Intensity distortions due to phase-only spatial light modulation: Characterization for applications in electronic speckle-pattern interferometry.

Electronic speckle-pattern interferometry (ESPI) is a powerful tool for precise, full-field, and non-contact contouring of optically rough surfaces. Due to the interferometric principle, the sensitivity of ESPI is directly related to the involved wavelengths and is thereby a global parameter. Surfaces with a broad variation of phase gradients, as, for instance, a target with both smooth and comparatively steep areas, result in just partially resolvable fringe interferograms. In recent studies, spatial light modulators (SLMs) have been implemented to adapt the interferometric reference phase front to the measurement task and broaden or squeeze the fringe spacing locally in critical areas. This method is limited by diffraction effects, observable for all types of phase-only SLMs. We demonstrate a straight-forward model, describing the diffraction-based intensity distortions occurring in interferograms after wavefront adaptation. The aim is to characterize the intensity distortions by means of the proposed model and minimize their impact, especially with regard to phase-only spatial light modulation in ESPI. For validation, the modeled behavior of SLMs is compared to the experimental results, obtained for two different SLM designs. Finally, experiments are presented, which demonstrate a successful adaptation of the interferometric reference phase front in compliance with the boundary conditions determined by the model.

Structure-based models of cadherin-mediated cell adhesion: the evolution continues.

Cadherins are glycoproteins that are responsible for homophilic, Ca2+-dependent cell-cell adhesion and play crucial roles in many cellular adhesion processes ranging from embryogenesis to the formation of neuronal circuits in the central nervous system. Many different experimental approaches have been used to unravel the molecular basis for cadherin-mediated adhesion. In particular, several high-resolution structures have provided models for cadherin-cadherin interactions that are illuminative in many respects yet contradictory in others. This review gives an overview of the structural studies of cadherins over the past decade while focusing on recent developments that reconcile some of the earlier findings.

Homophilic adhesion by cadherins.

Cadherins mediate cell-cell adhesion through homophilic interactions. High-resolution structures have greatly enhanced our understanding of this phenomenon over the past few years. Nonetheless, some of the original concepts about cadherin interactions need revision, with the new structural and additional mutagenesis data currently available. Furthermore, in vivo studies on cadherins have provided supplementary information.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation.

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

Reconstruction of surfaces from phase-shifting speckle interferometry: Bayesian approach.

The reconstruction of surfaces from speckle interferometry data is a demanding data-analysis task that involves edge detection, edge completion, and image reconstruction from noisy data. We present an approach that makes optimal use of the experimental information to minimize the hampering influence of the noise. The experimental data are then analyzed with a combination of wavelet transform and Bayesian probability theory. Nontrivial examples are presented to illustrate the proposed technique.

Spinalin, a new glycine- and histidine-rich protein in spines of Hydra nematocysts.

Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst's capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.

Calcium binding and homoassociation of E-cadherin domains.

Cadherins are single pass transmembrane glycoproteins which mediate calcium dependent cell-cell adhesion by homophilic interactions. To reveal the molecular details of calcium binding and homoassociation, we recombinantly expressed in Escherichia coli a domain pair consisting of the first two domains of E-cadherin (ECAD12) and the single domains 1, 2, and 5. ECAD12 encompasses the most N-terminal of the four putative calcium-binding pockets in the extracellular region of E-cadherin. Equilibrium dialysis experiments revealed that the single domains do not bind Ca2+, but ECAD12 was found to bind three calcium ions. ECAD12 dimerizes (Kd = 0.08 +/- 0.02 mM) in the presence of Ca2+ as we could demonstrate by analytical ultracentrifugation. Calcium binding to ECAD12 induces conformational changes which were monitored by electrophoretic mobility and by circular dichroism. By analyzing our equilibrium dialysis data with a single binding site model, we found an average Kd of 460 microM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical Kds of 330 microM and a third much higher Kd of 2 mM. The entire extracellular region of E-cadherin, which was recombinantly expressed in mammalian cells, binds nine Ca2+ with a much lower average Kd of 30 microM. Therefore, we conclude that the four calcium binding pockets are not identical. Since binding to ECAD12 occurs at Ca2+ concentrations close to those in the extracellular space, we suggest that the N-terminal domain pair might be involved in calcium regulation of E-cadherin mediated cell-cell adhesion.

Approach for the evaluation of speckle deformation measurements by application of the wavelet transformation.

We introduce a new, to our knowledge, method using wavelets and probability theory for the evaluation of speckle interference patterns for quantitative out-of-plane deformation measurements of rough surfaces of nontransparent solids. The experiment uses a conventional Twyman-Green interferometer setup. The speckle interference patterns are obtained by the common method of subtraction of images taken before and after a surface deformation. The data are processed by a wavelet transformation, which analyzes the image structures on different length scales. Thus it is possible to separate the interference fringes from the noise. From the locations of the interference fringes, the deformation of the surface can be reconstructed by means of probability theory.

New calibration method for the determination of the absolute density of CH radicals through laser-induced fluorescence.

Laser-induced fluorescence (LIF) was applied at the B-X transition of the CH radical to measure the absolute densities of CH radicals in an electron-cyclotron resonance methane plasma. The absolute experimental uncertainty is only approximately 30% as a result of a new calibration procedure. The experimental setup was calibrated through the comparison of the LIF signal of N(2)(+) with that of CH. The absolute N(2)(+) density was derived from the spatially resolved N(2)(+) LIF signal and the line-averaged electron density as measured with microwave interferometry.