Speeding up active mesh segmentation by local termination of nodes.

This article outlines a procedure for speeding up segmentation of images using active mesh systems. Active meshes and other deformable models are very popular in image segmentation due to their ability to capture weak or missing boundary information; however, where strong edges exist, computations are still done after mesh nodes have settled on the boundary. This can lead to extra computational time whilst the system continues to deform completed regions of the mesh. We propose a local termination procedure, reducing these unnecessary computations and speeding up segmentation time with minimal loss of quality

Long-term imaging of the photosensitive, reef-building coral Acropora muricata using light-sheet illumination

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Laissue, P. P., Roberson, L., Gu, Y., Qian, C., & Smith, D. J. Long-term imaging of the photosensitive, reef-building coral Acropora muricata using light-sheet illumination. Scientific Reports, 10(1), (2020):10369, doi:10.1038/s41598-020-67144-w.Coral reefs are in alarming decline due to climate emergency, pollution and other man-made disturbances. The numerous ecosystem services derived from coral reefs are underpinned by the growth and physical complexity of reef-forming corals. Our knowledge of their fundamental biology is limited by available technology. We need a better understanding of larval settlement and development, skeletogenesis, interactions with pathogens and symbionts, and how this biology interacts with environmental factors such as light exposure, temperature, and ocean acidification. We here focus on a fast-growing key coloniser, Acropora muricata (Linnaeus, 1758). To enable dynamic imaging of this photosensitive organism at different scales, we developed light-sheet illumination for fluorescence microscopy of small coral colonies. Our approach reveals live polyps in previously unseen detail. An imaging range for Acropora muricata with no measurable photodamage is defined based upon polyp expansion, coral tissue reaction, and photobleaching. We quantify polyp retraction as a photosensitive behavioural response and show coral tissue rupture at higher irradiance with blue light. The simple and flexible technique enables non-invasive continuous dynamic imaging of highly photosensitive organisms with sizes between 1 mm3 and 5 cm3, for eight hours, at high temporal resolution, on a scale from multiple polyps down to cellular resolution. This live imaging tool opens a new window into the dynamics of reef-building corals.This work was made possible through a Royal Society Research Grant [RG120274], an innovation voucher from the University of Essex [DBF6000], a Royal Society Industry Fellowship [IF150018] and two Whitman Center Fellowships from the Marine Biological Laboratory (Woods Hole, USA) to PPL. PPL would like to thank Russell Smart for aquarium maintenance and Tony Jordan for production of customised parts. PPL also thanks the open-source communities OpenSPIM and µManager for support, as well as Cairn Research, 89North, Nikon Instruments UK, Alex Gardiol from Olympus Keymed UK, and Scott Young, Matt Preston and Daniel Croucher from Teledyne Photometrics for equipment loans. PPL is grateful to Amy Gladfelter, Hari Shroff, Abhishek Kumar, Louis Kerr, Philip M. Mullineaux, Marino Exposito-Rodriguez and Jean A. Laissue for support and critical discussions

Speeding Up Active Mesh Segmentation by Local Termination of Nodes

This article outlines a procedure for speeding up segmentation of images using active mesh systems. Active meshes and other deformable models are very popular in image segmentation due to their ability to capture weak or missing boundary information; however, where strong edges exist, computations are still done after mesh nodes have settled on the boundary. This can lead to extra computational time whilst the system continues to deform completed regions of the mesh. We propose a local termination procedure, reducing these unnecessary computations and speeding up segmentation time with minimal loss of quality

Identification of Quantitative Trait Loci responsible for embryonic lethality in mice assessed by ultrasonography

Chantier qualité GAInternational audienceRecurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In ~50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with diverse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA

Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells.

Phototoxicity is a significant constraint for live cell fluorescence microscopy. Excessive excitation light intensities change the homeostasis of the observed cells. Erroneous and misleading conclusions may be the problematic consequence of observing such light-induced pathophysiology. In this study, we assess the effect of blue light, as commonly used for GFP and YFP excitation, on a motile mammalian cell line. Tracking PC3 cells at different light doses and intensities, we show how motility can be used to reliably assess subtle positive and negative effects of illumination. We further show that the effects are a factor of intensity rather than light dose. Mitotic delay was not a sensitive indicator of phototoxicity. For early detection of the effect of blue light, we analysed the expression of genes involved in oxidative stress. This study addresses the need for relatively simple and sensitive methods to establish a dose-response curve for phototoxicity in mammalian cell line models. We conclude with a working model for phototoxicity and recommendations for its assessment

Multidimensional en-face OCT imaging of the retina.

Fast T-scanning (transverse scanning, en-face) was used to build B-scan or C-scan optical coherence tomography (OCT) images of the retina. Several unique signature patterns of en-face (coronal) are reviewed in conjunction with associated confocal images of the fundus and B-scan OCT images. Benefits in combining T-scan OCT with confocal imaging to generate pairs of OCT and confocal images similar to those generated by scanning laser ophthalmoscopy (SLO) are discussed in comparison with the spectral OCT systems. The multichannel potential of the OCT/SLO system is demonstrated with the addition of a third hardware channel which acquires and generates indocyanine green (ICG) fluorescence images. The OCT, confocal SLO and ICG fluorescence images are simultaneously presented in a two or a three screen format. A fourth channel which displays a live mix of frames of the ICG sequence superimposed on the corresponding coronal OCT slices for immediate multidimensional comparison, is also included. OSA ISP software is employed to illustrate the synergy between the simultaneously provided perspectives. This synergy promotes interpretation of information by enhancing diagnostic comparisons and facilitates internal correction of movement artifacts within C-scan and B-scan OCT images using information provided by the SLO channel

