OpenSPIM - an open access platform for light sheet microscopy

Light sheet microscopy promises to revolutionize developmental biology by enabling live in toto imaging of entire embryos with minimal phototoxicity. We present detailed instructions for building a compact and customizable Selective Plane Illumination Microscopy (SPIM) system. The integrated OpenSPIM hardware and software platform is shared with the scientific community through a public website, thereby making light sheet microscopy accessible for widespread use and optimization to various applications.Comment: 7 pages, 3 figures, 6 supplementary videos, submitted to Nature Methods, associated public website http://openspim.or

Blowup of Jang's equation at outermost marginally trapped surfaces

The aim of this paper is to collect some facts about the blowup of Jang's equation. First, we discuss how to construct solutions that blow up at an outermost MOTS. Second, we exclude the possibility that there are extra blowup surfaces in data sets with non-positive mean curvature. Then we investigate the rate of convergence of the blowup to a cylinder near a strictly stable MOTS and show exponential convergence near a strictly stable MOTS.Comment: 15 pages. This revision corrects some typo

BigFUSE: Global Context-Aware Image Fusion in Dual-View Light-Sheet Fluorescence Microscopy with Image Formation Prior

Light-sheet fluorescence microscopy (LSFM), a planar illumination technique that enables high-resolution imaging of samples, experiences defocused image quality caused by light scattering when photons propagate through thick tissues. To circumvent this issue, dualview imaging is helpful. It allows various sections of the specimen to be scanned ideally by viewing the sample from opposing orientations. Recent image fusion approaches can then be applied to determine in-focus pixels by comparing image qualities of two views locally and thus yield spatially inconsistent focus measures due to their limited field-of-view. Here, we propose BigFUSE, a global context-aware image fuser that stabilizes image fusion in LSFM by considering the global impact of photon propagation in the specimen while determining focus-defocus based on local image qualities. Inspired by the image formation prior in dual-view LSFM, image fusion is considered as estimating a focus-defocus boundary using Bayes Theorem, where (i) the effect of light scattering onto focus measures is included within Likelihood; and (ii) the spatial consistency regarding focus-defocus is imposed in Prior. The expectation-maximum algorithm is then adopted to estimate the focus-defocus boundary. Competitive experimental results show that BigFUSE is the first dual-view LSFM fuser that is able to exclude structured artifacts when fusing information, highlighting its abilities of automatic image fusion.Comment: paper in MICCAI 202

Reducing Library Characterization Time for Cell-aware Test while Maintaining Test Quality

Cell-aware test (CAT) explicitly targets faults caused by defects inside library cells to improve test quality, compared with conventional automatic test pattern generation (ATPG) approaches, which target faults only at the boundaries of library cells. The CAT methodology consists of two stages. Stage 1, based on dedicated analog simulation, library characterization per cell identifies which cell-level test pattern detects which cell-internal defect; this detection information is encoded in a defect detection matrix (DDM). In Stage 2, with the DDMs as inputs, cell-aware ATPG generates chip-level test patterns per circuit design that is build up of interconnected instances of library cells. This paper focuses on Stage 1, library characterization, as both test quality and cost are determined by the set of cell-internal defects identified and simulated in the CAT tool flow. With the aim to achieve the best test quality, we first propose an approach to identify a comprehensive set, referred to as full set, of potential open- and short-defect locations based on cell layout. However, the full set of defects can be large even for a single cell, making the time cost of the defect simulation in Stage 1 unaffordable. Subsequently, to reduce the simulation time, we collapse the full set to a compact set of defects which serves as input of the defect simulation. The full set is stored for the diagnosis and failure analysis. With inspecting the simulation results, we propose a method to verify the test quality based on the compact set of defects and, if necessary, to compensate the test quality to the same level as that based on the full set of defects. For 351 combinational library cells in Cadence’s GPDK045 45nm library, we simulate only 5.4% defects from the full set to achieve the same test quality based on the full set of defects. In total, the simulation time, via linear extrapolation per cell, would be reduced by 96.4% compared with the time based on the full set of defects

Meeting in the Middle: Towards Successful Multidisciplinary Bioimage Analysis Collaboration

With an increase in subject knowledge expertise required to solve specific biological questions, experts from different fields need to collaborate to address increasingly complex issues. To successfully collaborate, everyone involved in the collaboration must take steps to "meet in the middle". We thus present a guide on truly cross-disciplinary work using bioimage analysis as a showcase, where it is required that the expertise of biologists, microscopists, data analysts, clinicians, engineers, and physicists meet. We discuss considerations and best practices from the perspective of both users and technology developers, while offering suggestions for working together productively and how this can be supported by institutes and funders. Although this guide uses bioimage analysis as an example, the guiding principles of these perspectives are widely applicable to other cross-disciplinary work

The area of horizons and the trapped region

This paper considers some fundamental questions concerning marginally trapped surfaces, or apparent horizons, in Cauchy data sets for the Einstein equation. An area estimate for outermost marginally trapped surfaces is proved. The proof makes use of an existence result for marginal surfaces, in the presence of barriers, curvature estimates, together with a novel surgery construction for marginal surfaces. These results are applied to characterize the boundary of the trapped region.Comment: 44 pages, v3: small changes in presentatio

Regulation of neurocoel morphogenesis by Pard6γb

AbstractThe Par3/Par6/aPKC protein complex plays a key role in the establishment and maintenance of apicobasal polarity, a cellular characteristic essential for tissue and organ morphogenesis, differentiation and homeostasis. During a forward genetic screen for liver and pancreas mutants, we identified a pard6γb mutant, representing the first known pard6 mutant in a vertebrate organism. pard6γb mutants exhibit defects in epithelial tissue development as well as multiple lumens in the neural tube. Analyses of the cells lining the neural tube cavity, or neurocoel, in wildtype and pard6γb mutant embryos show that lack of Pard6γb function leads to defects in mitotic spindle orientation during neurulation. We also found that the PB1 (aPKC-binding) and CRIB (Cdc-42-binding) domains and the KPLG amino acid sequence within the PDZ domain (Pals1-and Crumbs binding) are not required for Pard6γb localization but are essential for its function in neurocoel morphogenesis. Apical membranes are reduced, but not completely absent, in mutants lacking the zygotic, or both the maternal and zygotic, function of pard6γb, leading us to examine the localization and function of the three additional zebrafish Pard6 proteins. We found that Pard6α, but not Pard6β or Pard6γa, could partially rescue the pard6γbs441 mutant phenotypes. Altogether, these data indicate a previously unappreciated functional diversity and complexity within the vertebrate pard6 gene family

A versatile cortical pattern-forming circuit based on Rho, F-actin, Ect2 and RGA-3/4

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, “chases” Rho waves in an F-actin–dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics

Invaginating endoderm pulls on the extending ectoderm

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.This work was funded by a Biotechnology and Biological Sciences Research Council grant BB/ J010278/1 to GBB, RJA and BS, a Wellcome Trust Investigator Award 099234/Z/12/Z to BS and a Herchel Smith Postdoctoral Fellowship from the University of Cambridge to C