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The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies. © 2012 Elsevier Ltd.

Fixational eye movements remain a major cause of artifacts in optical coherence tomography (OCT) images despite the increases in acquisition speeds. One approach to eliminate the eye motion is to stabilize the ophthalmic imaging system in real-time. This paper describes and quantifies the performance of a tracking OCT system, which combines a phase-stabilized optical frequency domain imaging (OFDI) system and an eye tracking scanning laser ophthalmoscope (TSLO). We show that active eye tracking minimizes artifacts caused by eye drift and micro saccades. The remaining tracking lock failures caused by blinks and large saccades generate a trigger signal which signals the OCT system to rescan corrupted B-scans. Residual motion artifacts in the OCT B-scans are reduced to 0.32 minutes of arc (~1.6 μm) in an in vivo human eye enabling acquisition of high quality images from the optic nerve head and lamina cribrosa pore structure. © 2012 Optical Society of America.

Protein-induced DNA looping is crucial for many genetic processes such as transcription, gene regulation and DNA replication. Here, we use tethered-particle motion to examine the impact of DNA bending and twisting rigidity on loop capture and release, using the restriction endonuclease FokI as a test system. To cleave DNA efficiently, FokI bridges two copies of an asymmetric sequence, invariably aligning the sites in parallel. On account of the fixed alignment, the topology of the DNA loop is set by the orientation of the sites along the DNA. We show that both the separation of the FokI sites and their orientation, altering, respectively, the twisting and the bending of the DNA needed to juxtapose the sites, have profound effects on the dynamics of the looping interaction. Surprisingly, the presence of a nick within the loop does not affect the observed rigidity of the DNA. In contrast, the introduction of a 4-nt gap fully relaxes all of the torque present in the system but does not necessarily enhance loop stability. FokI therefore employs torque to stabilise its DNA-looping interaction by acting as a 'torsional' catch bond. © 2012 The Author(s).

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology. © 2012 The Author(s).

To cope with the deleterious effects of excess illumination, photosynthetic organisms have developed photoprotective mechanisms that dissipate the absorbed excess energy as heat from the antenna system. In cyanobacteria, a crucial step in the process is the activation, by blue-green light, of a soluble protein, known as orange carotenoid protein (OCP), which binds the carotenoid 3′-hydroxyechinenone as its only pigment. While the spectroscopic properties of the inactive form of OCP have been described, the nature of the excited states in the active form still awaits elucidation. We applied transient absorption spectroscopy to the dark and the light activated forms of OCP to study and compare the excited state dynamics of both forms. We show that excitation of the photoactivated OCP leads to the population of new carotenoid excited states. One of these states populated shortly after excitation is characterized by a very pronounced charge transfer character and a lifetime of about 0.6 ps. When the illuminated sample is exposed to a dark relaxation period, it responds to excitation as the original dark sample, showing that photoactivation and decay of the photoactivated state are fully reversible. Thus OCP functions as a light-powered molecular switch that modulates its spectroscopic properties as a response to specific changes in light environment. We discuss the importance of this switch in cyanobacteria photoprotection and propose a mechanism wherein the red state of OCP echinenone acts as an energy dissipator via its charge transfer state. © 2012 American Chemical Society.

BLUF domains are flavin-binding photoreceptors that can be reversibly switched from a dark-adapted state to a light-adapted state. Proton-coupled electron transfer (PCET) from a conserved tyrosine to the flavin that results in a neutral flavin semiquinone/tyrosyl radical pair constitutes the photoactivation mechanism of BLUF domains. Whereas in the dark-adapted state PCET occurs in a sequential fashion where electron transfer precedes proton transfer, in the light-adapted state the same radical pair is formed by a concerted mechanism. We propose that the altered nature of the PCET process results from a hydrogen bond switch between the flavin and its surrounding amino acids that preconfigures the system for proton transfer. Hence, BLUF domains represent an attractive biological model system to investigate and understand PCET in great detail. © 2011 American Chemical Society.

Heterogeneity of regional coronary blood flow is caused in part by heterogeneity in O2 demand in the normal heart. We investigated whether myocardial O2 supply/demand mismatching is associated with the myocardial depression of sepsis. Regional blood flow (microspheres) and O2 uptake ([13C]acetate infusion and analysis of resultant NMR spectra) were measured in about nine contiguous tissue samples from the left ventricle (LV) in each heart. Endotoxemic pigs ( n = 9) showed hypotension at unchanged cardiac output with a fall in LV stroke work and first derivative of LV pressure relative to controls ( n = 4). Global coronary blood flow and O2 delivery were maintained. Lactate accumulated in arterial blood, but net lactate extraction across the coronary bed was unchanged during endotoxemia. When LV O2 uptake based on blood gas versus NMR data were compared, the correlation was 0.73 ( P = 0.007). While stable over time in controls, regional blood flows were strongly redistributed during endotoxin shock, with overall flow heterogeneity unchanged. A stronger redistribution of blood flow with endotoxin was associated with a larger fall in LV function parameters. Moreover, the correlation of regional O2 delivery to uptake fell from r = 0.73 ( P < 0.001) in control to r = 0.18 ( P = 0.25, P = 0.009 vs. control) in endotoxemic hearts. The results suggest a redistribution of LV regional coronary blood flow during endotoxin shock in pigs, with regional O2 delivery mismatched to O2 demand. Mismatching may underlie, at least in part, the myocardial depression of sepsis.

Architectural proteins have an important role in shaping the genome and act as global regulators of gene expression. How these proteins jointly modulate genome plasticity is largely unknown. In archaea, one of the most abundant proteins, Alba, is considered to have a key role in organizing the genome. Here we characterize the multimodal architectural properties and interplay of the Alba1 and Alba2 proteins using single-molecule imaging and manipulation techniques. We demonstrate that the two paralogues can bridge and rigidify DNA and that the interplay between the two proteins influences the balance between these effects. Our data yield a structural model that explains the multimodal behaviour of Alba proteins and its impact on genome folding., How the genome is physically organized is less understood in archaea than in eubacteria or eukaryotes. Laurens et al. measure DNA binding by the Sulfolobus solfataricus proteins Alba1 and Alba2 using single-molecule techniques and conclude that the presence of Alba2 leads to more bridging between DNA.

Mitochondria organize their genome in proteinĝ€"DNA complexes called nucleoids. The mitochondrial transcription factor A (TFAM), a protein that regulates mitochondrial transcription, is abundant in these nucleoids. TFAM is believed to be essential for mitochondrial DNA compaction, yet the exact mechanism has not been resolved. Here we use a combination of single-molecule manipulation and fluorescence microscopy to show the nonspecific DNA-binding dynamics and compaction by TFAM. We observe that single TFAM proteins diffuse extensively over DNA (sliding) and, by collisions, form patches on DNA in a cooperative manner. Moreover, we demonstrate that TFAM induces compaction by changing the flexibility of the DNA, which can be explained by local denaturation of the DNA (melting). Both sliding of TFAM and DNA melting are also necessary characteristics for effective, specific transcription regulation by TFAM. This apparent connection between transcription and DNA organization clarifies how TFAM can accomplish two complementary roles in the mitochondrial nucleoid at the same time. © 2012 Macmillan Publishers Limited. All rights reserved.

We investigate experimentally whether self-organized criticality (SOC) occurs in granular piles composed of different grains, namely, rice, lentils, quinoa, and mung beans. These four grains were selected to have different aspect ratios, from oblong to oblate. As a function of aspect ratio, we determined the growth (β) and roughness (α) exponents, the avalanche fractal dimension (D), the avalanche size distribution exponent (τ), the critical angle (γ), and its fluctuation. At superficial inspection, three types of grains seem to have power-law-distributed avalanches with a well-defined τ. However, only rice is truly SOC if we take three criteria into account: a power-law-shaped avalanche size distribution, finite size scaling, and a universal scaling relation relating characteristic exponents. We study SOC as a spatiotemporal fractal; in particular, we study the spatial structure of criticality from local observation of the slope angle. From the fluctuation of the slope angle we conclude that greater fluctuation (and thus bigger avalanches) happen in piles consisting of grains with larger aspect ratio. © 2012 American Physical Society.

Proton transfer is an elementary process in biology. Green fluorescent protein (GFP) has served as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. We have used pump-dump-probe spectroscopy to study how proton transfer through the 'proton-wire' around the chromophore is affected by a combination of mutations in a modern GFP variety (sGFP2). The results indicate that in H(2)O, after absorption of a photon, a proton is transferred (A* → I*) in 5 ps, and back-transferred from a ground state intermediate (I → A) in 0.3 ns, similar to time constants found with GFPuv, although sGFP2 shows less heterogeneous proton transfer. This suggests that the mutations left the proton-transfer largely unchanged, indicating the robustness of the proton-wire. We used pump-dump-probe spectroscopy in combination with target analysis to probe suitability of the sGFP2 fluorophore for super-resolution microscopy.

