Separate Stacks Research Articles

Golgi clustering by the deficiency of COPI-SNARE in Drosophila photoreceptors.

A comprehensive study of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the fly genome by RNAi in Drosophila photoreceptors indicated that knockdown of any of the COPI-SNAREs, Syx18, Sec20, and Use1, resulted in the same characteristic phenotypes: Golgi stacks gathering on their trans-side, laterally expanded Golgi cisternae, and a reduced number of discrete Golgi stacks. These Golgi stacks are reminiscent of mammalian Golgi ribbons and Brefeldin A (BFA)-bodies in Drosophila S2 cells. As previously reported, BFA suppresses trans-Golgi network (TGN) fission and Golgi stack separation to form a BFA-body, which is a cluster of Golgi stacks cored by recycling endosomes. We found that the impairing each of COPI-SNAREs results in clustered Golgi stacks similar to BFA-bodies, indicating that COPI-SNAREs have a role to separate clustered Golgi stacks. These results further support the idea that the movement of Golgi stacks and the balance of fusion and fission of the TGN determine the level of clustering and ribbon formation of Golgi stacks within cells.

Transforming Thermally Activated Delayed Fluorescence to Room-Temperature Phosphorescence through Modulation of the Donor in Charge-Transfer Cocrystals.

A cocrystallization strategy is used through incorporation of 1,2,4,5-tetracyanobenzene (TCNB) as an acceptor with halogen-substituent thioxanthone (TX) derivatives as donors. The resulting cocrystals TT-R (R = H, F, Cl, Br, or I) transform the thermally activated delayed fluorescence emission in the TT-H, TT-F, and TT-Cl cocrystals to room-temperature phosphorescence in the TT-Br and TT-I cocrystals. Definite crystal packing structures demonstrate a 1:1 alternative donor-acceptor stacking in the TT-H cocrystal, a 2:1 alternative donor-acceptor stacking in the TT-F and TT-Cl cocrystals, and a separate stacking of donor and acceptor in the TT-Br and TT-I cocrystals. A transformation law can be revealed that with an increase in atomic number from H, F, Cl, Br, to I, the cocrystals show the structural transformation of the number of aggregated TX-R molecules from monomers to dimers and finally to multimers. This work will facilitate an understanding of the effect of halogen substituents on the crystal packing structure and luminescence properties in the cocrystals.

A targeted search for FRB counterparts with Konus-Wind

ABSTRACT We present results of the search for hard X-ray/soft gamma-ray emission in coincidence with publicly reported (via Transient Name Server, TNS1) fast radio bursts (FRBs). The search was carried out using continuous Konus-Wind data with 2.944 s time resolution. We perform a targeted search for each individual burst from 581 FRBs, along with a stacking analysis of the bursts from eight repeating sources in our sample and a separate stacking analysis of the bursts from the non-repeating FRBs. We find no significant associations in either case. We report upper bounds on the hard X-ray (20–1500 keV) flux assuming four spectral models, which generally describe spectra of short and long gamma-ray bursts (GRBs), magnetar giant flares, and the short burst, coincident with FRB 200428 from a Galactic magnetar. Depending on the spectral model, our upper bounds are in the range of (0.1–2) × 10−6 erg cm−2. For 18 FRBs with known distances, we present upper bounds on the isotropic equivalent energy release and peak luminosity. For the nearest FRB 200120E, we derive the most stringent upper bounds of Eiso ≤ 2.0 × 1044 erg and Liso ≤ 1.2 × 1044 erg s−1. Furthermore, we report lower bounds on radio-to-gamma-ray fluence ratio Eradio/Eiso ≥ 10−11–10−9 and compare our results with previously reported searches and theoretical predictions for high-energy counterparts to FRBs.

Emergent fermionic gauge theory and foliated fracton order in the Chamon model

The Chamon model is an exactly solvable spin Hamiltonian exhibiting nontrivial fracton order. In this paper, we dissect two distinct aspects of the model. First, we show that it exhibits an emergent fractonic gauge theory coupled to a fermionic subsystem symmetry-protected topological state under four stacks of ${\mathbb{Z}}_{2}$ planar symmetries. Second, we show that the Chamon model hosts 4-foliated fracton order by describing an entanglement renormalization group transformation that exfoliates four separate stacks of 2D toric codes from the bulk system.

