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The transfer of electronic charge in the reaction center of Photosystem II is one of the key building blocks of the conversion of sunlight energy into chemical energy within the cascade of the photosynthetic reactions. Since the charge transfer dynamics is mixed with the energy transfer dynamics, an effective tool for the direct resolution of charge separation in the reaction center is still missing. Here, we use experimental two-dimensional optical photon echo spectroscopy in combination with the theoretical calculation to resolve its signature. A global fitting analysis allows us to clearly and directly identify a decay pathway associated to the primary charge separation. In particular, it can be distinguished from regular energy transfer and occurs on a time scale of 1.5 ps under ambient conditions. This technique provides a general tool to identify charge separation signatures from the energy transport in two-dimensional optical spectroscopy.

International audience; Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP(r)) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP(r). This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP(r), enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.

DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

Carotenes, found in a diversity of fruit-containing foods, are important sources of antioxidants; a good example is apricot jam. In the study described in this paper, both the total carotenoid content (TCC) as well as the content of $$\upbeta $$ -carotene in six different apricot jams were quantified using traditional (UV–VIS) spectrophotometry (SP), high-performance liquid chromatography (HPLC), laser photoacoustic spectroscopy (LPAS), and the optothermal window (OW) method. Unlike SP and HPLC, LPAS and the OW methods require the minimum of sample preparation and only a one time calibration step which enables practically direct quantification of the TCC. Results were verified versus data obtained with SP as the reference technique. It was shown that LPAS and the OW method (at 473 nm) provide satisfactory results with $$R^{2}=0.9884$$ and 0.9766 for LPAS and OW, respectively.

This report adds to the ongoing vivid dispute on the fate of carotenes in tomato upon thermal processing. Although many papers dealing with changes in the raw tomatoes during industrial treatment have already appeared, data on the fate of finished, processed tomato products when they are additionally heated (e.g., when used in the household) is scarce. In this study, effects of heating and storage on a commercial, double concentrated tomato pur ee were examined spectrophotometrically. Our results indicate that upon exposing unopened cans with double concentrated tomato pur ee to thermal treatments between 100 and 135 ^o C during specific time intervals spectral profile of lycopene remained preserved. Likewise, a slight hypsochromic shift of lycopene peak III did not occur up to 135 ^o C. However, significant (20%) initial loss of lycopene content was induced by thermal treatment for 20 min at 100 ^o C. During the more intensive treatments that followed the lycopene content was first leveling off and then slightly increased. After storage of thermally treated samples at - 18 ^o C the content of lycopene was found to increase. All these results suggest simultaneous working of several mechanisms: possible autooxidation and isomerization processes of carotenes taking place, in addition to the Maillard reaction and enhanced extractability of carotenes at increased temperatures. Results acquired from hexane solutions of samples treated at temperatures of 120 and 135 ^oC obtained at different time points, confirmed severe isomerization in organic solvent and/or photo-oxidative degradation of lycopene.

Complex multi-exponential fluorescence decay kinetics observed in various photosynthetic systems like photosystem II (PSII) have often been explained by the reversible quenching mechanism of the charge separation taking place in the reaction center (RC) of PSII. However, this description does not account for the intrinsic dynamic disorder of the light-harvesting proteins as well as their fluctuating dislocations within the antenna, which also facilitate the repair of RCs, state transitions, and the process of non-photochemical quenching. Since dynamic fluctuations result in varying connectivity between pigment-protein complexes, they can also lead to non-exponential excitation decay kinetics. Based on this presumption, we have recently proposed a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer pathways. In the current work, this model is further developed and then applied to describe fluorescence kinetics originating from very diverse antenna systems, ranging from PSII of various sizes to LHCII aggregates and even the entire thylakoid membrane. In all cases, complex multi-exponential fluorescence kinetics are perfectly reproduced on the entire relevant time scale without assuming any radical pair equilibration at the side of the excitation quencher, but using just a few parameters reflecting the mean excitation energy transfer rate as well as the overall average organization of the photosynthetic antenna.

The achievable time resolution of camera-based single-molecule detection is often limited by the frame rate of the camera. Especially in experiments utilizing single-molecule Fo¨rster resonance energy transfer (smFRET) to probe conformational dynamics of biomolecules, increasing the frame rate by either pixel-binning or cropping the field of view decreases the number of molecules that can be monitored simultaneously. Here, we present a generalised excitation scheme termed stroboscopic alternating-laser excitation (sALEX) that significantly improves the time resolution without sacrificing highly parallelised detection in total internal reflection fluorescence (TIRF) microscopy. In addition, we adapt a technique known from diffusion-based confocal microscopy to analyse the complex shape of FRET efficiency histograms. We apply both sALEX and dynamic probability distribution analysis (dPDA) to resolve conformational dynamics of interconverting DNA hairpins in the millisecond time range.

Challenge tests are used to assess the resilience of human beings to perturbations by analyzing responses to detect functional abnormalities. Well known examples are allergy tests and glucose tolerance tests. Increasingly, metabolomics analysis of blood or serum samples is used to analyze the biological response of the individual to these challenges. The information content of such metabolomics challenge test data involves both the disturbance and restoration of homeostasis on a metabolic level and is thus inherently different from the analysis of steady state data. It opens doors to study the variation of resilience between individuals beyond the classical biomarkers; preferably in terms of underlying biological processes. We review challenge tests in which metabolomics was used to analyze the biological response. Specifically, we describe strategies to perform statistical analyses on the responses and we will show some examples of these strategies applied to a postprandial challenge that was used to study a diet with anti-inflammatory properties. Finally we discuss open issues and give recommendation for further research. © 2014, Springer Science+Business Media New York.

