Search

The main aim of this work was to stimulate bone-forming cells to produce three-dimensional networks of mineralized proteins such as those occurring in bones. This was achieved by a novel approach using a specific type of mesenchymal progenitor cells (i.e., primary fibroblast cells from human hair roots) seeded on to polymer scaffolds. We wrote polymer microstructures with one or more levels of quadratic pores on to a flexible substrate by means of two-photon polymerization using a Ti-sapphire femtosecond laser focused into a liquid acrylate-based resin containing a photoinitiator. Progenitor cells, differentiated into an osteogenic lineage by the use of medium supplemented with biochemical stimuli, can be seeded on to the hydrophilic three-dimensional scaffolds. Due to confinement to the microstructures and/or mechanical interaction with the scaffold, the cells are stimulated to produce high amounts of calcium-binding proteins, such as collagen type I, and show an increased activation of the actin cytoskeleton. The best results were obtained for quadratic pore sizes of 35 µm: the pore volumes become almost filled with both cells in close contact with the walls of the structure and with extracellular matrix material produced by the cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 891-899, 2017. [ABSTRACT FROM AUTHOR]

Optical injection of electrons from CdSe nanocrystals into amorphous semiconductors can be used to optically control the electron density in trap-rich conduction bands. For the specific example of polycrystalline C60, we show that important parameters of the density of trap states below the mobility edge can be retrieved. [ABSTRACT FROM AUTHOR]

The race toward engineering metamaterials comprising of negative refractive indexes in the optical range started with the realization of negative-index materials for gigahertz frequencies six years ago. Sheer miniaturization of the gigahertz resonant structures is one approach. Alternative designs make use of localized plasmon resonant metal nanoparticles or nanoholes in metal films. Following this approach, a negative refractive index has been realized in the optical range very recently. We review these recent results and summarize how to unambiguously retrieve the effective refractive index of thin layers from data accessible to measurements. Numerical simulations show that a composite material comprising of silver strips and a gain-providing material can have a negative refractive index of -1.3 and 100% transmission, simultaneously. [ABSTRACT FROM PUBLISHER]

We report on efficient resonant energy transfer in bilayers of water-soluble CdTe quantum dots. The bilayers of CdTe nanocrystals of two different sizes capped by short-chain thiols were formed by layer-by-layer assembly. Temporally and spectrally resolved fluorescence spectroscopy reveals spectral diffusion of the fluorescence signal for quantum dots within one layer as well as rapid (254 ps) energy transfer from layers of small dots to layers of larger dots, which is fast for nanocrystal pairs. Subspecies within the inhomogeneous distribution of donor nanocrystals even show energy transfer rates of (134 ps)-1 due to a large spectral overlap with acceptor nanocrystals. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]

A concept to electrically control the scattering of light is introduced. The idea is to embed noble metal nanoparticles in an electro-optical material such as a liquid crystal in order to induce a spectral shift of the particle plasmon resonance by applying an electric field. Light scattering experiments on single gold nanoparticles show that spherically shaped nanoparticles become optically spheroidal when covered by an anisotropic liquid crystal. The two particle plasmon resonances of the optically spheroidal gold nanoparticles can be spectrally shifted by up to 50 meV when electric fields of more than 10 kV/cm are applied. [ABSTRACT FROM AUTHOR]

We demonstrate that an offset stimulated emission depletion (STED) beam breaks the diffraction barrier of fluorescence microscopy in both the lateral and the axial directions. A 2.5-fold axial reduction of the focal spot is accomplished through the ear-shaped lobes of the diffraction maximum of the STED beam. The effect of the minima and side maxima of the STED beam on the lateral and axial resolution is shown to be in remarkable agreement with theory. Conditions are given for which a regular STED beam reduces the axial extent of a confocal spot from 490±36 to 175±18 nm, and simultaneously from 183±12 to 70±8 nm along the direction of the offset. The latter establishes the lowest reported value in far-field fluorescence microscopy. [ABSTRACT FROM AUTHOR]