Structural Damage Identification Using Ensemble Deep Convolutional Neural Network Models

The existing strategy for evaluating the damage condition of structures mostly focuses on feedback supplied by traditional visual methods, which may result in an unreliable damage characterization due to inspector subjectivity or insufficient level of expertise. As a result, a robust, reliable, and repeatable method of damage identification is required. Ensemble learning algorithms for identifying structural damage are evaluated in this article, which use deep convolutional neural networks, including simple averaging, integrated stacking, separate stacking, and hybrid weighted averaging ensemble and differential evolution (WAE-DE) ensemble models. Damage identification is carried out on three types of damage. The proposed algorithms are used to analyze the damage of 4585 structural images. The effectiveness of the ensemble learning techniques is evaluated using the confusion matrix. For the testing dataset, the confusion matrix achieved an accuracy of 94 percent and a minimum recall of 92 percent for the best model (WAE-DE) in distinguishing damage types as flexural, shear, combined, or undamaged.

Buddy Stacks: Protecting Return Addresses with Efficient Thread-Local Storage and Runtime Re-Randomization

Shadow stacks play an important role in protecting return addresses to mitigate ROP attacks. Parallel shadow stacks, which shadow the call stack of each thread at the same constant offset for all threads, are known not to support multi-threading well. On the other hand, compact shadow stacks must maintain a separate shadow stack pointer in thread-local storage (TLS) , which can be implemented in terms of a register or the per-thread Thread-Control-Block (TCB) , suffering from poor compatibility in the former or high performance overhead in the latter. In addition, shadow stacks are vulnerable to information disclosure attacks. In this paper, we propose to mitigate ROP attacks for single- and multi-threaded server programs running on general-purpose computing systems by using a novel stack layout, called a buddy stack (referred to as Bustk ), that is highly performant, compatible with existing code, and provides meaningful security. These goals are met due to three novel design aspects in Bustk . First, Bustk places a parallel shadow stack just below a thread’s call stack (as each other’s buddies allocated together), avoiding the need to maintain a separate shadow stack pointer and making it now well-suited for multi-threading. Second, Bustk uses an efficient stack-based thread-local storage mechanism, denoted STK-TLS , to store thread-specific metadata in two TLS sections just below the shadow stack in dual redundancy (as each other’s buddies), so that both can be accessed and updated in a lightweight manner from the call stack pointer rsp alone. Finally, Bustk re-randomizes continuously (on the order of milliseconds) the return addresses on the shadow stack by using a new microsecond-level runtime re-randomization technique, denoted STK-MSR . This mechanism aims to obsolete leaked information, making it extremely unlikely for the attacker to hijack return addresses, particularly against a server program that sits often tens of milliseconds away from the attacker. Our evaluation using web servers, Nginx and Apache Httpd , shows that Bustk works well in terms of performance, compatibility, and security provided, with its parallel shadow stacks incurring acceptable memory overhead for real-world applications and its STK-TLS mechanism costing only two pages per thread. In particular, Bustk can protect the Nginx and Apache servers with an adaptive 1-ms re-randomization policy (without observable overheads when IO is intensive, with about 17,000 requests per second). In addition, we have also evaluated Bustk using other non-server applications, Firefox , Python , LLVM , JDK and SPEC CPU2006 , to demonstrate further the same degree of performance and compatibility provided, but the protection provided for, say, browsers, is weaker (since network-access delays can no longer be assumed).

Data-Driven Compressive Strength Prediction of Fly Ash Concrete Using Ensemble Learner Algorithms

Concrete is one of the most popular materials for building all types of structures, and it has a wide range of applications in the construction industry. Cement production and use have a significant environmental impact due to the emission of different gases. The use of fly ash concrete (FAC) is crucial in eliminating this defect. However, varied features of cementitious composites exist, and understanding their mechanical characteristics is critical for safety. On the other hand, for forecasting the mechanical characteristics of concrete, machine learning approaches are extensively employed algorithms. The goal of this work is to compare ensemble deep neural network models, i.e., the super learner algorithm, simple averaging, weighted averaging, integrated stacking, as well as separate stacking ensemble models, and super learner models, in order to develop an accurate approach for estimating the compressive strength of FAC and reducing the high variance of the predictive models. Separate stacking with the random forest meta-learner received the most accurate predictions (97.6%) with the highest coefficient of determination and the lowest mean square error and variance.

