([lambda]) = f^sub i^([lambda])I^sub s^([lambda]) (28), where I^sub i^([lambda]) represents the DAS of the ith component, I^sub s^([lambda]) is the fluorescence intensity of the steady-state spectrum and f^sub i^([lambda]) is the mean fractional intensity of a fluorescence lifetime component.

Deconvolution of spectra. The computerized analyses of the spectra were carried out with the GRAMS program. The second derivatives were used to assist the Gaussian deconvolution of the spectra. To make comparisons with the lifetime measurements obtained, it was most important to find variations in fluorescence intensity of the different components. Therefore, the peak position was not allowed to vary more than or -1 nm, and the half-bandwidth was locked to a maximum of 12 nm. The deconvolution was made in a wavenumber function and then transferred to the wavelength function for representation in the figures. The sum of the Gaussian components deviated from the experimental spectra by less than 1%. All calculations were made on mean values of 10-15 spectra.