Nanoscale nuclear magnetic resonance with chemical resolution.

Nuclear magnetic resonance (NMR) spectroscopy is a key analytical technique in chemistry, biology, and medicine. However, conventional NMR spectroscopy requires an at least nanoliter-sized sample volume to achieve sufficient signal. We combined the use of a quantum memory and high magnetic fields with a dedicated quantum sensor based on nitrogen vacancy centers in diamond to achieve chemical shift resolution in 1H and 19F NMR spectroscopy of 20-zeptoliter sample volumes. We demonstrate the application of NMR pulse sequences to achieve homonuclear decoupling and spin diffusion measurements. The best measured NMR linewidth of a liquid sample was ~1 part per million, mainly limited by molecular diffusion. To mitigate the influence of diffusion, we performed high-resolution solid-state NMR by applying homonuclear decoupling and achieved a 20-fold narrowing of the NMR linewidth.

Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

All-Optical Preparation of Coherent Dark States of a Single Rare Earth Ion Spin in a Crystal.

All-optical addressing and coherent control of single solid-state based quantum bits is a key tool for fast and precise control of ground-state spin qubits. So far, all-optical addressing of qubits was demonstrated only in a very few systems, such as color centers and quantum dots. Here, we perform high-resolution spectroscopic of native and implanted single rare earth ions in solid, namely, a cerium ion in yttrium aluminum garnet (YAG) crystal. We find narrow and spectrally stable optical transitions between the spin sublevels of the ground and excited optical states. Utilizing these transitions we demonstrate the generation of a coherent dark state in electron spin sublevels of a single Ce^{3+} ion in YAG by coherent population trapping.

Multiple regulatory safeguards confine the expression of the GATA factor Serpent to the hemocyte primordium within the Drosophila mesoderm.

serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. Here, we find that srp is sufficient to transform trunk mesoderm into hemocytes. We identify two disjoint cis-regulatory modules that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium.

Mapping spin coherence of a single rare-earth ion in a crystal onto a single photon polarization state.

We report on optical detection of a single photostable Ce(3+) ion in an yttrium aluminium garnet (YAG) crystal and on its magneto-optical properties at room temperature. The spin quantum state of the emitting level of a single cerium ion in YAG can be initialized by a circularly polarized laser pulse. Coherent precession of the electron spin is read out by observing temporal behavior of circularly polarized fluorescence of the ion. This implies direct mapping of the spin quantum state of Ce(3+) ion onto the polarization state of the emitted photon and represents the quantum interface between a single spin and a single photon.

A vertex model of Drosophila ventral furrow formation.

UNLABELLED: Ventral furrow formation in Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline (the ventral furrow). Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a "tug-of-war" between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought. SUMMARY: For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their tips and then fold inwards into the interior of the embryo. Although numerous genes have been identified that are critical for ventral furrow formation, it is an open question how cells work together to elicit this tissue rearrangement. We use a computational model to mimic the physical properties of cells in the ventral epithelium and simulate the formation of the furrow. We find that the ventral furrow can form through stochastic self-organisation and that previous experimental observations can be readily explained in our model by considering forces that arise when cells execute contractions while being coupled to each other in a mechanically coherent epithelium. The model highlights the importance of a physical perspective when studying tissue morphogenesis and shows that only a minimal genetic regulation may be required to drive complex processes in embryonic development.

Single defect center scanning near-field optical microscopy on graphene.

We present a scanning-probe microscope based on an atomic-size emitter, a single nitrogen-vacancy center in a nanodiamond. We employ this tool to quantitatively map the near-field coupling between the NV center and a flake of graphene in three dimensions with nanoscale resolution. Further we demonstrate universal energy transfer distance scaling between a point-like atomic emitter and a two-dimensional acceptor. Our study paves the way toward a versatile single emitter scanning microscope, which could image and excite molecular-scale light fields in photonic nanostructures or single fluorescent molecules.

A dual role for the ßPS integrin myospheroid in mediating Drosophila embryonic macrophage migration.

Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main ßPS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards wounds.

Boosting nanodiamond fluorescence: towards development of brighter probes.

A novel approach for preparation of ultra-bright fluorescent nanodiamonds (fNDs) was developed and the thermal and kinetic optimum of NV center formation was identified. Combined with a new oxidation method, this approach enabled preparation of particles that were roughly one order of magnitude brighter than particles prepared with commonly used procedures.

