Arabidopsis ͉ LHCII ͉ NPQ ͉ two-photon excitation P lants are exposed to sunlight intensities varying over several orders of magnitude during a typical day (1). Under low light conditions, almost all absorbed sunlight photons are used for the primary photosynthetic reaction steps. However, under high light conditions the photosynthetic apparatus must be protected from excess excitation energy, because it may lead to deleterious side-effects. Balancing between efficient utilization of solar energy under restrictive light conditions and dissipation of excess energy when the absorbed light exceeds the photosynthetic capacity is therefore essential for the survival and fitness of plants (2). It is known that light-induced increase of the pH gradient across the thylakoid membrane (3, 4) and the presence of the protein PsbS (5) are necessary for the down-regulation of the photosynthetic activity under excess light and that Zea is simultaneously formed from violaxanthin (Vio) through the enzymatic xanthophyll cycle (6). However, although many different studies have been undertaken to elucidate the details of this important regulation, a complete picture of its mechanisms is still missing. Several different regulation models have been proposed and indeed it cannot be excluded that different mechanisms contribute more or less to plants adaptation to varying light conditions. However, at present even the regulation site and photophysical mechanisms are unresolved, because the models are at least partly contradicting each other (5, 7-15).The most important measurable signature of plants regulation activity is its varying residual Chl fluorescence intensity (16), which is proportional to the regulated amount of excitation energy in the photosynthetic apparatus. The actual extent of adaptation-dependent quenching of Chl singlet excited state energy, known as nonphotochemical quenching (NPQ), is typically quantified by the parameterwhich reflects the reduction in the residual Chl fluorescence of plants, FЈ m , brought about by the unknown excitation energy dissipation mechanisms, in comparison to the residual Chl fluorescence observable from the completely dark adapted plant, F m , in which no photoprotective energy dissipation is operating. FЈ m and F m are usually measured using short, intense light flashes that saturate the photosynthetic reaction center chemistry. This guarantees that the observed differences in FЈ m and F m reflect only the extent of energy dissipation through photoprotective channels, without affecting the adaptation status of the plant (16). It is long known that carotenoids play an important role in the regulation mechanisms and several different types of electronic interactions between carotenoids and Chls have been proposed to play a key role as dissipation valves for excess excitation energy (9, 10, 12). However, so far it was difficult to quantify the extent of these interactions and to investigate directly their involvement in the flow of excitation energy and its regulation, especially in living...

The Car S 1 ? Chl energy transfer efficiency, U Transfer , in xanthophyll-cycle mutants of living plants and LHC II was investigated by selective Car S 1 two-photon excitation. Before high-light illumination U Transfer , of the violaxanthin deficient mutant npq2 is $30% smaller than the corresponding value for wild type plants. For the zeaxanthin deficient mutant, npq1, U Transfer is $30% larger. Wild type Arabidopsis thaliana is the only variant which is capable of a light-dependent decrease of up to 40% and complete recovery to the original U Transfer values. In contrast, U Transfer remains constant during dark adaptation in both mutants. Surprisingly, changes in U Transfer of LHC II preparations were less than 5% only, when substituting violaxanthin by zeaxanthin.

Two-color two-photon (2c2p) excitation fluorescence is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by subtilisin. Fluorescence is generated by irradiation with spatially and temporally overlapping femtosecond laser beams resulting in simultaneous absorption of an 800 and a 400 nm photon. Thereby, excitation of the fluorescent amino acid tryptophan present in BSA corresponds to an effective one-photon wavelength of 266 nm. The progress of protein cleavage is monitored by time-resolved fluorescence analysis. The fluorescence lifetime of tryptophan decreases during the reaction. This demonstrates a novel label-free multiphoton observation technique for conformational changes of proteins containing tryptophan. Due to the strong 2c2p fluorescence signal it is suitable for fast evaluation and monitoring of protein reactions. The course of the reaction is monitored simultaneously by gel electrophoresis. In contrast to conventional one-photon techniques, 2c2p excitation enables label-free protein fluorescence studies without irradiating the sample with UV light. Due to the dependence of the excitation on the power of both laser beams, excitation is limited to a relatively small focal volume. This results in dramatically reduced overall photodamage compared to direct UV irradiation. This method can be easily extended to microscopy imaging techniques.

The capability of using ultrafast detection technologies for a fast analysis of biomolecular reactions has been explored. As an example, the ultrafast response of tetramethylrhodamine (TMR)-labeled bovine serum albumin (BSA) as a function of different extents in proteolytic cleavage was investigated. The authors compared 4 samples of masses differing over several orders of magnitude: untreated, TMR-labeled BSA (66 kDa), TMR-labeled BSA treated with elastase (6-33 kDa) and with subtilisin (< 3 kDa), and the pure label TMR (0.4 kDa). A direct comparison with gel electrophoresis revealed that various ultrafast parameters give robust information about the progress of the proteolytic cleavage. The authors found the ratio of the transient absorption signal observed at 0 psec and 50 psec after excitation (lambda(Pump) = 540 nm, lambda(Probe) = 570 nm) to be the most precise parameter for determining the cleavage. This parameter allowed determining the mass accurately within 1 sec (Z' factor of 0.83) or 600 msec (Z' factor of 0.64), measuring time per sample. This indicates that many of the known ultrafast detection technologies might be used for monitoring biochemical reactions, probably even without any labeling procedure. The authors also discuss briefly which ultrafast processes contribute to the signals and how they are affected by changes in the biomolecular environment.