Anoxic conditions should hamper the transport of sugar in the phloem, as this is an active process. The canopy is a carbohydrate source and the roots are carbohydrate sinks.By fumigating the shoot with N2 or flooding the rhizosphere, anoxic conditions in the source or sink, respectively, were induced. Volume flow, velocity, conducting area and stationary water of the phloem were assessed by non-invasive magnetic resonance imaging (MRI) flowmetry. Carbohydrates and d13C in leaves, roots and phloem saps were determined. Following flooding, volume flow and conducting area of the phloem declined and sugar concentrations in leaves and in phloem saps slightly increased. Oligosaccharides appeared in phloem saps and after 3 d, carbon transport was reduced to 77%. Additionally, the xylem flow declined and showed finally no daily rhythm. Anoxia of the shoot resulted within minutes in a reduction of volume flow, conductive area and sucrose in the phloem sap decreased. Sugar transport dropped to below 40% by the end of the N2 treatment. However, volume flow and phloem sap sugar tended to recover during the N2 treatment. Both anoxia treatments hampered sugar transport. The flow velocity remained about constant, although phloem sap sugar concentration changed during treatments. Apparently, stored starch was remobilized under anoxia.

We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30¿min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50¿Hz sine wave) at three magnetic flux densities (5¿µT, 300¿µT, and 500¿µT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50¿Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

Photosystem II (PSII) is the powerhouse of all organisms that perform oxygen-generating photosynthesis. It uses sunlight energy to extract electrons and protons from water, which fuel their metabolism. It also provides the oxygen needed by most organisms to extract energy from biomass. On page 815 of this issue, Su et al. (1) report the cryo-electron microscopy structure of the largest PSII complex of vascular plants at 2.7-Å resolution. The structure shows the details and overall organization of 28 proteins, 203 pigments, 34 lipids, and 8 other cofactors, providing the basis for understanding the functional behavior of the PSII supercomplex.

Scope: Theobromine is a major active compound in cocoa with allegedly beneficial effect on high-density-lipoprotein-cholesterol (HDL-CH). We have investigated the effect of theobromine (TB) consumption on the concentrations of triglyceride (TG) and cholesterol (CH) in various lipoprotein (LP) subclasses.Methods: In a randomized, double-blind, placebo-controlled, cross-over study, 44 apparently healthy women and men (age: 60 +/- 6 years, BMI: 29 +/- 3 kg/m(2)) with low baseline HDL-CH concentrations consumed a drink supplemented with 500 mg/d theobromine for 4 weeks. TG and CH concentrations in 15 LP subclasses were predicted from diffusion-edited H-1 NMR spectra of fasting serum.Results: The LP phenotype of the subjects was characterized by low CH concentrations in the large HDL particles and high TG concentrations in large VLDL and chylomicron (CM) particles, which clearly differed from a LP phenotype of subjects with normal HDL-CH. TB only reduced CH concentrations in the LDL particles by 3.64 and 6.79%, but had no effect on TG and CH in any of the HDL, VLDL and CM subclasses.Conclusion: TB was not effective on HDL-CH in subjects with a LP phenotype characterized by low HDL-CH and high TG in VLDL.

We introduce a nanofluidic mixing device entirely fabricated in glass for the fluorescence detection of single molecules. The design consists of a nanochannel T-junction and allows the continuous monitoring of chemical or enzymatic reactions of analytes as they arrive from two independent inlets. The fluorescently labeled molecules are tracked before, during and after they enter the mixing region, and their reactions with each other are observed by means of optical readout such as Förster Resonance Energy Transfer (FRET). Our method can be used for analyzing the kinetics of DNA annealing in a high-parallelized fashion.

During the last decades, fluorescence spectroscopy has emerged as a powerful tool in the fields of biophysics, biotechnology, biochemistry, cellular biology and the medical sciences. These techniques are highly sensitive, and allow us to study the structure and dynamics of (bio)molecular systems (Valeur 2001). A significant advantage of fluorescence techniques is that they can often be non-invasive and measurements can be performed in real time. In this thesis different advanced fluorescence methods will be used to study two important biological processes: (1) DNA dynamics and (2) plant photosynthesis. The first part aims at improving the smFRET technique for the analysis of DNA dynamics and other fast conformational changes. This improvement is made by combining and developing instrumentation and data evaluation tools. The second part is the continuous development of time-resolved fluorescence spectroscopy methods, as well their application in the field of photosynthesis to study ultrafast processes in thylakoid membranes and leaves. The two fluorescence techniques are technically and conceptually very different, but they are both designed for analysis of biomolecular systems. In this thesis, the techniques are applied to study energy transfer and dynamical changes in DNAs, thylakoid membranes and leaves. REFERENCE: VALEUR B 2001. Molecular Fluorescence: Principles and Applications. 1 ed: Wiley-VCH.