Process evaluation and kinetic analysis of 3D-printed monoliths comprised of CaO and Cr/H-ZSM-5 in combined CO2 Capture-C2H6 oxidative dehydrogenation to C2H4

In this study, dual-function materials (DFMs) comprised of CaO and Cr/H-ZSM-5 were formulated in 3D-printed monolithic structures and investigated in a combined process for capture and utilization of CO2in oxidative dehydrogenation of C2H6 to C2H4 (CO2-ODHE). Various formulation strategies were employed to fabricate these DFM structures. Two bed designs were considered: i) a layered-bed in which adsorbent (CaO) and catalyst were printed separately and stacked on top of each other, and ii) a single-layer bed where the adsorbent-catalyst materials were 3D-printed into a singular monolith and loaded into the bed as DFMs. Between these, the layered-bed displayed enhanced performance as compared to the composite DFMs, as this configuration generated a 3.73 mmol/g CO2 adsorption capacity, 42.5% C2H6 conversion, 90.6% C2H4 selectivity and 38.6% C2H4 yield compared to 2.4 mmol/g CO2 adsorption capacity, 37.9% C2H6 conversion, 89% C2H4 selectivity and 33.8% C2H4 yield in the best singular monolith configuration. The enhanced performance of this configuration was attributed to a higher degree of adsorptive site accessibility and a lesser degree of active site blockage from intraparticle binding (as evident from the textural properties). This work also assessed the effects of monolith cell density on CO2 adsorption and utilization for ODHE by varying the cells per square inch (cpsi) from 200 to 600 cpsi. These experiments revealed that increasing the cell density from 200 to 600 cpsi enhanced the overall mass transfer coefficient from 0.40 × 10-2 to 1.01 × 10-2 s-1 due to a sizable enhancement in film mass transfer between the two geometric designs. Such enhancement improved the C2H4 selectivity and yield to 92 and 45.1% in the 600 cpsi sample. As such, this work also indicated that higher cell densities lead to better performance of the layered-bed configuration, where the optimum configuration is separate stacking of the adsorbent and catalyst phases. Overall, these findings provide a deeper understanding of DFM materials, bed configuration, and establish a groundwork which can be used to optimize their structures for CO2 adsorption-reaction processes.

Investigating Mechanical Properties of Carbon Glass Jute Fiber based Composite

Hybrid composites are made by combining natural and synthetic fibers with an effective matrix, which usually means they’ve received additional strengthening, such as epoxy, to create the additional material properties you can’t obtain on their own. To attain the desirable tensile modulus, compressive modulus, and so on, a fiber composite needs to be added to the FRP (Fiber Reinforced Polymer). Polymer matrix composites are light and cost-effective to manufacture, but they still friendly to the environment and have viable applications, which is why they are often used in various commercial applications. Unidirectional fibers and bidirectionally reinforced with epoxy (SikaDur is a composite medium) carbon fibers are two-way reinforced with unidirectional (use unidirectional) Before we developed test procedures for preparing the test specimens, the testing lab implemented the layup method according to ASTM standards. Ten separate stacking sequences were tested and four different intensity sequences were used in testing the compressive structures according to ASTM D15. The results of the study indicate that hybridization helps natural fiber-reinforced polymer composites to increase their mechanical properties We would use natural fibers rather than synthetic ones since the natural ones make comparable strength when hybridized with synthetic ones.

Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms

Continuous monitoring of large specimens for long durations requires fast volume imaging. This is essential for understanding the processes occurring during the developmental stages of multicellular organisms. One of the key obstacles of fluorescence based prolonged monitoring and data collection is photobleaching. To capture the biological processes and simultaneously overcome the effect of bleaching, we developed single- and multi-color lightsheet based OVSS imaging technique that enables rapid screening of multiple tissues in an organism. Our approach based on OVSS imaging employs quantized step rotation of the specimen to record 2D angular data that reduces data acquisition time when compared to the existing light sheet imaging system (SPIM). A co-planar multicolor light sheet PSF is introduced to illuminate the tissues labelled with spectrally-separated fluorescent probes. The detection is carried out using a dual-channel sub-system that can simultaneously record spectrally separate volume stacks of the target organ. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged the Drosophila larvae and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish for a prolonged duration (5 days) were carried out to understand muscle structuring (Dystrophin, microfibers), primitive Macrophages (in yolk-sac) and inter-dependent lipid and protein-based metabolism. The volume-based study, intensity line-plots and inter-dependence ratio analysis allowed us to understand the transition from lipid-based metabolism to protein-based metabolism during early development (Pharyngula period with a critical transition time, tau _c = 50 h post-fertilization) in Zebrafish. The advantage of multicolor lightsheet illumination, fast volume scanning, simultaneous visualization of multiple organs and an order-less photobleaching makes OVSS imaging the system of choice for rapid monitoring and real-time assessment of macroscopic biological organisms with microscopic resolution.

AN EFFICIENT NOVEL APPROACH FOR SOCIETAL COMMUNICATION OF Q&A COMMUNITY SYSTEM USING TOPIC MODELING TECHNIQUES

The emerging trend in technical research is to use customer-generated data collected by community media to probe community opinion and scientific communication on employment and care issues. This review of the collected data, the launch of a question-and-answer social website, is a separate stack for exploring the key factors that influence public preferences for technical knowledge and opinions. by means of a web search engine, topic modeling, and regression data modeling, this study quantified the effect of the response textual and auxiliary functions on the number of votes received with the response. Compared to previous studies based on open estimates, the model results show that Quora users are more likely to only talk about technology. It can fail when the keywords in the query do not match the text content of large documents that contain relevant questions of existing methods, ie. CNNMF and NMF, as well as some restrictions are not enough. Also, users are often not experts and provide ambiguous queries leading to mixed results and encountering problems with existing methods. To address this problem, in this article we propose a Hadoop model, distributed using semantics, non-negative matrix factorization (HDiSANNMF), to find topics for short texts. It effectively incorporates the semantic correlations of the word context into the model, where the semantic connections between words and their context are learned by omitting the grammatical view of the corpus. The researchers are trying to reorganize the main results and present modern techniques for modeling distributed themes to address technologies and platforms with increasing attributes, as well as how much time and space it takes to generate the model. This document briefly describes the structure of public questions and answers around the world and tracks the development of the main topics Housing and employment opportunities for next generation technologies in the world in real time.

Investigation of the crystal structures and magnetic features of two bis(dithiolato)nickelate salts with flexible organic cations

Two bis(dithiolato)nickel salts with different flexible ammonium counterions, [Et3MeN][Ni(dmit)2] (1) and [(i-Bu)Et3N][Ni(dmit)2] (2) (dmit2− = 2-thioxo-1,3-dithiole-4,5-dithiolate, Et3MeN+ = triethylmethylammonium, (i-Bu)Et3N+ = triethylisobutylammonium), are prepared and identified by powder X-ray diffraction (PXRD) patterns and single-crystal X-ray diffraction. Salt 1 crystallizes in the triclinic space group P − 1 at 293 K. The anions and cations in 1 form alternating layered arrangements along the a-axis direction. Salt 2 belongs to the monoclinic crystal system and space group P21/n. The anions and cations in 2 form separate columnar stacks along bc-plane direction. The neighboring anions are stacked as dimers in the anion columnar stacks of 2. The magnetic features of salts 1 and 2 show 1D alternating spin chain magnetic exchange behavior, and the magnetic experimental data are well fitted through a spin chain magnetic model. The difference in the crystal structures and magnetic properties between the two new salts 1 and 2 fully demonstrates that the magnetic properties are dependent on the alignment of the [Ni(dmit)2]− anions, which are related to the flexible organic cations.