We report the three-dimensional imaging of the vibrational, solvent, orientational, and electronic relaxation in organic fluorescent samples at 200-500 nm spatial resolution. This is achieved in steady-state recordings by exciting the fluorophore with a femtosecond pulse and subsequent quenching with a time-delayed, redshifted femtosecond pulse through stimulated emission. Temporal resolution of 380 fs is solely determined by the pulse widths and is further reducible. Images of submicron structures revealing vibrational and solvent relaxation gradients are shown. Furthermore, we introduce contrast modes based on stimulated emission depletion and apply them to cellular imaging. © 2000 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic steady state of actin network emerges from biochemical and structural feedbacks., Competing Interests: AM, TI, CG, TK, RB, MT, LB, AM No competing interests declared, (© 2019, Manhart et al.)

The growth of branched actin networks powers cell-edge protrusions and motility. A heterogeneous density of actin, which yields to a tunable cellular response, characterizes these dynamic structures. We study how actin organization controls both the rate and the steering during lamellipodium growth. We use a high-resolution surface structuration assay combined with mathematical modeling to describe the growth of a reconstituted lamellipodium. We demonstrate that local monomer depletion at the site of assembly negatively impacts the network growth rate. At the same time, network architecture tunes the protrusion efficiency, and regulates the rate of growth. One consequence of this interdependence between monomer depletion and network architecture effects is the ability of heterogeneous network to impose steering during motility. Therefore, we have established that the general principle, by which the cell can modulate the rate and the direction of a protrusion, is by varying both density and architecture of its actin network.Protrusive cellular structures contain a heterogeneous density of actin, but whether this influences motility is not known. Using an in vitro system and modelling, here the authors show that local actin monomer depletion and network architecture can tune the rate of network growth to impose steering during motility.

Nature has always served as an inspiration for scientists, helping them to solve a large diversity of technical problems. In our case, we are interested in the directional transport of oily liquids and as a model for this application we used the flat bug Dysodius lunatus . In this report, we present arrays of drops looking like polymer microstructures produced by the two-photon polymerization technique that mimic the micro-ornamentation from the bug's cuticle. A good directionality of oil transport was achieved, directly controlled by the direction of the pointed microstructures at the surface. If the tips of the drop-like microstructures are pointing towards the left side, the liquid front moves to the right and vice versa. Similar effects could be expected for the transport of oily lubricants. These results could, therefore, be interesting for applications in friction and wear reduction.

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

Unbiased gold nanoparticles are negatively charged in aqueous solution but not hydrated. Optical spectroscopy of voltage-clamped single gold nanoparticles reveals evidence that anion adsorption starts at positive potentials above the point of zero charge, causing severe but reversible plasmon damping in combination with a spectral red shift exceeding the linear double layer charging effect. Plasmon damping by adsorbate is relevant for the use of nanoparticles in catalysis, in biodiagnostics, and in surface enhanced Raman scattering., (© 2012 American Chemical Society)

DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.

Transition metal complexes such as biotinylated ruthenium(II) tris(bipyridyl) and palladium(II) porphyrin show an increase in luminescence intensity and lifetime upon binding to streptavidin in aqueous solution. Here we show that this increase of luminescence lifetime and intensity are caused by the shielding of the transition metal complexes from dissolved oxygen through streptavidin rather than by hydrophobicity effects as recently claimed.

We show that plasmonic nanoresonators composed of two gold nanoparticles change not only the intensity but also the spectral shape of the emission of fluorescent molecules. The plasmonic resonance frequency can be tuned by varying the distance between the nanoparticles, which allows us to selectively favor transitions of a fluorescent molecule to a specific vibrational ground state. Experimental data from correlated scattering and fluorescence microscopy agree well with calculations in the framework of generalized Mie theory. Our results show that the widely used description of a dye molecule near a metal surface as a mere two-level system is inadequate.

