Trait-dependent resemblance of the flowering phenology and floral morphology of the allopolyploid Cardamine flexuosa to those of the parental diploids in natural habitats

Allopolyploids possess complete sets of genomes derived from different parental species and exhibit a range of variation in various traits. Reproductive traits may play a key role in the reproductive isolation between allopolyploids and their parental species, thus affecting the thriving of allopolyploids. However, empirical data, especially in natural habitats, comparing reproductive trait variation between allopolyploids and their parental species remain rare. Here, we documented the flowering phenology and floral morphology of the allopolyploid wild plant Cardamine flexuosa and its diploid parents C. amara and C. hirsuta in their native range in Switzerland. The flowering of C. flexuosa started at an intermediate time compared with those of the parents and the flowering period of C. flexuosa overlapped with those of the parents. Cardamine flexuosa resembled C. hirsuta in the size of flowers and petals and the length/width ratio of petals, while it resembled C. amara in the length/width ratio of flowers. These results provide empirical evidence of the trait-dependent variation of allopolyploid phenotypes in natural habitats at the local scale. They also suggest that the variation in some reproductive traits in C. flexuosa is associated with self-fertilization. Therefore, it is helpful to consider the mating system in furthering the understanding of the processes that may have shaped trait variation in polyploids in nature

A quantitative analysis of the interplay of environment, neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell-intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems-level studies of single-cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell-intrinsic and cell-extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies

Balanced Super-resolution Optical Fluctuation Imaging (bSOFI)

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing higher-order cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wide range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders. Balanced super-resolution optical fluctuation imaging (bSOFI), extends SOFI by the combination of several cumulant orders to map fluorescence-related molecular statistics, such as molecular state lifetimes, concentrations and brightness distributions with super-resolution. Since these parameters are often linked to the chemical microenvironment of the fluorophores, they report on static differences and/or dynamic changes within cells and thus add a “functional” dimension to super-resolution microscopy based on stochastic switching. Furthermore, the information obtained can be used to correct for the nonlinear brightness and blinking response of cumulants. We show experimental results of Alexa647-labeled microtubules in fixed HeLa cells with an up to five-fold resolution improvement compared to diffraction-limited widefield microscopy. Using a total-internal-reflection illumination scheme, we obtain depth information through the estimation of the spatial distributions of the molecular brightness as well as the blinking on-ratio

A quantitative analysis of the interplay of environment, neighborhood, and cell state in 3D spheroids

Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. To analyze the interplay of environment, local neighborhood, and internal cell state effects on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging analysis of up to 240 pooled spheroid microtissues. We quantified the contributions of environment, neighborhood, and intracellular state to marker variability in single cells of the spheroids. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. The contributions of cell‐intrinsic and environmental factors to marker variability are hierarchically interdependent, a finding that we propose has general implications for systems‐level studies of single‐cell phenotypic variability. By the overexpression of 51 signaling protein constructs in subsets of cells, we also identified proteins that have cell‐intrinsic and cell‐extrinsic effects. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.ISSN:1744-429

Characterization of Nanoparticles Using Non-linear Correlation Spectroscopy

Nanoparticles are interesting for biomedical imaging as novel non- fluorescent non-bleaching labels. We present the characterization of freely diffusing nanoparticles using third harmonic generation correlation spectroscopy by illumination with a focussed pulsed femtosecond laser. We present the optical system as well as first spectroscopic results on nanoparticles

Nonlinear Correlation Spectroscopy (NLCS)

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO3 show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments

Triplet imaging of oxygen consumption during the contraction of a single smooth muscle cell (A7r5)

Triplet imaging is a novel optical technique that allows investigating oxy- gen metabolism at the single cell and the sub-cellular level. The method combines high temporal and spatial resolution, which are required for the monitoring of fast kinetics of oxygen concentration in living cells. Calibration and validation is demon- strated with a titration experiment using L-Ascorbic Acid with the enzyme Ascor- base oxidase. The method was applied to a biological cell system, employing as reporter a cytosolic fusion protein of β-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg8]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays mono- exponentially with time. This is in good agreement with previously reported mea- surements of oxygen consumption in skeletal muscle fibers

Nonlinear Correlation Spectroscopy (NLCS)

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO₃ show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments

The clumpy absorber in the high-mass X-ray binary Vela X-1

Bright and eclipsing, the high-mass X-ray binary Vela X-1 offers a unique opportunity to study accretion onto a neutron star from clumpy winds of O/B stars and to disentangle the complex accretion geometry of these systems. In Chandra-HETGS spectroscopy at orbital phase 0.25, when our line of sight towards the source does not pass through the large-scale accretion structure such as the accretion wake, we observe changes in overall spectral shape on timescales of a few kiloseconds. This spectral variability is, at least in part, caused by changes in overall absorption and we show that such strongly variable absorption cannot be caused by unperturbed clumpy winds of O/B stars. We detect line features from high and low ionization species of silicon, magnesium, and neon whose strengths and presence depend on the overall level of absorption. These features imply a co-existence of cool and hot gas phases in the system, which we interpret as a highly variable, structured accretion flow close to the compact object such as has been recently seen in simulations of wind accretion in high-mass X-ray binaries.V.G. is thankful for support of her work through the ESA internal research fellowship. Part of this work was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Support for this work was provided by NASA through the Smithsonian Astrophysical Observatory (SAO) contract SV3-73016 to MIT for Support of the Chandra X-Ray Center (CXC) and Science Instruments; CXC is operated by SAO for and on behalf of NASA under contract NAS8-03060. I.E.M. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie SkłlCodowska-Curie grant agreement No. 665501 with the Research Foundation Flanders (FWO). A.A.C.S. is supported by the Deutsche Forschungsgemeinschaft (DFG) under grant HA 1455/26. J.N. acknowledges support from NASA through the Hubble Postdoctoral Fellowship Program, grant HST-HF2-51343.001-A. S.M.N. acknowledges support by research project ESP2016-76683-C3-1-R.Peer Reviewe