A novel use of twisted continuous carbon fibers in additive manufacturing of composites

3D printing of composites is one of the trending topics in the field of additive manufacturing. Several research papers on carbon fiber reinforced composites, used carbon fiber tows directly or sized carbon fibers as reinforcement for the composite, which is time consuming and not reliable as frequent clogging occurs. This paper presents a simple method by introducing the novel use of twisted carbon fiber filament and analyze its effect on the additive manufactured composites. The matrix material used is PLA. The commercially available 3D printers for printing continuous fibers, use double nozzle setups for printing the matrix and reinforcement separately in a layer by layer manner. However, there are no reports on the use of twisted continuous carbon fiber as filament and its effect on the printed parts. This paper uses twisted carbon fibers with single nozzle setup such that the material coming out of the nozzle will be ready to use CCF-PLA composite. Thus, the need of separate stacking of each layer of the matrix and fiber can be avoided. A separate path process planning has been done for printing the twisted carbon fibers in consideration of its continuity and the anisotropy. Standard mechanical tests are conducted, and the morphological study of the composite is done.

Moduli of formal torsors

We construct the moduli stack of torsors over the formal punctured disk in characteristic p > 0 p>0 for a finite group isomorphic to the semidirect product of a p p -group and a tame cyclic group. We prove that the stack is a limit of separated Deligne-Mumford stacks with finite and universally injective transition maps.

The layout strategy of container yard and comparative analysis under double cycling process

In recent years, to adapt to the rapid growth of container throughput, the new loading and unloading technology of double cycling is beginning to be used in ports, because it can improve the loading and unloading efficiency of the terminal. However, the ports are currently lack of a corresponding and suitable container yard storage strategy, and according to the characteristics of the new loading and unloading process of double cycling, this paper proposes two strategies of the separate stacking strategy of import and export containers in the same vessel with different container areas and the mixed stacking strategy in the same vessel with the same container areas. The mixed stacking strategy of the same vessel with the same container area can be divided into two methods: the same vessel with the same ship-bay positions and the same vessel with different ship-bay positions, and the cost of the latter method is higher than the former, because the yard cranes and the trucks need to move back and forth frequently. Therefore, this paper focuses on the comparison and analysis of the two strategies of the same vessel with different container areas and the same vessel with the same ship-bay positions. The FlexTerm simulation modelling test shows that it is not easy to generate traffic congestion and the loading and unloading operation time is shorter if the strategy of the same vessel with different container areas is applied.

The relationship between the pH value of dilute effluent streams and system durability in the separate bed electrodeionization process

The successful application of electrodeionization (EDI) for the preparation of ultra-pure water has been encouraging researchers to explore its feasibility in the treatment of heavy metal electroplating wastewaters. Based on the separate bed membrane stack, the relationship between the pH of dilute effluent streams and its long-term operation stability was investigated. The pH, conductivity, electric current and voltage were recorded on-line with cupric ions contents measured off-line. The microstructure and morphology of ion exchange resins and membranes were studied with infrared spectroscopy and scanning electron microscopy. It is the adsorption saturation in the cation exchange resins-filled compartment (CR-cell) that results in the acidity of dilute effluent waters due to the consumption of hydroxide ions in the anion exchange resins-filled compartment (AR-cell). The periodic change of acidity and alkalinity of dilute effluents was caused by the alternative processes of adsorption saturation and hydrolysis regeneration in CR-cell. The conversion of quaternary amines into tertiary ones of the anion resins and membranes owing to hygro-thermal aging weakened alkaline and shortened the acid-base change period. Increasing stack voltage and intermittent leaching play an important role in prolonging the service life of EDI, and make it feasible to remove heavy metal ions from industrial wastewaters.

Crystal structure of imidazo[1,5-a]pyridinium-based hybrid salt (C13H12N3)2[MnCl4

A new organic-inorganic hybrid salt [L]2[MnCl4] (I) where L + is the 2-methyl-3-(pyridin-2-yl)imidazo[1,5-a]pyridinium cation, is built of discrete organic cations and tetra-chlorido-manganate(II) anions. The L + cation was formed in situ in the oxidative cyclo-condensation of 2-pyridine-carbaldehyde and CH3NH2·HCl in methanol. The structure was refined as a two-component twin using PLATON (Spek, 2020 ▸) to de-twin the data. The twin law (-1 0 0 0-1 0 0.5 0 1) was applied in the refinement where the twin component fraction refined to 0.155 (1). The compound crystallizes in the space group P21/c with two crystallographically non-equivalent cations in the asymmetric unit, which possess similar structural conformations. The fused pyridinium and imidazolium rings of the cations are virtually coplanar [dihedral angles are 0.89 (18) and 0.78 (17)°]; the pendant pyridyl rings are twisted by 36.83 (14) and 36.14 (13)° with respect to the planes of the remaining atoms of the cations. The tetra-hedral MnCl4 2- anion is slightly distorted with the Mn-Cl distances falling in the range 2.3469 (10)-2.3941 (9) Å. The distortion value of 0.044 relative to the ideal tetra-hedron was obtained by continuous shape measurement (CShM) analysis. In the crystal, the cations and anions form separate stacks propagating along the a-axis direction. The organic cations display weak π-π stacking. The anions, which are stacked identically one above the other, demonstrate loose packing; the minimum Mn⋯Mn separation in the cation stack is approximately 7.49 Å. The investigation of the fluorescent properties of a powdered sample of (I) showed no emission. X-band EPR data for (I) at 293 and 77 K revealed broad fine structure signals, indicating moderate zero-field splitting.