The water-binding capacities (WBCs) of pea protein isolate, soy protein isolate, lupin protein concentrate and vital wheat gluten particles were investigated by hydrating them in excess water, centrifuging these dispersions, and calculating the WBCs from the weight of the pellets. It was found that, except for pea proteins, the pellet consisted of a notable amount of interstitial water. Furthermore, it seems that when particles were largely deformable a (semi-)continuous protein network was formed in which individual particles could not be distinguished anymore. Then, the WBC of the pellet did not represent the WBC of the original particles anymore. Consequently, it was concluded that the WBC of the pellet (WBC-P) differs from the WBC of the particles. Therefore, the characteristics of the particles and their pellets were further investigated with, among others, time domain nuclear magnetic resonance (TD NMR). TD NMR turned out to be a useful additional tool to do this, and has the potential to give an indication of the amount of water present in each water domain. From the information obtained about the characteristics of the particles and their pellets, it could be concluded that variations in the WBC-P were the result of differences in the deformability of the particles (i.e., their capability to swell and to withstand the centrifugal force), and their ability to bind water interstitially.

Human health is impacted by a complex network of interactions between biological pathways, mechanisms, processes, and organs, which need to be able to adapt to a continuously changing environment to maintain health. This adaptive ability is called ‘phenotypic flexibility’. It is thought that health is compromised and diseases develop when these adaptive processes fail. As the product of interactions between several factors such as genetic makeup, diet, lifestyle, environment and the gut microbiome, the ‘metabolic phenotype’ provides a readout of the metabolic state of an individual. Understanding these relationships will be one of a major challenges in nutrition and health research in the next decades. To address this challenge, the development of high-throughput omics tools combined with the application of elaborate statistical analyses will help characterize the complex relationship of (bio) chemicals in human systems and their interaction with other variables including environment and lifestyle to produce the measured phenotype. An important aim of this thesis was to identify phenotype shifts by looking at effect of prolonged resistance-type exercise training on skeletal muscle tissue in older subjects and the possible shift toward the features of younger subjects as a reference for a healthier phenotype. A second aim was to identify phenotype shifts by looking at the response to a challenge in obese subjects and the possible shift toward lean subjects as a reference for a healthier phenotype. Chapter 2 and 3 of this thesis show how the significant remaining plasticity of ageing skeletal muscle can adapt to resistance-type exercise training. The data indicate that frail and healthy older subjects have two distinct phenotypes according to the skeletal muscle tissue metabolite profiles and that exercise training shifts aged muscle towards a younger phenotype. We showed that the effect of exercise on amino acid derived acylcarnitines (AAAC’s) in older subjects points towards decreased branched chain amino acid catabolism, likely due to compromised activation of the branched chain α-keto acid hydrogenase (BCKDH) complex. Furthermore, we found that the protocadherin gamma gene cluster might be involved in aged-muscle denervation and re-innervation. Finally, plasma was found to be a poor indicator of muscle metabolism, emphasizing the need for direct assessment of metabolites in muscle tissue. Chapter 4 of this thesis examines whether a mixed meal challenge response provides a readout for a shift in phenotype upon weight loss in obese male subjects. We concluded that weight loss moderately affects the mixed meal challenge response of both plasma metabolome and transcriptome of peripheral blood mononuclear cells in obese subjects. Measurements at the fasted and postprandial state also provide us with a different type of information. In Chapter 5 it is demonstrated that the global testing of pathways could provide a concise summary of the multiple univariate testing approach used in Chapter 4. In Chapter 6 it is discussed how the findings of this thesis increase our understanding of how to measure phenotypic flexibility as a proxy of health. In this thesis it is shown that the correlations between tissue and plasma metabolites are rather weak, emphasising the need to perform organ-specific studies. Availability of less invasive/painful sampling techniques and the use of small amounts of tissue would enable larger scale human studies on adipose tissue and skeletal muscle to more accurately define phenotypical shifts due to diet or lifestyle interventions. With respect to the assessment of phenotypical flexibility by omics approaches, significant complications can be expected in trying to relate plasma metabolism to PBMC gene expression. Organ-focussed approaches that integrate multiple omics levels using system biology approaches are considered to be a lot more promising.

Quantitative magnetic resonance imaging (qMRI) is a versatile, non-destructive and non-invasive tool in life, material, and medical sciences. When multiple components contribute to the signal in a single pixel, however, it is difficult to quantify their individual contributions and characteristic parameters. Here we introduce the concept of phasor representation to qMRI to disentangle the signals from multiple components in imaging data. Plotting the phasors allowed for decomposition, unmixing, segmentation and quantification of our in vivo data from a plant stem, a human and mouse brain and a human prostate. In human brain images, we could identify 3 main T 2 components and 3 apparent diffusion coefficients; in human prostate 5 main contributing spectral shapes were distinguished. The presented phasor analysis is model-free, fast and accurate. Moreover, we also show that it works for undersampled data.

Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for in vitro studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete in vivo conditions in leaf disks of F. benjamina and A. platanoides and in suspensions of the green alga Chlamydomonas reinhardtii and blue-green alga Synechocystissp.PCC 6803. In leaf disks of Ficus benjamina and Acer platanoides, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in F. benjamina but not in A. platanoides) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of Synechocystissp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different.

The key to link intake of polyphenols to health benefits is the quantitative and kinetic assessment of their metabolites in circulation. Current analytical approaches have only provided limited quantitative coverage of the internal polyphenol metabolome, this in particular pertains to conjugated microbial catabolites. Current modelling approaches to describe the kinetic appearance of polyphenol metabolites into circulation mostly neglect the multi-compartment nature of polyphenol metabolism in the body. Furthermore, inter-individual variation is mostly neglected in current modelling approaches. These gaps hamper clear definition of the modulating effects of metabotypes and food matrix on the internal polyphenol metabolome.

