Engineered Nanoparticles Interacting with Cells: Size Matters

With the rapid advancement of nanoscience and nanotechnology, detailed knowledge of interactions between engineered nanomaterials and cells, tissues and organisms has become increasingly important, especially in regard to possible hazards to human health. This review intends to give an overview of current research on nano-bio interactions, with a focus on the effects of NP size on their interactions with live cells. We summarize common techniques to characterize NP size, highlight recent work on the impact of NP size on active and passive cellular internalization and intracellular localization. Cytotoxic effects are also discussed

Determination of mechanical stress distribution in Drosophila wing discs using photoelasticity

Morphogenesis, the process by which all complex biological structures are formed, is driven by an intricate interplay between genes, growth, as well as intra- and intercellular forces. While the expression of different genes changes the mechanical properties and shapes of cells, growth exerts forces in response to which tissues, organs and more complex structures are shaped. This is exemplified by a number of recent findings for instance in meristem formation in Arabidopsis and tracheal tube formation in Drosophila. However, growth not only generates forces, mechanical forces can also have an effect on growth rates, as is seen in mammalian tissues or bone growth. In fact, mechanical forces can influence the expression levels of patterning genes, allowing control of morphogenesis via mechanical feedback. In order to study the connections between mechanical stress, growth control and morphogenesis, information about the distribution of stress in a tissue is invaluable. Here, we applied stress-birefringence to the wing imaginal disc of Drosophila melanogaster, a commonly used model system for organ growth and patterning, in order to assess the stress distribution present in this tissue. For this purpose, stress-related differences in retardance are measured using a custom-built optical set-up. Applying this method, we found that the stresses are inhomogeneously distributed in the wing disc, with maximum compression in the centre of the wing pouch. This compression increases with wing disc size, showing that mechanical forces vary with the age of the tissue. These results are discussed in light of recent models proposing mechanical regulation of wing disc growth

Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors

Research Update: Interfacing ultrasmall metal nanoclusters with biological systems

Metal nanoclusters (NCs), a new type of nanomaterial with unique physicochemical properties, show great potential in many biomedical applications. Understanding their behavior in the complex biological environment is critical not only for designing highly efficient NC-based nanomedicines but also for elucidating the biological impact (e.g., toxicity) of these emerging nanomaterials. In this review, we give an overview of recent progress in exploring interactions of metal NCs with biological systems, including protein adsorption onto NCs, NC interactions with cells, and also the in vivo behavior of NCs. We also discuss the biological responses to the interactions, key parameters defining the interactions, and current challenges in the exploration of NCs in the complex biological environment

Wavelet-based background and noise subtraction for fluorescence microscopy images

Fluorescence microscopy images are inevitably contaminated by background intensity contributions. Fluorescence from out-of-focus planes and scattered light are important sources of slowly varying, low spatial frequency background, whereas background varying from pixel to pixel (high frequency noise) is introduced by the detection system. Here we present a powerful, easy-to-use software, wavelet-based background and noise subtraction (WBNS), which effectively removes both of these components. To assess its performance, we apply WBNS to synthetic images and compare the results quantitatively with the ground truth and with images processed by other background removal algorithms. We further evaluate WBNS on real images taken with a light-sheet microscope and a super-resolution stimulated emission depletion microscope. For both cases, we compare the WBNS algorithm with hardware-based background removal techniques and present a quantitative assessment of the results. WBNS shows an excellent performance in all these applications and significantly enhances the visual appearance of fluorescence images. Moreover, it may serve as a pre-processing step for further quantitative analysis

Pulsed interleaved excitation-based line-scanning spatial correlation spectroscopy (PIE-lsSCS)

We report pulsed interleaved excitation (PIE) based line-scanning spatial correlation spectroscopy (PIElsSCS), a quantitative fluorescence microscopy method for the study of dynamics in free-standing lipid bilayer membranes. Using a confocal microscope, we scan multiple lines perpendicularly through the membrane, each one laterally displaced from the previous one by several ten nanometers. Scanning through the membrane enables us to eliminate intensity fluctuations due to membrane displacements with respect to the observation volume. The diffusion of fluorescent molecules within the membrane is quantified by spatial correlation analysis, based on the fixed lag times between successive line scans. PIE affords dual-color excitation within a single line scan and avoids channel crosstalk. PIE-lsSCS data are acquired from a larger membrane region so that sampling is more efficient. Moreover, the local photon flux is reduced compared with single-point experiments, resulting in a smaller fraction of photobleached molecules for identical exposure times. This is helpful for precise measurements on live cells and tissues. We have evaluated the method with experiments on fluorescently labeled giant unilamellar vesicles (GUVs) and membrane-stained live cells

Electron-hole pairs during the adsorption dynamics of O2 on Pd(100) - Exciting or not?

During the exothermic adsorption of molecules at solid surfaces dissipation of the released energy occurs via the excitation of electronic and phononic degrees of freedom. For metallic substrates the role of the nonadiabatic electronic excitation channel has been controversially discussed, as the absence of a band gap could favour an easy coupling to a manifold of electronhole pairs of arbitrarily low energies. We analyse this situation for the highly exothermic showcase system of molecular oxygen dissociating at Pd(100), using time-dependent perturbation theory applied to first-principles electronic-structure calculations. For a range of different trajectories of impinging O2 molecules we compute largely varying electron-hole pair spectra, which underlines the necessity to consider the high-dimensionality of the surface dynamical process when assessing the total energy loss into this dissipation channel. Despite the high Pd density of states at the Fermi level, the concomitant non-adiabatic energy losses nevertheless never exceed about 5% of the available chemisorption energy. While this supports an electronically adiabatic description of the predominant heat dissipation into the phononic system, we critically discuss the non-adiabatic excitations in the context of the O2 spin transition during the dissociation process.Comment: 20 pages including 7 figures; related publications can be found at http://www.fhi-berlin.mpg.de/th/th.html [added two references, changed V_{fsa} to V_{6D}, modified a few formulations in interpretation of spin asymmetry of eh-spectra, added missing equals sign in Eg.(2.10)

Lef1 regulates caveolin expression and caveolin dependent endocytosis, a process necessary for Wnt5a/Ror2 signaling during Xenopus gastrulation

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/β-catenin pathway stimulates expression of β-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements