Thick Sections Research Articles

Size design and nonlinear stress-strain relationship of parallel bamboo strand lumbers in tension and compression

Based on the current national test standard of wood structures (China), tensile specimens with different section thickness and effective length were designed to investigate the size design on the tensile property of the parallel bamboo strand lumber (PBSL). At the same time, compression components with different length were designed to study the effect of slenderness ratio on the compression performance of the PBSL. Four nonlinear constitutive models were applied to model the compressive stress-strain curves. The results show that the tensile property of the PBSL is tightly related to the section thickness. It is recommended that section thickness of 10 mm and effective length of 60 mm might be the most appropriate specimen size for the tensile experiment. The compressive strength increases with reducing the slenderness ratio. Among the four nonlinear constitutive models, the Ramberg-Osgood relationship could most consistently fit the compression stress-strain relationship of the PBSL.

Cross-Sectional Preparation of Challenging Devices and Their Scale-Bridging Micro- and Nanoanalytical Characterization

Complex devices like batteries, fuel cells or organic sensors and solar cells are commonly assembled from multiple functional components with different materials properties. To gain a fundamental understanding of the microstructure-property relations of such complex macroscopic structures, their degradation and failure mechanisms by complementary scale-bridging microscopic and spectroscopic techniques, it is desirable to prepare cross sections of entire devices or parts. Most challenging is the generation of electron transparent cross-sectional samples for investigation by (cryo) transmission electron microscopy (TEM); preparing TEM samples of devices consisting of different material classes may be utmost difficult. Therefore, TEM sample preparation is often preceded by the disassembly of a device down to individual components rendering an investigation of relations between the individual components impossible.(Cryo-)ultramicrotomy as well as plasma-FIB as advanced cross-sectioning techniques for challenging samples may solve those issues. Both are capable to generate ultra-thin, electron transparent cross sections of hundreds of micrometers in size being larger than the typical thickness of modern devices.In this contribution, we demonstrate the capabilities of modern (cryo-)ultramicrotomy in conjunction with advanced TEM to characterize such sensitive or reactive and thus challenging devices down to the atomic scale. Examples include battery parts, PEM fuel cells and novel flexible all-organic electronics. The achieved thickness of those cross sections is commonly of the order of a few 10 nm, which even allows for atomic-resolution imaging and advanced spectroscopy for microstructure and defect analyses as well as compositional and chemical-bond investigation, respectively. Hepp et al., Trained Laser-Patterned Carbon as High-Performance Mechanical Sensors, NPJ Flex. Electron. 6 (2022), 3, DOI: 10.1038/s41528-022-00136-0 Beaupain et al., Reaction of Li1.3Al0.3Ti1.7(PO4)3 and LiNi0.6Co0.2Mn0.2O2 in Co-sintered Composite Cathodes for Solid-State Batteries, ACS Appl. Mater. Inter. 13 (2021) 47488-47498, DOI: 10.1021/acsami.1c11750 Li et al., Atomic structure of sensitive battery materials and interfaces revealed by cryo-electron microscopy, Science 358 (2017), 506–510, DOI: 10.1126/science.aam6014 Figure 1

Effects of arc interaction on lack-of-fusion and porosity in tandem NG-GMAW of 5083 Al-Mg alloy

The tandem narrow-gap gas metal arc welding (NG-GMAW) is an advanced high efficient technology for joining thick section metals. However, the common defects such as lack-of-fusion (LOF) at groove sidewalls, porosity in weld bead are still hard to be avoided, especially for joining thick section of aluminum alloy. In this study, a pulsed tandem NG-GMAW process was applied to join thick 5083 Al-Mg alloy plates. The LOF and its formation mechanism at varied arc length were investigated by observing the tandem arc interaction behaviors with high-speed photography and by synchronized recording the corresponding welding signals. The results indicate, the arc heat distribution, which is likely influenced by the tandem arc arrangement, is a critical factor to the formation of LOF. However, a properly adjusted arc length can improve the arc interaction and the weld formation. Moreover, a proper arc interaction behavior can change the direction of arc force, which makes contribution to the stirring effect on molten pool. It is beneficial to reduce porosity. The oxygen enriched pores (pore-II) caused by short-circuiting transfer were found when arc length correction coefficient (ALCC) is −15, which needs to be avoided during the welding process.