Lipoproteins and their subfraction profiles have been associated to diverse diseases including CardioVascular Disease (CVD). There is thus a great demand for measuring and quantifying the lipoproteinprofile in an efficient and accurate manner.Nuclear Magnetic Resonance (NMR) spectroscopy is uniquely able to measure the lipoprotein profile ofa blood sample non-destructively due to its sensitivity to both lipid chemistry and lipid-micellar physics.However, the NMR spectra must be scaled/regressed to a primary method of reference, such as ultracentrifugation,using multivariate regression methods.This review provides an overview of the field and explains the methods at stake.© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

In this paper we propose an energy dissipation mechanism that is completely reliant on changes in the aggregation state of the phycobilisome light-harvesting antenna components. All photosynthetic organisms regulate the efficiency of excitation energy transfer (EET) to fit light energy supply to biochemical demands. Not many do this to the extent required of desert crust cyanobacteria. Following predawn dew deposition, they harvest light energy with maximum efficiency until desiccating in the early morning hours. In the desiccated state, absorbed energy is completely quenched. Time and spectrally resolved fluorescence emission measurements of the desiccated desert crust Leptolyngbya ohadii strain identified (i) reduced EET between phycobilisome components, (ii) shorter fluorescence lifetimes, and (iii) red shift in the emission spectra, compared with the hydrated state. These changes coincide with a loss of the ordered phycobilisome structure, evident from small-angle neutron and X-ray scattering and cryo-transmission electron microscopy data. Based on these observations we propose a model where in the hydrated state the organized rod structure of the phycobilisome supports directional EET to reaction centers with minimal losses due to thermal dissipation. In the desiccated state this structure is lost, giving way to more random aggregates. The resulting EET path will exhibit increased coupling to the environment and enhanced quenching.

The microstructure of fat spreads is of fundamental importance to their sensorial properties such as texture, mouthfeel and spreadability. Fat spreads are water in oil emulsions,with a continuous phase supported by a fat crystal network. Confocal Raman microscopy offers the possibility for the spatialmapping of themicrostructure of fat spreads: It can distinguish the solid and liquid phases of the lipids and the emulsifiers present at the water–oil interface. A constraint multivariate curve resolution with an alternating least squares approach using a fixed set of pure component spectra is a suitable method to extract the spatial distribution information of each individual component present in the fat spread. A new normalization method provides semi-quantitative concentration maps, allowing good microstructural comparison. The technique was able to determine the spatial distribution of sunflower oil, water, emulsifier and solid fat in various investigated spreads. This method can be applied to a wide range of different food emulsions such as butter, margarine, mayonnaise and salad dressings. Copyright © 2017 John Wiley & Sons, Ltd.

Properties and consumer appreciation of fat containing food products are greatly influenced by the growth of thefat crystalline domains and their morphology. Various analytical methods can be exploited to provide insight into the multilengthscale microstructure of fat networks, but very few are able to discriminate structural features between different ways of processingfats. We selected in this work methods that highlight the mesostructure of fat crystalline systems. In this respect, 1H spindiffusionNMR and XRD methods were employed to determine the fat crystallite thicknesses (crystalline domain size). This isthe first attempt to quantify the mesostructure domains of triglyceride-based fat crystals by means of 1H spin-diffusion NMR experiments. The crystalline domain sizes determined by spin-diffusion NMR were found to be in good agreement with the crystallite thickness determined by the Scherrer analysis of the first order diffraction line from SAXS data. These resultsdemonstrate the ability of the NMR technique to characterize the mesostructure of fats in a quantitative manner. This method is of particular interest for the structure analysis of fats, especially because of the possibility to quantify the size of the crystalline domains in diluted systems where scattering techniques struggle with the amount of diffracting material.

Whey protein microparticles (CG MPs) were made with a cold gelation method. Without shearing or mixing during gelation spherical CG MPs were formed, while shearing or mixing resulted in smaller irregularly shaped CG MPs. The water-binding capacity of pellets (WBC-P) that were obtained after centrifuging CG MP dispersions was remarkably large (11–18 g water/g dry matter), though this value decreased at larger centrifugation speeds. Microscopy images hinted at the presence of two water domains in the CG MP pellets: water within and between the CG MPs. The images also imply that the amount of water within the CG MPs was determined by the centrifugation speed. The amount of water between CG MPs seemed to be defined by the amount of particle-particle interactions that were present, as suggested by the effects of the particles' size and the inhibition of the disulphide bridge formation on the WBC-P. Although microscopy images showed two water domains, only one main peak was found with time domain nuclear magnetic resonance. This was explained by water diffusion from one water domain to the other within the measuring time. This fast diffusion implies that the CG MPs had a relatively open structure. Overall, it was concluded that water-binding by CG MPs was affected by various factors and that a good understanding of the water-binding requires the use of a range of measurements.

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.

The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10–22 and 82–91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.

Photosynthetic organisms are crucial for life on Earth as they provide food and oxygen and are at the basis of most energy resources. They have a large variety of light-harvesting strategies that allow them to live nearly everywhere where sunlight can penetrate. They have adapted their pigmentation to the spectral composition of light in their habitat, they acclimate to slowly varying light intensities and they rapidly respond to fast changes in light quality and quantity. This is particularly important for oxygen-producing organisms because an overdose of light in combination with oxygen can be lethal. Rapid progress is being made in understanding how different organisms maximize light harvesting and minimize deleterious effects. Here we summarize the latest findings and explain the main design principles used in nature. The available knowledge can be used for optimizing light harvesting in both natural and artificial photosynthesis to improve light-driven production processes. © 2014 Nature America, Inc.