Mineral formation in the primary polyps of pocilloporoid corals

In reef-building corals, larval settlement and its rapid calcification provides a unique opportunity to study the bio-calcium carbonate formation mechanism involving skeleton morphological changes. Here we investigate the mineral formation of primary polyps, just after settlement, in two species of the pocilloporoid corals: Stylophora pistillata (Esper, 1797) and Pocillopora acuta (Lamarck, 1816). We show that the initial mineral phase is nascent Mg-Calcite, with rod-like morphology in P. acuta, and dumbbell morphology in S. pistillata. These structures constitute the first layer of the basal plate which is comparable to Rapid Accretion Deposits (Centers of Calcification, CoC) in adult coral skeleton. We found also that the rod-like/dumbbell Mg-Calcite structures in subsequent growth step will merge into larger aggregates by deposition of aragonite needles. Our results suggest that a biologically controlled mineralization of initial skeletal deposits occurs in three steps: first, vesicles filled with divalent ions are formed intracellularly. These vesicles are then transferred to the calcification site, forming nascent Mg-Calcite rod/pristine dumbbell structures. During the third step, aragonite crystals develop between these structures forming spherulite-like aggregates

ROS-dependent signaling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes

Abstract Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signaling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidizing equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signaling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signaling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signaling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signaling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells

Morphogenesis of a filamentous fungus : dynamics of the actin cytoskeleton and control of hyphal integrity in "Ashbya gossypii"

PART I - THE DYNAMIC ACTIN CYTOSKELETON OF ASHBYA GOSSYPII: Polarized growth is an intriguing aspect in a continuously elongating organism like A. gossypii. We therefore attempted a detailed study of the live actin cytoskeleton in this model filamentous fungus. We analyse the different components of the actin cytoskeleton tagged with Green Fluorescent Protein (GFP) by means of rapid, multi-dimensional video microscopy, studying their structural and dynamic properties. Cap1p and Cap2p are the subunits making up capping protein, a heterodimer which binds the barbed end of actin filaments. GFP-labelled variants of each were studied. Cap1-GFP and Cap2-GFP colocalize with actin patches in rhodamine-phalloidin stainings. They are highly enriched in the first six micrometers from the tip, mostly cortical, and at sites of septation and branch formation. Cap1p-GFP and Cap2p-GFP patches moved at 224 (+-98) nm/s over distances of 0.8 μm (+/-0.7μm) and generally had a lifetime of 14 seconds ((+/-6.5). Sequential recordings of the entire hypha were analysed, suggesting that these particles undergo a pattern of movement consistent with their role in endocytosis. That is, following an initial non-motile stage, actin patches undergo random movement near their site of formation, often followed by a secondary, linear retrograde movement away from the tip. Co-stainings with the endocytosis marker FM4-64 show partial colocalization, further supporting the notion that actin patches are involved in endocytosis. A second movement type is that of retrograde patches returning to the tip, resulting in a cycling pattern. This suggests maintenance of polarization by endocytic recycling, a mechanism which was corroborated by experiments concerning lateral diffusion in the apical membrane. Application of Latrunculin A results in depolarized, spherical tips. The combination of these results suggests that apart from their role in endocytosis, Cap-GFP patches are charged with the task of maintaining polarization by endocytic recycling. Actin cables and actin rings were made visible by using a GFP tagged variant of Abp140p, an F-actin binding and crosslinking protein. Abp140p-GFP colocalizes fully with actin cables, actin patches and actin rings in rhodamine phalloidin stainings. Abp140p-GFP cables are mostly cortical, often helical, can be as long as 40μm and are highly motile. The different fluorescent intensities indicate existence of actin bundles with different numbers of cables. Elongation of the tip of a cable is 184 (+/-62) nm/s. Fine cables in the apical zone often feature Abp140p-GFP patches moving to the tip, where they desintegrate. This is strongly reminiscent of the short, straight actin cables in S. cerevisiae, which have been shown to transport exocytic vesicles to the site where a new cell wall is formed. We conclude with a model of the hyphal organisation of the actin cytoskeleton in A. gossypii. PART II - FAR11P IS REQUIRED TO PREVENT PREMATURE HYPHAL ABSCISSION IN THE FILAMENTOUS FUNGUS ASHBYA GOSSYPII: AgFar11p belongs to the Far proteins which have diverse functions. In the budding yeast Saccharomyces cerevisiae, the syntenic homolog ScFar11p links pheromone response to the cell cycle. In the filamentous fungus Neurospora crassa, the Far11p homolog (NcHAM-2) is required for hyphal fusion. While this process is important for communication and homeostasis in filamentous fungi, it has not been observed in A.gossypii. We investigated the structure and role of AgFar11p. It is a putative transmembrane protein and bears conserved domains found in the homologs of S.cerevisiae and N.crassa. Deletion of the FAR11 gene in Ashbya gossypii leads to premature hyphal abscission at septa and lysis of hyphal compartments. This chain of events occurs in wild type only at the end of the life cycle, when spores are released from hyphal compartments. We conclude that hyphal abscission in far11Δ strains is premature and suggest that in A.gossypii, Far11p is involved in the timing of sporangium formation