Sealing Ability and Adaptability of Nano Mineral Trioxide Aggregate as a Root-End Filling Material

Aim: Comparison between Nano MTA & MTA as a root-end filling materials regarding adaptability and sealing ability.
 Materials and Methods: Forty extracted human maxillary incisors with straight roots were used. After root canals preparation and obturation, the apical 3 mm of each root was resected perpendicular to the long axis of the tooth. Root end cavities were prepared to a depth of 3mm parallel to the long axis of the tooth. The teeth were randomly divided into two main equal groups of 20 samples each according to the root-end filling material used either MTA or Nano MTA. Ten samples from each group were sectioned longitudinally into two equal halves to measure the sealing ability and another ten samples from each group were sectioned transversally to obtain 1 mm thick section to measure the adaptability of both materials. All samples were photographed under the SEM at three different magnifications (×1000). The gap thickness between the root end filling material and the retro cavity dentine walls were measured at seven selected points at the material-dentine interface in micrometers (µm).
 Results: Nano MTA and MTA showed no statistically significant difference in the gap thickness between dentin-material interface in both longitudinal and transverse sections. Regarding the sealing ability, the mean value in MTA was (3.27±0.77), while the mean in Nano-MTA was (3.15±0.71). Regarding the adaptability, the mean value in MTA was (2.46±0.60), while the mean in Nano-MTA was (2.05±0.712). Both materials showed good sealing ability and good adaptation to the dentinal wall.
 Conclusion: Nano MTA revealed good sealing ability and adaptability comparable to MTA when used as a retrograde filling material.

Generating Optimized Trajectories for Robotic Spray Painting

In the manufacturing industry, spray painting is often an important part of the manufacturing process. Especially in the automotive industry, the perceived quality of the final product is closely linked to the exactness and smoothness of the painting process. For complex products or low batch size production, manual spray painting is often used. But in large scale production with a high degree of automation, the painting is usually performed by industrial robots. There is a need to improve and simplify the generation of robot trajectories used in industrial paint booths. A novel method for spray paint optimization is presented, which can be used to smooth out a generated initial trajectory and minimize paint thickness deviations from a target thickness. The smoothed out trajectory is found by solving, using an interior point solver, a continuous non-linear optimization problem. A two-dimensional reference function of the applied paint thickness is selected by fitting a spline function to experimental data. This applicator footprint profile is then projected to the geometry and used as a paint deposition model. After generating an initial trajectory, the position and duration of each trajectory segment are used as optimization variables. The primary goal of the optimization is to obtain a paint applicator trajectory, which would closely match a target paint thickness when executed. The algorithm has been shown to produce satisfactory results on both a simple 2-dimensional test example, and a non-trivial industrial case of painting a tractor fender. The resulting trajectory is also proven feasible to be executed by an industrial robot. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Note to Practitioners</i> —The work is motivated by the need to generate well performing robot trajectories in robotized spray-painting booths. The described method applies to cases where robotic spray painting is to be used for painting a surface with an even layer of paint at a specified thickness. The method generates and optimizes robot trajectories and is shown to be able to generate a satisfactory paint cover for simple test cases as well as more realistic industrial cases. For a user it could be implemented as is, or be obtained as a standalone service, but there are some prerequisites that need to be fulfilled to make use of the optimization method. It is assumed that the surface to be painted is available as a CAD-model and that physical testing has been performed to determine the characteristics of the paint and nozzle. These physical tests amount to spraying paint on a flat piece of material at a few different distances from the surface and measuring the cross section of the paint thickness. The resulting trajectory can be executed on any industrial painting robot that can handle linear motion commands.

Hydatidiform Mole; Histological Types and Age Distribution in Basrah

Background Hydatidiform mole (HM) is a placental pathology, which is a form of gestational trophoblastic disease (GTD). HM is classified into complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM). The incidence of molar pregnancy varies according to the geographical area. It is found to be higher in developing countries. Age is a risk factor for developing HM. In this study, we aim to determine the frequency of hydatidiform mole among cases of early trimester evacuation specimens and its relation to patient's age at Basrah maternity and paediatric hospital and Al- Mosawi private hospital. Patients and Methods This was a descriptive retrospective study for a four-year period; all cases of early trimester evacuation specimens were from January 1, 2017, to December 31, 2020. All specimens were fixed in 10% formalin and dehydrated in graduated alcohol, (5) micron thickness sections were obtained, stained with hematoxylin and eosin and examined, and cases of CHM, PHM and RPOG were analyzed. Results A total of 216 evacuation specimens were examined, and the patients age ranged from 14–50 years. Among these, 78.2% of patients were between 20 and 30 years. The percentage of RPOC was 54.2%, while that of CHM was 19.4% and that of PHM was 26.4%. The maximum cases of complete and partial mole were in the 20–30 years age group. Conclusions The frequency of HM was high compared to many other studies.

To analyse the mitotic and keratinisation correlation with bcl-2 expression in varying grades of oral epithelial dysplasia and squamous cell carcinoma.