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited auto-fluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content.

In this paper we present a pore-scale model describing the multiphysics occurring during the rehydration of freeze-dried vegetables. This pore-scale model is part of a multiscale simulation model, which should explain the effect of microstructure and pre-treatments on the rehydration rate. Simulation results are compared to experimental data, obtained by MRI and XRT. Time scale estimates based on the pore-scale model formulation agree with the experimental observations. Furthermore, the pore-scale simulation model provides a plausible explanation for the strongly increased rehydration rate, induced by the blanching pre-treatment. Industrial relevance The increased insight in the physical processes governing the rehydration of porous or freeze-dried foods gives more rationale for optimizing all processing steps. Industry is seeking for means to give dried fruits and vegetables more convenience, but also higher quality concerning health and texture. This study shows that blanching pretreatment prior to freeze-drying strongly enhances the rehydration, while the loss of nutrients is hardly affected.

Gut microbial catabolites of black tea polyphenols (BTPs) have been proposed to exert beneficial cardiovascular bioactivity. This hypothesis is difficult to verify because the conjugation patterns and pharmacokinetics of these catabolites are largely unknown. The objective of our study was to identify, quantify, and assess the pharmacokinetics of conjugated BTP metabolites in plasma of healthy humans by means of an a priori untargeted LC–MS-based metabolomics approach. In a randomized, open, placebo-controlled, crossover study, 12 healthy men consumed a single bolus of black tea extract (BTE) or a placebo. The relative and, in several cases, absolute concentrations of a wide range of metabolites were determined using U(H)PLC-LTQ-Orbitrap-FTMS. Following BTE consumption, a kinetic response in plasma was observed for 59 BTP metabolites, 11 of these in a quantitative manner. Conjugated and unconjugated catechins appeared in plasma without delay, at 2–4 h, followed by a range of microbial catabolites. Interindividual variation in response was greater for gut microbial catabolites than for directly absorbed BTPs. The rapid and sustained circulation of conjugated catabolites suggests that these compounds may be particularly relevant to proposed health benefits of BTE. Their presence and effects may depend on individual variation in catabolic capacity of the gut microbiota.

Molecular dynamics (MD) simulations over a 30 ns trajectory have been carried out on apoflavodoxin from Azotobacter vinelandii to compare with the published, experimental time-resolved fluorescence anisotropy results of F¨orster Resonance Energy Transfer (FRET) between the three tryptophan residues. MD analysis of atomic coordinates yielding both the time course of geometric parameters and the time-correlated second-order Legendre polynomial functions reflects immobilization of tryptophans in the protein matrix. However, one tryptophan residue (Trp167) undergoes flip-flop motion on the nanosecond timescale. The simulated time-resolved fluorescence anisotropy of tryptophan residues in apoflavodoxin implying a model of two unidirectional FRET pathways is in very good agreement with the experimental time-resolved fluorescence anisotropy, although the less efficient FRET pathway cannot be resolved and is hidden in the contribution of a slow protein motion.

The dynamics of rigid dendrimer and flexible PEG probes in sodium caseinate dispersions and acid gels, including both translational diffusion and rotational diffusion, were studied by NMR. Above the onset of the close-packing limit (C ~ 10 g/100 g H2O), translational diffusion of the probe depended on its flexibility and on the fluctuations of the matrix chains. The PEG probe diffused more rapidly than the spherical dendrimer probe of corresponding hydrodynamic radius. The greater conformational flexibility of PEG facilitated its motion through the crowded casein matrix. Rotational diffusion was, however, substantially less hindered than the translational diffusion and depended on the local protein–probe friction which became high when the casein concentration increased. The coagulation of the matrix led to the formation of large voids, which resulted in an increase in the translational diffusion of the probes, whereas the rotational diffusion of the probes was retarded in the gel, which could be attributed to the immobilized environment surrounding the probe. Quantitative information from PFG-NMR and SEM micrographs have been combined for characterizing microstructural details in SC acid gels.

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Forster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.

Photosystem II (PSII) is the pigment-protein complex which converts sunlight energy into chemical energy by catalysing the process of light-driven oxidation of water into reducing equivalents in the form of protons and electrons. Three-dimensional structures from x-ray crystallography have been used extensively to model these processes. However, the crystal structures are not necessarily identical to those of the solubilised complexes. Here we compared picosecond fluorescence of solubilised and crystallised PSII core particles isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The fluorescence of the crystals is sensitive to the presence of artificial electron acceptors (K3Fe(CN)(3)) and electron transport inhibitors (DCMU). In PSII with reaction centres in the open state, the picosecond fluorescence of PSII crystals and solubilised PSII is indistinguishable. Additionally we compared picosecond fluorescence of native PSII with PSII in which Ca-2 in the oxygen evolving complex (OEC) is biosynthetically replaced by Sr2+. With the Sr2+ replaced OEC the average fluorescence decay slows down slightly (81 ps to 85 ps), and reaction centres are less readily closed, indicating that both energy transfer/trapping and electron transfer are affected by the replacement. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.

