Bruce P. Wittmershaus

Spectral Properties of Single BODIPY Dyes in Polystyrene Microspheres and in Solutions

The absorption, fluorescence, fluorescence quantum yield, and photostability of five BODIPY dyes are characterized and compared as single dyes in two environments, in 40-nm polystyrene spheres and in solution. The absorption and fluorescence spectra of the dyes in spheres are similar in profile but shifted to lower energies compared to those in solution. All the dyes are highly fluorescent, with three having fluorescence quantum yields of 1.0. For three of the five dyes, the yields were the same in spheres as in solution (1.00, 1.00, and 0.73). The high concentration of these dyes in spheres does not quench their fluorescence. For two other dyes the yields dropped, from 1.00 to 0.55 in one case and 0.83 to 0.50 in another, comparing the dyes in solution versus in spheres. The photodegradation of the dyes decreases in spheres compared to in solution in all but one case. For one dye, it decreases as much as 800-fold. Dyes overlooked because of low fluorescence or stability in solution could become useful fluorescent materials in the microsphere environment.

Spectral resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 through direct excitation

We have measured fluorescence spectra from Photosystem I (PS I) on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 at room temperature as a function of excitation wavelength. Our data show a gradual enhancement of long-wavelength fluorescence at 710 nm as the excitation wavelength is increased from 695 to 720 nm. This verifies the presence of low-energy chlorophylls (LE Chls), antenna Chls with energy levels below that of the primary electron donor, P700. The change in fluorescence with excitation wavelength is attributed to the finite time it takes for equilibration of excitations between the bulk and LE Chls. The spectra were deconvoluted into the sum of two basis spectra, one an estimate for fluorescence from the majority or bulk Chls and the other, the LE Chls. The bulk Chl spectrum has a major peak at 688 nm and a lower amplitude vibrational band around 745 nm and is assumed independent of excitation wavelength. The LE Chl spectrum has a major peak at 710 nm, with shoulders at 725 and 760 nm. The relative amplitude of emission at the vibrational side bands increases slightly as the excitation wavelength increases. The ratio of the fluorescence yields from LE Chls to that from bulk Chls ranges from 0.3 to 1.3 for excitation wavelengths of 695 to 720 nm, respectively. These values are consistent with a model where the LE Chls are structurally close to P700 allowing for direct transfer of excitations from both the bulk and LE Chls to P700.

FLUORESCENCE FROM LOW-ENERGY CHLOROPHYLLS IN PHOTOSYSTEM I OF SYNECHOCYSTIS SP. PCC 6803 AT PHYSIOLOGICAL TEMPERATURES

Fluorescence spectra from Photosystem I (PS I) are measured from 25 to −5 °C on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Emission from antenna chlorophylls (Chls) with energy levels below that of the reaction center, or low-energy Chls (LE Chls), is resolved verifying their presence at physiological temperatures. The 25°C spectrum is characterized by peaks at 688 and 715 nm. As temperature decreases, fluorescence at 688 nm decreases while at 715 nm it increases. The total fluorescence yield does not change. The temperature dependent spectra are fit to a sum of two basis spectra. At 25°C, the first basis spectrum has a major peak at 686 nm and a minor peak at 740 nm. This is attributed to fluorescence from the majority or bulk antenna Chls. The second basis spectrum has a major peak at 712 nm, with shoulders at 722 and 770 nm. It characterizes fluorescence from a small number of LE Chls. A progressive shift to the red in the fluorescence spectra occurs as the temperature is decreased. The temperature dependence in the relative amount of fluorescence from the bulk and LE Chls is fit using a two-component energy transfer model at thermal equilibrium.