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440–670 nm; the two-photon excitation range is 900–1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.

Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.

Capsid maturation with large-scale subunit reorganization occurs in virtually all viruses that use a motor to package nucleic acid into preformed particles. A variety of ensemble studies indicate that the particles gain greater stability during this process, however, it is unknown which material properties of the fragile procapsids change. Using Atomic Force Microscopy-based nano-indentation, we study the development of the mechanical properties during maturation of bacteriophage HK97, a λ -like phage of which the maturation-induced morphological changes are well described. We show that mechanical stabilization and strengthening occurs in three independent ways: ( i ) an increase of the Young’s modulus, ( ii ) a strong rise of the capsid’s ultimate strength, and ( iii ) a growth of the resistance against material fatigue. The Young’s modulus of immature and mature capsids, as determined from thin shell theory, fit with the values calculated using a new multiscale simulation approach. This multiscale calculation shows that the increase in Young’s modulus isn’t dependent on the crosslinking between capsomers. In contrast , the ultimate strength of the capsids does increase even when a limited number of cross-links are formed while full crosslinking appears to protect the shell against material fatigue. Compared to phage λ , the covalent crosslinking at the icosahedral and quasi threefold axes of HK97 yields a mechanically more robust particle than the addition of the gpD protein during maturation of phage λ . These results corroborate the expected increase in capsid stability and strength during maturation, however in an unexpected intricate way, underlining the complex structure of these self-assembling nanocontainers.

Photochemical charge separation in isolated reaction center-light harvesting 1 (RC-LH1) complexes from Rhodobacter sphaeroides was examined using time-resolved mid-infrared pump-probe spectroscopy. Absorption difference spectra were recorded between 1760 and 1610 cm(-1) with subpicosecond time resolution to characterize excited-state and radical pair dynamics in these complexes, via the induced absorption changes in the keto carbonyl modes of the bacteriochlorophylls and bacteriopheophytins. Experiments on RC-LH1 complexes with and without the polypeptide PufX show that its presence is required to achieve generation of the radical pair P(+)Q(A)(-) under mildly reducing conditions. In the presence of PufX, the final radical pair formed over a ~3 ns period was P(+)Q(A)(-), but in its absence the corresponding radical pair was P(+)H(A)(-), implying that Q(A) was either absent in these PufX-deficient complexes or was prereduced. However, P(+)Q(A)(-) could be generated in PufX-deficient complexes following addition of the oxidant DMSO, showing that Q(A) was present in these complexes and allowing the conclusion that under mildly reducing conditions charge separation was blocked after P(+)H(A)(-) due to the presence of an electron on Q(A). The data provide strong support for the hypothesis that one of the functions of PufX is to regulate the stability of Q(B)(-), ensuring the oxidation of Q(A)(-) in the presence of a reduced quinone pool and so preserving efficient photochemical charge separation under anaerobic conditions.

The energy migration dynamics of the major light-harvesting antenna (LHCII) of plants was experimentally studied in the chlorophyll Qy region (630–700 nm). Using polarized transient absorption (TA) spectroscopy at 77 K we obtain information unaccessible to conventional isotropic TA on the energy transfer dynamics between isoenergetic Chlorophylls (Chls). The data are simultaneously analyzed with a target analysis and interpreted in the context of the exciton model for LHCII. The transients at 652 and 662 nm excitation show that the memory of polarization is completely lost with a (3.3–5.0 ps)−1 rate over the whole Qy range, which is attributed to equilibration within monomers and between the three subunits of the LHCII trimer.

The capsids of icosahedral viruses are closed shells assembled from a hexagonal lattice of proteins with fivefold angular defects located at the icosahedral vertices. Elasticity theory predicts that these disclinations are subject to an internal compressive prestress, which provides an explanation for the link between size and shape of capsids. Using a combination of experiment and elasticity theory we investigate the question of whether macromolecular assemblies are subject to residual prestress, due to basic geometric incompatibility of the subunits. Here we report the first direct experimental test of the theory: by controlled removal of protein pentamers from the icosahedral vertices, we measure the mechanical response of so-called "whiffle ball" capsids of herpes simplex virus, and demonstrate the signature of internal prestress locked into wild-type capsids during assembly. © 2012 American Physical Society.