The alternating-laser excitation (ALEX) scheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Förster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables the sorting of fluorescently labelled species based on the number and type of fluorophores present. ALEX also provides a convenient way of accessing the correction factors necessary for determining accurate molecular distances. Here, we provide a comprehensive overview of the concept and current applications of ALEX and we explicitly discuss how to obtain fully corrected distance information across the entire FRET range. We also present new ideas for applications of ALEX which will push the limits of smFRET-based experiments in terms of temporal and spatial resolution for the study of complex biological systems.

article i nfo Rehydration kinetics by two modes of imbibition is studied in pieces of freeze-dried winter carrot, after different thermal pre-treatments. Water ingress at room temperature is measured in real time by in situ MRI and NMR relaxometry. Blanched samples rehydrate substantially faster compared to non-blanched samples, independent of their porous microstructure. It is proposed that for non-blanched tissues immobilized sugars result in nearly complete swelling of the solid matrix, hindering the ingress of water through the porous network. Non- blanched carrot pieces frozen at �28 °C rehydrate faster compared to those frozen at �150 °C, due to blocking of smaller pores by swelling. In blanched tissues the mobilization of sugars results in a more homogeneous sugar distribution, leading to less swelling of the solid matrix and allowing fast ingress of water via capillary suction. Industrial relevance: The dried fruits and vegetables that arecurrently available on themarketare a poorcompro- mise betweenconvenience(rehydration kinetics) and sensorial quality. Thisis a major bottleneck for consumers to "Make the Healthy Choice the Easy Choice" and this also negatively impacts market growth. Currently, rational optimization of drying processes is impeded by lack of insight which structural features determine rehydration kinetics (convenience) and texture (sensorial quality) upon rehydration. We therefore started a program to quantitatively assess and model microstructural features and rehydration behavior of freeze-dried carrots as a model system.

Thermophysical properties of foods are of considerable relevance to food industry. The One among less explored thermophysical quantities is the thermal effusivity. In this paper the front variant of the photopyroelectric method was applied to determine thermal effusivity of both, fresh hen egg¿s yolk and white as well as of their blends. The amount of egg yolk added to the blend affected the thermal effusivity of egg¿s white. Thermal effusivity of mixtures containing pork meat and lard was also investigated. Addition of lard reduces significantly thermal effusivity of pork meat (the 1% increase of lard content leads to 6.93 Ws½m-2K-1 drop of thermal effusivity).

The beneficial health effects of fruits and vegetables have been attributed to their polyphenol content. These compounds undergo many bioconversions in the body. Modeling polyphenol exposure of humans upon intake is a prerequisite for understanding the modulating effect of the food matrix and the colonic microbiome. This modeling is not a trivial task and requires a careful integration of measuring techniques, modeling methods and experimental design. Moreover, both at the population level as well as the individual level polyphenol exposure has to be quantified and assessed. We developed a strategy to quantify polyphenol exposure based on the concept of nutrikinetics in combination with population-based modeling. The key idea of the strategy is to derive nutrikinetic model parameters that summarize all information of the polyphenol exposure at both individual and population level. This is illustrated by a placebo-controlled crossover study in which an extract of wine/grapes and black tea solids was administered to twenty subjects. We show that urinary and plasma nutrikinetic time-response curves can be used for phenotyping the gut microbial bioconversion capacity of individuals. Each individual harbours an intrinsic microbiota composition converting similar polyphenols from both test products in the same manner and stable over time. We demonstrate that this is a novel approach for associating the production of two gut-mediated ¿-valerolactones to specific gut phylotypes. The large inter-individual variation in nutrikinetics and ¿-valerolactones production indicated that gut microbial metabolism is an essential factor in polyphenol exposure and related potential health benefits

Oxygenic photosynthesis is driven by photosystems I (PSI) and II (PSII). In plants the number of chlorophylls of PSI versus PSII is adjusted to the light irradiance spectrum. On a timescale of days, this is regulated at the level of protein concentration. Instead, on a timescale of minutes, it is regulated by the dynamic association of light-harvesting complex II with either PSI or PSII. Thus far very diverse values have been reported for the PSI/PSII chlorophyll ratio, ranging from 0.54 to 1.4. The methods used require the isolation of chloroplasts and are time consuming. We present a fluorescence lifetime imaging approach that quantifies the PSI/PSII Chl ratio of chloroplasts directly in their natural leaf environment. In wild type Arabidopsis thaliana plants, grown under white light, the PSI/PSII chlorophyll ratio appeared to be 0.99 ± 0.09 at the adaxial side and 0.83 ± 0.05 at the abaxial side of the leaf. When these plants were acclimated to far red light for several days the PSI/PSII chlorophyll ratio decreased by more than a factor of 3 to compensate for the ineffective far red light absorption of PSII. This shows how plants optimize their light-harvesting capacity to the specific light conditions they encounter. Zooming in on single chloroplasts inside the leaf allowed to study the grana/stroma membrane network and their PSI/PSII chlorophyll ratios. The developed method will be useful to study dynamic processes in chloroplasts in intact leaves which involve changes in the grana and the stroma membranes such as state transitions.