Overexpression of AtHsfB4 induces specific effects on root development of Arabidopsis.

The functions of plant class B-heat shock factors (Hsfs) are not well understood. Hsfs belonging to this group differ from class A-Hsfs in structural features of the oligomerization domain and by the absence of a typical AHA motif for transcriptional activation. AtHsfB4 is expressed in different parts of the plants with highest levels in root tissue. Transgenic Arabidopsis plants overexpressing (OE) HsfB4 by CaMV-35S-promoter showed massively enhanced levels of Hsf mRNAs. The root surface of OE-plants was rough and cells became detached. Crossings with cell type specific root marker lines and confocal laser scanning microscopy provided clear evidence for a duplication of cells in the ground tissue and ectopic layers of lateral root cap (LRC) cells in HsfB4-OE plants. A duplication of endodermis cells occurs already during embryonic development, while the ectopic LRC cells are only detected during postembryonic growth. The mutant phenotypes of Hsf-OE plants are without precedence and indicate that class B-Hsfs may play an important role in root development.

Super-resolution fluorescence quenching microscopy of graphene.

Lately, fluorescence quenching microscopy (FQM) has been introduced as a new tool to visualize graphene-based sheets. Even though quenching of the emission from a dye molecule by fluorescence resonance energy transfer (FRET) to graphene happens on the nanometer scale, the resolution of FQM so far is still limited to several hundreds of nanometers due to the Abbe limit restricting the resolution of conventional light microscopy. In this work, we demonstrate an advancement of FQM by using a super-resolution imaging technique for detecting fluorescence of color centers used in FQM. The technique is similar to stimulated emission depletion microscopy (STED). The combined "FRET+STED" technique introduced here for the first time represents a substantial improvement to FQM since it exhibits in principle unlimited resolution while still using light in the visible spectral range. In the present case we demonstrate all-optical imaging of graphene with resolution below 30 nm. The performance of the technique in terms of imaging resolution and contrast is well described by a theoretical model taking into account the general distance dependence of the FRET process and the distance distribution of donor centers with respect to the flake. In addition, the change in lifetime for partially quenched emitters allows extracting the quenching distance from experimental data for the first time.

The PDZ-GEF protein Dizzy regulates the establishment of adherens junctions required for ventral furrow formation in Drosophila.

The PDZ-GEF protein Dizzy (Dzy) and its downstream GTPase Rap1 have pleiotropic roles during development of the Drosophila embryo. Here, we show that maternally provided Dzy and Rap1 first function during ventral furrow formation (VFF) where they are critical to guarantee rapid apical cell constrictions. Contraction of the apical actomyosin filament system occurs independently of Dzy and Rap1, but loss of Dzy results in a delayed establishment of the apical adherens junction (AJ) belt, whereas in the absence of Rap1 only a fragmentary apical AJ belt is formed in the epithelium. The timely establishment of apical AJs appears to be essential for coupling actomyosin contractions to cell shape change and to assure completion of the ventral furrow. Immediately after VFF, the downregulation of Dzy and Rap1 is necessary to allow normal mesodermal development to continue after the epithelial-to-mesenchymal transition, as overexpression of Dzy or of constitutively active Rap1 compromises mesodermal migration and monolayer formation. We propose that Dzy and Rap1 are crucial factors regulating the dynamics of AJs during gastrulation.

Highly efficient FRET from a single nitrogen-vacancy center in nanodiamonds to a single organic molecule.

We show highly efficient fluorescence resonance energy transfer (FRET) between negatively charged nitrogen-vacancy (NV) centers in diamond as donor and dye molecules as acceptor, respectively. The energy transfer efficiency is 86% with particles of 20 nm in size. Calculated and experimentally measured energy transfer efficiencies are in excellent agreement. Owing to the small size of the nanocrystals and careful surface preparation, energy transfer between a single nitrogen-vacancy center and a single quencher was identified by the stepwise change of energy transfer efficiencies due to bleaching of single acceptor molecules. Our studies pave the way toward FRET-based scanning probe techniques using single NV donors.

High yield fabrication of fluorescent nanodiamonds.