The ability to visualize neurons inside living brain tissue is a fundamental requirement in neuroscience and neurosurgery. Especially the development of a noninvasive probe of brain morphology with micrometer-scale resolution is highly desirable, as it would provide a noninvasive approach to optical biopsies in diagnostic medicine. Two-photon laser-scanning microscopy (2PLSM) is a powerful tool in this regard, and has become the standard for minimally invasive high-resolution imaging of living biological samples. However, while 2PLSM-based optical methods provide sufficient resolution, they have been hampered by the requirement for fluorescent dyes to provide image contrast. Here we demonstrate high-contrast imaging of live brain tissue at cellular resolution, without the need for fluorescent probes, using optical third-harmonic generation (THG). We exploit the specific geometry and lipid content of brain tissue at the cellular level to achieve partial phase matching of THG, providing an alternative contrast mechanism to fluorescence. We find that THG brain imaging allows rapid, noninvasive label-free imaging of neurons, white-matter structures, and blood vessels simultaneously. Furthermore, we exploit THG-based imaging to guide micropipettes towards designated neurons inside live tissue. This work is a major step towards label-free microscopic live brain imaging, and opens up possibilities for the development of laser-guided microsurgery techniques in the living brain.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network. A key feature of newly-forming cortical and hippocampal networks during pre and early postnatal development is spontaneous, synchronized neuronal activity (Katz & Shatz, 1996; Khaziphov & Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic & Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry. In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network. © 2011 Journal of Visualized Experiments.

The human physiological system is stressed to its limits during endurance sports competition events. We describe a whole body computational model for energy conversion during bicycle racing. About 23 per cent of the metabolic energy is used for muscle work, the rest is converted to heat. We calculated heat transfer by conduction and blood flow inside the body, and heat transfer from the skin by radiation, convection and sweat evaporation, resulting in temperature changes in 25 body compartments. We simulated a mountain time trial to Alpe d'Huez during the Tour de France. To approach the time realized by Lance Armstrong in 2004, very high oxygen uptake must be sustained by the simulated cyclist. Temperature was predicted to reach 39 ° C in the brain, and 39.7 ° C in leg muscle. In addition to the macroscopic simulation, we analysed the buffering of bursts of high adenosine triphosphate hydrolysis by creatine kinase during cyclical muscle activity at the biochemical pathway level. To investigate the low oxygen to carbohydrate ratio for the brain, which takes up lactate during exercise, we calculated the flux distribution in cerebral energy metabolism. Computational modelling of the human body, describing heat exchange and energy metabolism, makes simulation of endurance sports events feasible.

The hippocampus is critical for a wide range of emotional and cognitive behaviors. Here, we performed the first genome-wide search for genes influencing hippocampal oscillations. We measured local field potentials (LFPs) using 64-channel multi-electrode arrays in acute hippocampal slices of 29 BXD recombinant inbred mouse strains. Spontaneous activity and carbachol-induced fast network oscillations were analyzed with spectral and cross-correlation methods and the resulting traits were used for mapping quantitative trait loci (QTLs), i.e., regions on the genome that may influence hippocampal function. Using genome-wide hippocampal gene expression data, we narrowed the QTLs to eight candidate genes, including Plcb1, a phospholipase that is known to influence hippocampal oscillations. We also identified two genes coding for calcium channels, Cacna1b and Cacna1e, which mediate presynaptic transmitter release and have not been shown to regulate hippocampal network activity previously. Furthermore, we showed that the amplitude of the hippocampal oscillations is genetically correlated with hippocampal volume and several measures of novel environment exploration. © 2011 Jansen et al.

We demonstrate that the focusing of a Bessel-correlated beam produces an intensity minimum at focus rather than a maximum. Varying the size of an iris changes this minimum into a maximum in a continuous manner.

