An Implementation of Combined Local-Global Optical Flow

Optical Flow (OF) approaches for motion estimation calculate vector fields for the apparent velocities of objects in image sequences. In 1981 Horn and Schunck (HS) introduced two basic assumptions: 'brightness value constancy' and 'smooth variation' to estimate a smooth OF field over the entire image -global approach-. In parallel, Lucas and Kanade (LK) assumed constant motion patterns for image patches, estimating piecewise-homogeneous OF fields -local approach-. Several variations of these approaches exist today. Here we present the combined local-global (CLG) approach by Bruhn et al. which encompasses properties of HS-OF and LK-OF, aiming to improve the OF accuracy for small-scale variations, while delivering the HS-OF dense and smooth fields. A multiscale implementation is provided for 2D images, together with two numerical solvers: Successive Over-Relaxation and the faster Pointwise-Coupled Gauss-Seidel by Bruhn et al.. The algorithm works on gray-scale (single channel) images, with color images being converted prior to the OF computation

dSTORM microscopy evidences in HeLa cells clustered and scattered gamma H2AX nanofoci sensitive to ATM, DNA-PK, and ATR kinase inhibitors

In response to DNA double-strand breaks (DSB), histone H2AX is phosphorylated around the lesion by a feed forward signal amplification loop, originating gamma H2AX foci detectable by immunofluorescence and confocal microscopy as elliptical areas of uniform intensity. We exploited the significant increase in resolution (similar to x 10) provided by single-molecule localization microscopy (SMLM) to investigate at nanometer scale the distribution of gamma H2AX signals either endogenous (controls) or induced by the radiomimetic bleomycin (BLEO) in HeLa cells. In both conditions, clustered substructures (nanofoci) confined to gamma H2AX foci and scattered nanofoci throughout the remnant nuclear area were detected. SR-Tesseler software (Voronoi tessellation-based segmentation) was combined with a custom Python script to first separate clustered nanofoci inside gamma H2AX foci from scattered nanofoci, and then to perform a cluster analysis upon each nanofoci type. Compared to controls, gamma H2AX foci in BLEO-treated nuclei presented on average larger areas (0.41 versus 0.19 mu m(2)), more nanofoci per focus (22.7 versus 13.2) and comparable nanofoci densities (similar to 60 nanofoci/mu m(2)). Scattered gamma H2AX nanofoci were equally present (similar to 3 nanofoci/mu m(2)), suggesting an endogenous origin. BLEO-treated cells were challenged with specific inhibitors of canonical H2AX kinases, namely: KU-55933, VE-821 and NU-7026 for ATM, ATR and DNA-PK, respectively. Under treatment with pooled inhibitors, clustered nanofoci vanished from super-resolution images while scattered nanofoci decreased (similar to 50%) in density. Residual scattered nanofoci could reflect, among other alternatives, H2AX phosphorylation mediated by VRK1, a recently described non-canonical H2AX kinase. In addition to H2AX findings, an analytical approach to quantify clusters of highly differing density from SMLM data is put forward

Drosophila Atlastin regulates synaptic vesicle mobilization independent of bone morphogenetic protein signaling

Abstract Background The endoplasmic reticulum (ER) contacts endosomes in all parts of a motor neuron, including the axon and presynaptic terminal, to move structural proteins, proteins that send signals, and lipids over long distances. Atlastin (Atl), a large GTPase, is required for membrane fusion and the structural dynamics of the ER tubules. Atl mutations are the second most common cause of Hereditary Spastic Paraplegia (HSP), which causes spasticity in both sexes’ lower extremities. Through an unknown mechanism, Atl mutations stimulate the BMP (bone morphogenetic protein) pathway in vertebrates and Drosophila. Synaptic defects are caused by atl mutations, which affect the abundance and distribution of synaptic vesicles (SV) in the bouton. We hypothesize that BMP signaling, does not cause Atl-dependent SV abnormalities in Drosophila. Results We show that atl knockdown in motor neurons (Atl-KD) increases synaptic and satellite boutons in the same way that constitutively activating the BMP-receptor Tkv (thick veins) (Tkv-CA) increases the bouton number. The SV proteins Cysteine string protein (CSP) and glutamate vesicular transporter are reduced in Atl-KD and Tkv-CA larvae. Reducing the activity of the BMP receptor Wishful thinking (wit) can rescue both phenotypes. Unlike Tkv-CA larvae, Atl-KD larvae display altered activity-dependent distributions of CSP staining. Furthermore, Atl-KD larvae display an increased FM 1–43 unload than Control and Tkv-CA larvae. As decreasing wit function does not reduce the phenotype, our hypothesis that BMP signaling is not involved is supported. We also found that Rab11/CSP colocalization increased in Atl-KD larvae, which supports the concept that late recycling endosomes regulate SV movements. Conclusions Our findings reveal that Atl modulates neurotransmitter release in motor neurons via SV distribution independently of BMP signaling, which could explain the observed SV accumulation and synaptic dysfunction. Our data suggest that Atl is involved in membrane traffic as well as formation and/or recycling of the late endosome

Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.Chilean Millennium Scientific Initiative ICM P09-015-F CONICYT PhD scholarships Comisión Nacional de Investigación Cientifica y Tecnológica (CONICYT) CONICYT FONDECYT 11170475 1181823 3160421 1161274 FONDECYT Ring Initiative ACT-1402 ACM170003 FONDAP 15150012 ANID Ring Initiative ACT-192015 Deutscher Akademischer Austausch Dienst (DAAD) 57220037 57168868 CORFO 16CTTS-6639