This thesis (Light on phloem transport – an MRI approach) aims to answer the question whether phloem transport can be a limiting factor for photosynthesis efficiency (and ultimately causing a bottleneck towards achieving higher yields). To answer this key question, we manipulated the source: sink ratio within tomato (Solanum lycopersicum L.) while measuring phloem transport with magnetic resonance imaging (MRI) flowmetry. Additionally we compared phloem flow characteristics of two potato plants (Solanum tuberosum L.) which differed in source : sink ratio. In Chapter 2, the source strength was manipulated by varying the light intensity. An increase in phloem sap volume flow under higher light intensities was observed. However, under all light intensities applied, the phloem flow velocity was found to be constant (as has previously been suggested in other studies) although a clear diurnal pattern was observed. This finding does not fit in current models to describe the mechanism of phloem transport and a different mechanism must be at play. The results of this chapter demonstrate that increased levels of photo-assimilates are transported in sieve tubes, which are activated when needed by the plant. This is the first study which shows that plants activate individual sieve tubes when more photo-assimilates are available, yet maintain constant velocity. Those observations were in a tomato plant with pruned fruit trusses (i.e., in a simplified system). In Chapter 3, we investigated whether tomato plants still exhibit constant phloem flow velocity (with a diurnal pattern) under normal conditions, i.e., with strong sinks (tomato fruits) still attached. This was tested for both a long and short photoperiod by measuring flow characteristics with MRI flowmetry. We simultaneously monitored other plant processes like xylem flow rates with a heat balance sensor, net photosynthesis with gas exchange and stem diameter changes with a linear motion potentiometer. With this integrated approach, we revealed a correlation between night phloem volume flow, dark respiration and stem growth. We also conclusively showed that phloem volume flow performs a diurnal pattern under a variety of source-sink ratios which appears to be a normal behaviour for tomato plants growing under moderately-high light conditions. In chapters 2 and 3 we learned that under higher source strength a greater amount of phloem sap is transported, but the changes in flow were not accompanied by changes in velocity. To further our understanding of the mechanisms driving phloem transport, it is of interest to know how the sucrose concentration in phloem sap relates to phloem flow. In Chapter 4 we used an average T2 relaxation time in the phloem vascular tissue region to reveal the plant’s phloem carbon status under source manipulation. In this chapter we demonstrated that T2 relaxation time, when measured in parallel with phloem flow, can provide additional information about phloem region carbon status, i.e., changes in the T2 relaxation time are correlated with changes in sucrose concentration in the whole phloem region. When studying phloem transport in plants with magnetic resonance imaging (MRI) flowmetry, plants which are relatively easy to manipulate (e.g. fruit pruning) like tomato have so far been used. However, tomato plants (used in all three previous chapters) have relatively low sink strength beneath the MRI measurement site. A potentially preferable approach is to work with plants with strong sinks beneath the measurement site. In Chapter 5 we studied potato as a potentially better test subject for MRI flowmetry as it possesses strong sink below the MRI measurement site (i.e., developing tubers). For that purpose we used two potato plants (cv. Desiree) both with several developing tubers. One of the plants overexpressed the StSWEET gene (35S:StSWEET) which appears to have altered its source : sink ratio. As a result, the 35S:StSWEET plant transported 60% more phloem sap than Desiree WT. Strikingly, the average phloem flow velocity in both plants was the same and the greater amount of transported phloem sap in the 35S:StSWEET plant was accommodated by more sieve tubes than in Desiree WT. This finding agrees with the hypothesis about the conserved nature of phloem flow velocity, where volume flow is regulated by the number of active sieve tubes (Chapter 2 and 3). In this chapter we also demonstrate that a potato plant with developing tubers represents a good subject to study phloem transport with MRI flowmetry. We concluded that under optimal conditions (which are commonly met in greenhouses) phloem transport is likely to reach its maximum capacity and therefore photosynthesis could be limited by the export and transport of photo-assimilates because of the finite number of sieve tubes and constant flow velocity.

Single-molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool for elucidating biological structure and mechanisms on the molecular level. Here, we focus on applications of smFRET to study interactions between DNA and enzymes such as DNA and RNA polymerases. SmFRET, used as a nanoscopic ruler, allows for the detection and precise characterisation of dynamic and rarely occurring events, which are otherwise averaged out in ensemble-based experiments. In this review, we will highlight some recent developments that provide new means of studying complex biological systems either by combining smFRET with force-based techniques or by using data obtained from smFRET experiments as constrains for computer-aided modelling.

Scope Circulating oxylipins may affect peripheral tissues and are assumed to play an important role in endothelial function. They are esterified in triglyceride-rich lipoproteins that are increased after a high-fat (HF) meal, depending on BMI and fatty acid (FA) type. Yet, it is unclear which oxylipins appear in circulation after HF meals differing in FA composition. Methods and results In a double-blind randomized crossover challenge study, we characterized the postprandial oxylipin response after different HF challenges in lean and obese men receiving HF milkshakes, either high in saturated FAs (SFA), monounsaturated FAs (MUFA), or omega 3 (n-3) polyunsaturated FAs (PUFA). Plasma oxylipin profiles were significantly altered at 2 and 4 h after shake consumption when compared to baseline. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) derived oxylipins increased after n-3 PUFA shake consumption. MUFA shake consumption increased levels of cytochrome P450 mediated oxylipins. SFA shake consumption led to strong increases in linoleic acid (LA) derived HODEs. No differences were observed between lean and obese individuals at baseline and after any shake consumption. Conclusion e are the first demonstrating acute effects on circulating oxylipins after HF meal challenges. These changes were strongly influenced by different dietary FAs and may affect endothelial function.