We show how to change optically the distance between two protein-linked gold nanoparticles by Raman-induced motion of the linker protein. Rayleigh scattering spectroscopy of the coupled-particle plasmon allows us to compare the inter-nanoparticle distance of individual protein-linked gold nanoparticle dimers before and after surface-enhanced Raman scattering (SERS). We find that low-intensity (50 microW/microm2) laser light in resonance with the nanoparticle-dimer plasmon provokes a change of the inter-nanoparticle distance on the order of 0.5 nm whenever SERS from the proteins connecting the nanoparticles can be observed.

Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.

X-ray crystallographic and functional analysis of the class I DNA photolyase from Thermus thermophilus revealed the binding of flavin mononucleotide (FMN) as an antenna chromophore. The binding mode of FMN closely coincides with the binding of a deazaflavin-like chromophore in the related class I DNA photolyase from Anacystis nidulans. Compared to the R46E mutant, which lacks a conserved arginine in the binding site for the antenna chromophore, the FMN-comprising holophotolyase exhibits an eightfold higher activity at 450 nm. The facile incorporation of the flavin cofactors 8-hydroxy-deazariboflavin and 8-iodo-8-demethyl-riboflavin into the binding site for the antenna chromophore paves the way for wavelength-tuning of the activity spectra of DNA photolyases by using synthetic flavins.

Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.

The fluorescence quantum yield of Cy5 molecules attached to gold nanoparticles via ssDNA spacers is measured for Cy5-nanoparticle distances between 2 and 16 nm. Different numbers of ssDNA per nanoparticle allow to fine-tune the distance. The change of the radiative and nonradiative molecular decay rates with distance is determined using time-resolved photoluminescence spectroscopy. Remarkably, the distance dependent quantum efficiency is almost exclusively governed by the radiative rate.

DNA photolyases use light energy to repair DNA that comprises ultraviolet-induced lesions such as the cis-syn cyclobutane pyrimidine dimers (CPDs). Here we report the crystal structure of a DNA photolyase bound to duplex DNA that is bent by 50 degrees and comprises a synthetic CPD lesion. This CPD lesion is flipped into the active site and split there into two thymines by synchrotron radiation at 100 K. Although photolyases catalyze blue light-driven CPD cleavage only above 200 K, this structure apparently mimics a structural substate during light-driven DNA repair in which back-flipping of the thymines into duplex DNA has not yet taken place.

The radiative and nonradiative decay rates of lissamine dye molecules, chemically attached to differently sized gold nanoparticles, are investigated by means of time-resolved fluorescence experiments. A pronounced fluorescence quenching is observed already for the smallest nanoparticles of 1 nm radius. The quenching is caused not only by an increased nonradiative rate but, equally important, by a drastic decrease in the dye's radiative rate. Assuming resonant energy transfer to be responsible for the nonradiative decay channel, we compare our experimental findings with theoretical results derived from the Gersten-Nitzan model.

We report on the generation of various hole-centered beams in the focal region of a lens and investigate their effectiveness to break the diffraction barrier in fluorescence microscopy by stimulated emission. Patterning of the phase of the stimulating beam across the entrance pupil of the objective lens produces point-spread-functions with twofold, fourfold, and circular symmetry, which narrow down the focal spot to 65-100 nm. Comparison with high-resolution confocal images exhibits a resolution much beyond the diffraction barrier. Particles that are only 65-nm apart are resolved with focused light.

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

We overcame the resolution limit of scanning far-field fluorescence microscopy by disabling the fluorescence from the outer part of the focal spot. Whereas a near-UV pulse generates a diffraction-limited distribution of excited molecules, a spatially offset pulse quenches the excited molecules from the outer part of the focus through stimulated emission. This results in a subdiffraction-sized effective point-spread function. For a 1.4 aperture and a 388-nm excitation wavelength spatial resolution is increased from 150 +/- 8 nm to 106 +/- 8 nm with a single offset beam. Superior lateral resolution is demonstrated by separation of adjacent Pyridine 2 nanocrystals that are otherwise indiscernible.