The bcl-2 proto-oncogene was discovered at the chromosomal breakpoint of t (14;18) found in follicular lymphoma. Histological changes in dysplasia are considered the earliest signs preceding the progression into squamous cell carcinoma. Serving as critical regulators of apoptotic pathways, bcl-2 prohibits programmed cell death and subsequently assists in uncontrolled neoplastic growth. This study included 48 cases, eight each of epithelial dysplasia and squamous cell carcinoma. Immunohistochemical staining using bcl-2 antibody was performed and different histological parameters were correlated with bcl-2 positive cells in all the cases. All 3 μm thick sections were stained with bcl-2 antibody. After identifying four representative fields at 40x, their images were obtained for assessment of bcl-2 labelled cells and their intensity along with different histological parameters in all the cases. The differences between different histological parameters were analysed using the Anova test, post hoc test and Bonferroni test. Pearson's Chi-square test was carried out to determine the level of correlation between the bcl-2 positive cells in both epithelial dysplasia and oral squamous cell carcinoma cases. Sequential increase in the bcl-2 expression was observed in increasing grades of epithelial dysplasia, whereas bcl-2 expression was significantly decreased in ascending stages of squamous cell carcinoma thus, suggesting a possible role of bcl-2 in disease progression from premalignancy to malignancy.

Solidification cracking of a nickel alloy during high-power keyhole mode laser welding

Nickel alloy Inconel 740H, a candidate material for use in ultra-supercritical power plants, is susceptible to solidification cracking during high power deep penetration laser welding. Here we examine how cracking is affected by welding variables and determine the locations where the cracks occur experimentally and theoretically. We use a solidification cracking model to calculate the effects of welding variables on cracking and the locations where the cracks form during high power laser keyhole mode welding of IN 740H. The parameters needed for the cracking model are obtained from a well-tested numerical heat transfer and fluid flow model for keyhole-mode welding. Model predictions of cracking and their locations for different welding conditions are verified by experiments. • Solidification cracking of nickel alloys during laser welding is poorly understood. • We propose a novel methodology to understand why, when, and where cracking occurs. • A heat and fluid flow and solidification model, and experimental data are used. • Model predictions of cracking locations and conditions are verified by experiments. • The work can be used to prevent weld failures in thick section laser keyhole welds.

Aerodynamic and Aeroacoustic Performance of a Wing with Structured Surface Inspired by Owl’s Wings

With the advent of various advanced materials, the idea of flying like birds has attracted considerable attention in recent years. In addition, aeroacoustics has become an important issue and is being widely studied. In this work, based on the shape of Owls’ wings, an attempt was made to improve the aeroacoustic and aerodynamic performances of conventional aircraft wings. For this purpose, wings with different elements, namely, square, triangular, and semicircular, on their top surface were examined. In addition, three different spatial distributions of the elements according to the Owl’s wings shape were considered. For incompressible airflow, aerodynamic and aeroacoustic parameters of wing with structured surfaces were investigated. Also, a wing with serrations was examined. The results indicate that wings with elements distributed starting from maximum section thickness and continuing up to the trailing edge are the most suitable case for both aerodynamic and aeroacoustic improvements. On the other hand, a two-sided serrated wing and a serrated wing in the trailing edge reduce the sound level significantly. In addition, the use of both elements and serrations delays wing stall and thus markedly increases the maximum lift coefficient.

Evaluation of Palatal Bone Thickness and Its Relationship with Palatal Vault Depth for Mini-Implant Insertion Using Cone Beam Computed Tomography Images.

The purpose of this study was to measure the thickness of the palatal bone using cone beam computed tomography images for placement of mini-screws and their relationship with palatal vault depth. This study was performed on 150 maxillary cone beam computed tomography images, 50% (n = 75) had deep palate and 50% (n = 75) had normal palate and 27.3% (n = 41) were male and 72.7% (n =109) were female. Coronal sections with a thickness of 1 mm were prepared at distances of 4 mm, 8 mm, 12 mm, 16 mm, 20 mm, and 24 mm from the posterior wall of the incisive foramen. Then, in each section, in the midsagittal line and at distances of 2 mm, 4 mm, 6 mm, 8 mm, and 10 mm from that to the lateral sides, the bone thickness was measured. The Korkhaus index was used to identify the patients with a high palatal vault. The results showed that at the posterior sections in the midsagittal and parasagittal area, a significant difference (P < .05) was observed between deep and normal palate, and in these points, the bone thickness in the normal palate was greater. Also, in the section of 4 mm and 8 mm, a significant difference was observed between males and females in most of these points, and those were greater in males than females. The maximum thickness of the palatal bone was observed first along the midsagittal line and then the paramedian and in the anterior section. Patients with deep palate had less palatal bone thickness in the posterior sections.