Item does not contain fulltext Silver nanoparticle aggregates have been shown to support very large enhancements of fluorescence intensity from organic dye molecules coupled with an extreme reduction in observed fluorescence lifetimes. Here we show that for the same type of aggregates, similar enhancement factors ( approximately 75x in intensity and approximately 3400x in lifetime compared to the native radiative lifetime) are observed for a ruthenium-based phosphorescent dye (when taking into account the effect of charge and the excitation/emission wavelengths). Additionally, the inherently long native phosphorescence lifetimes practically enable more detailed analyses of the distribution of lifetimes (compared with the case with fluorescence decays). It was thus possible to unambiguously observe the deviation from mono-exponential decay which we attribute to emission from a distribution of fluorophores with different lifetimes, as we could expect from a random aggregation process. We believe that combining phosphorescent dyes with plasmonic structures, even down to the single dye level, will offer a convenient approach to better characterize plasmonic systems in detail.

We report the use of pendant droplet evaporation for exchange of eluents for (1)H nuclear magnetic resonance ((1)H NMR) purposes. Analytes are fed and retained in 500 nL droplets, which are concentrated by evaporation and subsequently redissolved in deuterated solvent. Droplet size is monitored by machine vision (MV), and heating rates are adjusted concordingly to maintain a stable droplet volume. Evaporation control is independent of solvent properties, and the setup handles feed rates up to 7 μL min(-1). The interface is capable of exchanging up to 90% of solvent for deuterated solvent, with a good recovery and repeatability for tomato extracts (Solanum lycopersicum). The system was capable of handling both polar and nonpolar analytes in one run. Volatiles such as formate, acetate, and lactate and the thermosensitive compound epigallocatechin gallate were recovered without significant losses. Ethanol and propionate were recovered with significant losses due to the formation of a minimum boiling azeotrope. The current setup is ideally suited for on- and off-line hyphenation of liquid chromatography and NMR, as it is comprehensive, fully automated, and easy to operate.

LHCII, the most abundant membrane protein on earth, is the major light-harvesting complex of plants. It is generally accepted that LHCII is associated with Photosystem II and only as a short-term response to overexcitation of PSII a subset moves to Photosystem I, triggered by its phosphorylation (state1 to state2 transition). However, here we show that in most natural light conditions LHCII serves as an antenna of both Photosystem I and Photosystem II and it is quantitatively demonstrated that this is required to achieve excitation balance between the two photosystems. This allows for acclimation to different light intensities simply by regulating the expression of LHCII genes only. It is demonstrated that indeed the amount of LHCII that is bound to both photosystems decreases when growth light intensity increases and vice versa. Finally, time-resolved fluorescence measurements on the photosynthetic thylakoid membranes show that LHCII is even a more efficient light harvester when associated with Photosystem I than with Photosystem II.

Hydration properties of semi-diluted hyaluronan were studied by means of time domain nuclear magnetic resonance. Based on the transverse proton relaxation times T2, the plasticization of hyaluronan which was precipitated by isopropylalcohol and dried in the oven have been determined at water content 0.4 g of water per g of hyaluronan. Above this water content, the relaxation times increased and levelled off around 0.8 g of water per g of hyaluronan which agrees well with values determined earlier by differential scanning calorimetry and dielectric relaxometry. The freeze dried and oven dried samples showed differences in their physical structure such as glass transition, plasticization concentration and sample topography which influenced their kinetics and mechanisms of hydration. Results confirmed earlier hypothesis that some native biopolymer structures can be easily modified by manipulation of preparation conditions, e.g. drying, giving fractions with specific physicochemical properties without necessity of their chemical modification.

Photosystems I (PSI) and II (PSII) are two major pigment–protein complexes of photosynthetic organisms that function in series to convert sunlight energy into chemical energy. We have studied the picosecond fluorescence behaviour of the core of both photosystems in vivo by using two Synechocystis PCC 6803 mutants: BE cells contain PSI but are lacking both PSII and the light-harvesting complexes called phycobilisomes (PBs) whereas PAL cells contain both PSI and PSII but lack the PBs. Measurements were performed at room temperature and at 77 K. The fluorescence kinetics of PSI and PSII can nicely be separated, en passant providing the PSI/PSII ratio. At room temperature, the PSI kinetics are identical to those of isolated PSI whereas the PSII kinetics can equally well be described by the in vitro trap-limited model of Y. Miloslavina, M. Szczepaniak, M. G. Muller, J. Sander, M. Nowaczyk, M. Rogner and A. R. Holzwarth, Biophys J., 2009, 96(2), 621–631, and by the transfer-to-the-trap-limited model of C. D. van der Weij-de Wit, J. P. Dekker, R. van Grondelle and I. H. M. van Stokkum, J. Phys. Chem. A, 2011, 115(16), 3947–3956, albeit that the in vivo kinetics turn out to be somewhat slower. At 77 K several low-energy pigments are observed in both photosystems which complicate the overall dynamics but the PSII kinetics can still be described by both a trap-limited and a transfer-to-the-trap-limited model.

EPR-based Gd(III)-nitroxide distance measurements were performed on a series of membrane-incorporated orthogonally labeled WALP23 polypeptides. The obtained distance distributions were stable upon the change of detection frequency from 10 GHz (X-band) to 35 GHz (Q-band). The alpha-helical pitch of WALP23 polypeptide could be experimentally observed, despite the flexibility of the two spin labels. The spectroscopic properties of Gd(III) ions and nitroxide radicals allow detecting both types of paramagnetic species selectively in different EPR experiments. In particular, this spectroscopic selectivity allows for supplementing Gd(III)-nitroxide distance measurements with independent checks of polypeptide aggregation and with measurements of the local environment of the nitroxide spin labels. All mentioned additional checks do not require preparation of further samples, as it is the case in the experiments with pairs of identical nitroxide spin labels.