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The ‘phosphocreatine shuttle’ hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using ‘sloppy modeling’ ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 µM*s−1) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 µM*s−1, while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 µM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect., Author Summary Creatine kinase (CK) has several functions in cellular energy metabolism. It catalyzes the reversible transfer of high-energy phosphate from ATP to creatine, facilitating storage of energy in the form of phosphocreatine. In muscle cells, this extra energy buffer plays a pivotal role in maintaining ATP homeostasis. Another proposed function of CK is the transport of energy from ATP producing to ATP consuming sites via a shuttle mechanism involving a mitochondrial and a myofibrillar isoform of CK. The extent to which this ‘phosphocreatine shuttle’ mechanism is used in muscle and other tissues is hotly debated. We use a computational model of the CK system which can predict energy transport and buffering of high demand peaks to estimate the relative importance of both roles in heart muscle. We validate the model with multiscale data on the level of enzyme kinetic constants and with dynamic oxygen consumption measurements in rabbit hearts. Since model predictions can be strongly affected by changes in parameter values, we employ ‘sloppy’ ensemble modeling which allows to set confidence regions for predictions. Our results indicate that the main function of CK in heart muscle lies more in temporal energy buffering of high peaks in ATP consumption during cardiac contraction than in energy transportation.

The last decade showed an increased interest in Langevin equations for modeling time series recorded from complex dynamical systems. These equations allow to discriminate between deterministic (drift) and stochastic (diffusion) components of the recorded time series. In practice, the estimation of drift and diffusion is often based on approximations of the models' dynamics that are valid only for high sampling frequencies. Also, model assessment is not or only indirectly performed, potentially leading to false claims. In this study we compare the performance of an asymptotically unbiased estimation method with a generally used approximate method, demonstrating the necessity of using (asymptotically) unbiased estimators. Furthermore, we describe how confidence intervals for the unknown parameters can be constructed and how model assessment can be carried out. We apply the methodology to local field potentials recorded in vitro from mouse hippocampus from eight genetically different strains. The recorded field potentials turn out to be well described by linearly damped Langevin equations with parabolic diffusion. The modeling enables a dynamical interpretation of the spectral power of the field potentials. It reveals that observed spectral power differences in the field potentials across hippocampal regions are associated with differences in the deterministic component of the system, and it reveals transiently active current dipoles, which are not detectable by conventional methods. Also, all estimated parameters have significant heritabilities, which suggests that the Langevin equations capture biological relevant aspects of electrical hippocampal activity. © 2011 American Physical Society.

We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 μm over a 500 μm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a threedimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan. © 2012 Optical Society of America.

We present a full-range Fourier-domain optical coherence tomography (OCT) system that is capable of acquiring two-dimensional images of living tissue in a single shot. By using line illumination of the sample in combination with a two-dimensional imaging spectrometer, 1040 depth scans are performed simultaneously on a sub-millisecond timescale. Furthermore, we demonstrate an easy and flexible real-time single-shot technique for full-range (complex-conjugate cancelled) OCT imaging that is compatible with both two-dimensional as well as ultrahighresolution OCT. By implementing a dispersion imbalance between reference and sample arms of the interferometer, we eliminate the complex-conjugate signal through numerical dispersion compensation, effectively increasing the useful depth range by a factor of two. The system allows us to record 6.7 × 3.2 mm images at 5 μm depth resolution in 0.2 ms. Data postprocessing requires only 4 s. We demonstrate the capability of our system by imaging the anterior chamber of a mouse eye in vitro, as well as human skin in vivo. © 2009 Optical Society of America.

We demonstrate high-resolution coherent lensless imaging using ultra-broadband light sources. We exploit Fourier-transform techniques to remove all spectral bandwidth limitations, paving the way for efficient soft-X-ray microscopy with high-harmonic sources.

International audience; In this work the software application called Glotaran is introduced as a Java-based graphical user interface to the R package TIMP, a problem solving environment for fitting superposition models to multi-dimensional data. TIMP uses a command-line user interface for the interaction with data, the specification of models and viewing of analysis results. Instead, Glotaran provides a graphical user interface which features interactive and dynamic data inspection, easier -- assisted by the user interface -- model specification and interactive viewing of results. The interactivity component is especially helpful when working with large, multi-dimensional datasets as often result from time-resolved spectroscopy measurements, allowing the user to easily pre-select and manipulate data before analysis and to quickly zoom in to regions of interest in the analysis results. Glotaran has been developed on top of the NetBeans rich client platform and communicates with R through the Java-to-R interface Rserve. The background and the functionality of the application are described here. In addition, the design, development and implementation process of Glotaran is documented in a generic way.