IoTEF: A Federated Edge-Cloud Architecture for Fault-Tolerant IoT Applications

The evolution of Internet of Things (IoT) technology has led to an increased emphasis on edge computing for Cyber-Physical Systems (CPS), in which applications rely on processing data closer to the data sources, and sharing the results across heterogeneous clusters. This has simplified the data exchanges between IoT/CPS systems, the cloud, and the edge for managing low latency, minimal bandwidth, and fault-tolerant applications. Nonetheless, many of these applications administer data collection on the edge and offer data analytic and storage capabilities in the cloud. This raises the problem of separate software stacks between the edge and the cloud with no unified fault-tolerant management, hindering dynamic relocation of data processing. In such systems, the data must also be preserved from being corrupted or duplicated in the case of intermittent long-distance network connectivity issues, malicious harming of edge devices, or other hostile environments. Within this context, the contributions of this paper are threefold: (i) to propose a new Internet of Things Edge-Cloud Federation (IoTEF) architecture for multi-cluster IoT applications by adapting our earlier Cloud and Edge Fault-Tolerant IoT (CEFIoT) layered design. We address the fault tolerance issue by employing the Apache Kafka publish/subscribe platform as the unified data replication solution. We also deploy Kubernetes for fault-tolerant management, combined with the federated scheme, offering a single management interface and allowing automatic reconfiguration of the data processing pipeline, (ii) to formulate functional and non-functional requirements of our proposed solution by comparing several IoT architectures, and (iii) to implement a smart buildings use case of the ongoing Otaniemi3D project as proof-of-concept for assessing IoTEF capabilities. The experimental results conclude that the architecture minimizes latency, saves network bandwidth, and handles both hardware and network connectivity based failures.

Chapter 22 Upper and uppermost thrust sheets in the Caledonide orogen, Sweden: outboard oceanic and exotic continental terranes

Abstract Three separate stacks of thrust sheets (Köli Nappe Complex) constitute the Upper Allochthon in the Caledonide orogen, Sweden. This thrust complex is dominated by late Cambrian–Ordovician successions deposited in subduction-related, marginal oceanic basins. Magmatic activity at c. 488 Ma (Lower Köli) and c. 492–476 Ma (Middle Köli) is linked to rifted volcanic arcs and Zn–Cu–Fe–(Pb–Au–Ag) sulphide mineralization; serpentinite bodies with talc deposits are also conspicuous. Renewed magmatic activity, both plutonic (Upper and Middle Köli) and mafic volcanic (Middle and Lower Köli), occurred at c. 440–434 Ma during crustal extension. Late Ordovician shallow-marine sedimentation, deepening upwards into an early Silurian succession also prevailed (Lower Köli). Silurian ( c. 430 Ma and later) folding, eastwards-vergent thrusting and greenschist or lower amphibolite facies metamorphism preceded upright, orogen-parallel and orogen-transverse open folding. Juxtaposition of an arc-related terrane to an ancient continental margin, comprising slices of gneiss and marble, in the Middle Köli occurred prior to c. 437 Ma and the eastwards-vergent thrusting; remnants of an Ordovician amphibolite facies tectonothermal event are also preserved in the Upper Köli. The tectonic roof to the Köli complex contains amphibolite facies mica schist, gneiss and marble, derived from the Laurentian continental margin, and a major gabbroic pluton (Rödingsfjället Nappe Complex, Uppermost Allochthon).

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80-100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.