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Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.

The bleaching dynamics of reconstituted single light-harvesting chlorophyll a/b investigated. The complexes containing one histidine6 tag per monomeric subunit were immobilised predominantly in a defined orientation with their symmetry axis perpendicular to a Ni-ion-containing surface allowing for the first time the examination of single LHCIIb in an aqueous environment. Most complexes exhibit photobleaching in one step, indicating coupling between the monomeric subunits leading to an energy transfer between adjacent subunits. Differences in bleaching behaviour between these and previous observations with single LHCIIb are discussed.

The bleaching dynamics of reconstituted single light-harvesting chlorophyll a/b investigated. The complexes containing one histidine6 tag per monomeric subunit were immobilised predominantly in a defined orientation with their symmetry axis perpendicular to a Ni-ion-containing surface allowing for the first time the examination of single LHCIIb in an aqueous environment. Most complexes exhibit photobleaching in one step, indicating coupling between the monomeric subunits leading to an energy transfer between adjacent subunits. Differences in bleaching behaviour between these and previous observations with single LHCIIb are discussed.

Spectroscopic and polarization properties of single light-harvesting complexes of higher plants (LHC-II) were studied at both room temperature and T<5K. Monomeric complexes emit roughly linearly polarized fluorescence light thus indicating the existence of only one emitting state. Most probably this observation is explained by efficient triplet quenching restricted to one chlorophyll a (Chl a) molecule or by rather irreversible energy transfer within the pool of Chl a molecules. LHC-II complexes in the trimeric (native) arrangement bleach in a number of steps, suggesting localization of excitations within the monomeric subunits. Interpretation of the fluorescence polarization properties of trimers requires the assumption of transition dipole moments tilted out of the symmetry plane of the complex. Low-temperature fluorescence emission of trimers is characterized by several narrow spectral lines. Even at lowest excitation intensities, we observed considerable spectral diffusion most probably due to low temperature protein dynamics. These results also indicate weak interaction between Chls belonging to different monomeric subunits within the trimer thus leading to a localization of excitations within the monomer. The experimental results demonstrate the feasibility of polarization sensitive studies on single LHC-II complexes and suggest an application for determination of the Chl transition-dipole moment orientations, a key issue in understanding the structure-function relationships.

Individual antenna complexes from different photosynthetic units have been investigated by single molecule spectroscopy. In such energy transfer systems the polarization of the fluorescence emission gives valuable information about the nature of the emitting state, which is not readily available with other methods like fluorescence excitation or emission spectroscopy. The peripheral antenna light harvesting complex from purple bacterium Rhodopseudomonas Acidophila shows predominantly linear polarized fluorescence emission at low temperature, whereas at room temperature the fluorescence is randomly polarized. This is attributed to the fact, that at low temperature in the fluorescence emitting state the excitation energy is localized mainly on 4-5 chromophores. Analysis of the fluorescence emission of single peripheral antenna complexes of green plants indicate that for trimers of this species more than one Chlorophyll is responsible for the final fluorescence emission, which points towards a weak intermonomer coupling in the complex.

Single-molecule spectroscopy dealing with single photosynthetic aggregate provides a possibility to resolve the optical transition of individual pigment, even under non-selective excitation of antenna chlorophylls. We present the first results of single-molecule spectroscopy on reaction-center containing photosynthetic aggregate Photosystem I of Synechococcus elongatus. Spectroscopic properties of low-energy antenna pigments were probed in individual phtotosynthetic complexes. Temperature activated fluorescence quenching unravels energy transfer pathways within the red-most pigment pool and to the reaction center on individual complex level. Low-temperature spectroscopy indicates that two subgroup of pigments which are present in the red antenna pool differs not only by their spectral position but also by the strength of electron-phonon coupling. Possible dimeric nature of the strongly phonon coupled red- most state is discussed in context of hole-burning data (Rätsep at al., J. Phys. Chem. B 2000, 104: 836). Room-temperature bleaching dynamics shows that light energy absorbed by antenna chlorophylls is transferred to red-most pigments before being capturing in the reaction center. The present work demonstrates that fluorescence of single PS1 complexes can be detected at physiological temperatur.