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...

When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm) 2 image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, sPod). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, exPAn). this shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.In recent years the development of super-resolution techniques has had a profound impact on biology and other fields in which subdiffraction-limited resolution of fluorescently labeled samples is desired [1][2][3][4][5][6][7][8][9][10][11][12][13] . Prominent examples are stimulated emission depletion (STED) microscopy 1,4 , photoactivated localization microscopy (PALM) 3,5,6 and stochastic optical reconstruction microscopy (STORM) 2,7 . Generally, these techniques are based on reversible switching between two states. Whereas STED is based on a deterministic switching in a nanometric interference pattern, STORM and PALM are based on wide-field illumination and stochastic switching on the level of single isolated molecules, which are then localized. Other approaches, such as super-resolution optical fluctuation imaging (SOFI) 8 , reversible saturable optical fluorescence transitions (RESOLFT) microscopy 9,11 and saturated structured illumination microscopy (SSIM) 12,13 , are also based on stochastic or deterministic switching between two states and provide spatial resolution enhancement. Here we present an alternative approach that distinguishes adjacent molecules or nanoareas in the sample (arranged, for example, in a grid of 50 nm × 50 nm rectangular areas) by different average orientations of fluorescent dyes attached to rigid sample structures within these nanoareas. This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD). We also show that the range of polarization angles that results in effective excitation of differently oriented molecules can be substantially narrowed by rotating a second wide-field de-exciting stimulated emission beam of a polarization perpendicular to the excitation beam polarization (ExPAN), resulting in better spatial resolution ...

Efficient sunlight harvesting and re-directioning onto small areas has great potential for more widespread use of precious high-performance photovoltaics but so far intrinsic solar concentrator loss mechanisms outweighed the benefits. Here we present an antenna concept allowing high light absorption without high reabsorption or escape-cone losses. An excess of randomly oriented pigments collects light from any direction and funnels the energy to individual acceptors all having identical orientations and emitting ~90% of photons into angles suitable for total internal reflection waveguiding to desired energy converters (funneling diffuse-light re-directioning, FunDiLight). This is achieved using distinct molecules that align efficiently within stretched polymers together with others staying randomly orientated. Emission quantum efficiencies can be >80% and single-foil reabsorption <0.5%. Efficient donor-pool energy funneling, dipole re-orientation, and ~1.5–2 nm nearest donor–acceptor transfer occurs within hundreds to ~20 ps. Single-molecule 3D-polarization experiments confirm nearly parallel emitters. Stacked pigment selection may allow coverage of the entire solar spectrum.

a b s t r a c tThe aggregation dependent correlation between fluorescence quenching and the electronic carotenoidchlorophyll interactions, / Car S 1 ÀChl Coupling , as measured by comparing chlorophyll fluorescence observed after two-and one-photon excitation, has been investigated using native LHC II samples as well as mutants lacking Chl 2 and Chl 13. For native LHC II the same linear correlation between / Car S 1 ÀChl Coupling and the fluorescence quenching was observed as previously reported for the pH and Zea-dependent quenching of LHC II [1]. In order to elucidate which carotenoid-chlorophyll pair might dominate this correlation we also investigated the mutants lacking Chl 2 and Chl 13. However, also with these mutants the same linear correlation as for native LHC II was observed. This provides indication that these two chlorophylls play only a minor role for the observed effects. Nevertheless, we also conclude that this does not exclude that their neighboured carotenoids, lutein 1 and neoxanthin, might interact electronically with other chlorophylls close by.

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of nonmodulated back-ground intensity.