A new fabrication method to produce homogeneously fluorescent nanodiamonds with high yields is described. The powder obtained by high energy ball milling of fluorescent high pressure, high temperature diamond microcrystals was converted in a pure concentrated aqueous colloidal dispersion of highly crystalline ultrasmall nanoparticles with a mean size less than or equal to 10 nm. The whole fabrication yield of colloidal quasi-spherical nanodiamonds was several orders of magnitude higher than those previously reported starting from microdiamonds. The results open up avenues for the industrial cost-effective production of fluorescent nanodiamonds with well-controlled properties.

Mechanistic basis for differential inhibition of the F1Fo-ATPase by aurovertin.

The mitochondrial F(1)F(o)-ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F(1)F(o)-ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the beta subunits in the F(1) domain and inhibits F(1)F(o)-ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F(1)F(o)-ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K(i) values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F(1)F(o)-ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F(1)F(o)-ATPase catalyzes ATP synthesis and hydrolysis.

Fluorescence and spin properties of defects in single digit nanodiamonds.

This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, which is typical for the type Ib diamond from which the nanoparticle is made. We conclude that despite the tiny size of these nanodiamonds the photoactive nitrogen-vacancy color centers retain their bulk properties to the benefit of numerous exciting potential applications in photonics, biomedical labeling, and imaging.

Expression, function and regulation of Brachyenteron in the short germband insect Tribolium castaneum.

T-domain transcription factors are involved in many different processes during embryogenesis, such as mesoderm, heart or gut development in vertebrates and in invertebrates. In insects, the following five types of T-box genes are known: brachyenteron (byn), optomotor-blind (omb), optomotor-blind-related-gene-1 (org-1), dorsocross (doc) and H15. As all these classes are present in the genome of the fruit fly Drosophila melanogaster and the flour beetle Tribolium, the multiplicity of the five types of genes varies from dipterans to the beetle. In higher dipterans, a small cluster of three doc genes (doc1-doc3) exists, while the Tribolium genome contains a single Tc-doc gene only. Two H15 genes, Tc-H15a and Tc-H15b, are present in the Tribolium genome compared to a single H15 gene in Drosophila. We have analysed the expression and function of the Tribolium brachyenteron ortholog (Tc-byn). During embryogenesis, Tc-byn is exclusively expressed in the growth zone of the extending germband and later becomes confined to the distal proctodeum and the hindgut, a situation that parallels the expression pattern of byn in Drosophila. Tc-byn-RNAi treated embryos phenocopy Drosophila byn mutants and form no hindgut. In addition, we have characterised a regulatory element upstream of the Tc-byn transcription start site that confers specific gene expression in the developing hindgut of the Drosophila embryo. Our results demonstrate a highly conserved role for Brachyury-type transcriptional regulators in posterior gut development of insects at the level of expression, function and regulation.

The PDZ-GEF dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo.

In Drosophila embryos, macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The molecular mechanisms regulating this cell migration remain largely unknown. We identified the Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy as a component essential for normal macrophage migration. In mutants lacking Dizzy, macrophages have smaller cellular protrusions, and their migration is slowed down significantly. This phenotype appears to be cell-autonomous, as it is also observed in embryos with a dsRNA-induced reduction of dizzy function in macrophages. In a complementary fashion, macrophages overexpressing Dizzy are vastly extended and form very long protrusions. These cell shape changes depend on the function of the small GTPase Rap1: in rap1 mutants, Dizzy is unable to induce the large protrusions. Furthermore, forced expression of a dominant-active form of Rap1, but not of the wild-type form, induces similar cell shape changes as Dizzy does overexpression. These findings suggest that Dizzy acts through Rap1. We propose that integrin-dependent adhesion is a Rap1-mediated target of Dizzy activity: in integrin mutants, neither Dizzy nor Rap1 can induce cell shape changes in macrophages. These data provide the first link between a PDZ-GEF, the corresponding small GTPase and integrin-dependent cell adhesion during cell migration in embryonic development.

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.

Stepwise rotation of the gamma-subunit of EF(0)F(1)-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer.

The EF(0)F(1)-ATP synthase mutants bQ64C and gamma T106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the gamma-subunit. After reconstitution into liposomes, these double-labelled enzymes catalyzed ATP synthesis at a rate of 33 s(-1). Fluorescence of TMR and Cy5 was measured with a confocal set-up for single-molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET-states were identified, indicating stepwise rotation of the gamma-subunit of EF(0)F(1).