A morphological study of human pineal gland in post-mortem cases

The current study was conducted in the Department of Anatomy, Osmania Medical College, Koti, Hyderabad over a period of 1year. A total of 30 human pineal glands (16-males,11-females,3 excluded for post-mortem autolysis) from those bodies between the age groups of 1-80years were collected during the post-mortem study at the Mortuary of Osmania General Hospital, Koti, Hyderabad and were included in the study. Morphological parameters like weight and transverse diameter at the maximum width of the pineal glands were measured and then the pineal glands were processed for light microscopy. 4-5micron thick sections were cut and stained with H&E and Masson Trichrome for highlighting the connective tissue pattern of the pineal gland. Under light microscope, the capsular delineation, pattern of septation and lobulation, calcification, intracellular brown pigment, intraparenchymal inflammatory cells, fibrosis, gliosis and cystic changes in the pineal glands were observed in relation to age and gender.

Study on Different Shear Performance of Moso Bamboo in Four Test Methods.

Bamboo is recognized as a potential and sustainable green material. The longitudinal-splitting and shear strengths of bamboo are weak but critical to its utilizations. To discuss the different shear performances of bamboo, the shear strength and behaviors of bamboo culm were investigated by four test methods: the tensile-shear, step-shear, cross-shear, and short-beam-shear methods. Then, the different shear performance and mechanisms were discussed. Results indicated that the shear strength was significantly different in the four test methods and was highest in the step-shear-test method but lowest in the tensile-shear-test method. Moreover, the typical load-displacement curves were different across the shear methods but were similar to the curves of the respective loading modes. The axially aligned fiber bundles played an important role in all the shear performances. In the tensile-shear method, specimens fractured at the interface of the bamboo-fiber bundles. However, compress-shear behaviors were a combination of compression and shear. Then, the cross-shear method, in compress-shear, was lower than that of the step-shear method because of oval-shaped bamboo culm sections of different thickness. In the short-beam shear method, the behaviors and shearing characteristics were like bending with the fiber bundle pulled out.

Adaptive hybrid optimization method for valley functions in weight minimization problems for wind turbine blades

The article proposes an adaptive method for finding the minimum of an arbitrary smooth multivariable function. The method has been used to solve the benchmark optimization problem of a valley function. The essence of the proposed algorithm lies in the sequential approach to the bottom of the valley and the subsequent movement in the direction of decreasing the objective function. The comparison of the results of calculating the minimum point of the function is performed by using both non-gradient and gradient methods, namely: Powell, Hook-Jeeves, the steepest descent method and the method developed. It has been found that the effectiveness of the proposed method is greater than the usual search algorithms, but it is not without its drawbacks. The method that represents a number of hybrid methods, which form a hybrid coalition is proposed. The proposed hybrid algorithm does not provide a satisfactory result in the "single" search. The search algorithm reaches a point where all the values of the function at the surrounding points are greater than the values at the obtained point, and the algorithm cannot overcome the barrier. To solve the problem, it is necessary to take the obtained point as a new starting point and repeat the algorithm for finding the minimum of the function, that is, use the multistart method. The proposed method has been used to solve the problem of optimizing the blade of a wind turbine, which was reduced to the problem of unconditional optimization by using the method of penalty functions, but the goal function had a significantly valley structure. The optimal values of section thicknesses have been obtained, which makes it possible to build a blade with improved characteristics.

Crashworthiness analysis of bioinspired hierarchical gradient multicell tubes under axial impact

Hierarchical structures and gradient structures have become research hotspots due to their excellent crashworthiness. However, hierarchical gradient structures that combine hierarchical structures and gradient structures are rarely reported. Inspired by the gradient distribution of biological structure vascular bundles and the excellent energy absorption capacity of hierarchical structures, this paper innovatively combines hierarchical structures and gradient structures to propose a new type of bioinspired hierarchical gradient multicell tube (BHGMT). Crashworthiness studies have shown that the high-order BHGMT has a much better crashworthiness than the low-order BHGMT. The specific energy absorption of the 4th order BHGMT under the same mass is 216.26% that of the 0th order BHGMT. A comparison with other existing hierarchical structures under the same mass conditions shows that the proposed hierarchical gradient structure also has better crashworthiness. Subsequently, the systematic development of the influence of structural parameters such as hierarchy, section thickness, and crisscross rib thickness on the parameterization of BHGMT was carried out. Finally, theoretical research on the mean crushing force of the 3rd order BHGMT was carried out, and the theoretical solution was shown to be in agreement with the numerical simulation results.

Experimental Analysis of Steel Circular Hollow Section under Bending Loads: Comprehensive Study of Mechanical Performance

The present study aimed at evaluating the mechanical performance under bending loads of circular hollow sections of steel. Different bending tests have been carried out by applying two-point loads, to determine and examine the effects of the diameter, the thickness of the section, and the span of the beam on the performance of the steel tube. The effects of square opening and variation in the number of openings on the performance of these sections have also been examined. Ten samples of hollow circular beams of varying thickness (2 mm, 3 mm, and 6 mm), varying diameter (76.2 mm, 101.6 mm, and 219 mm), and varying span (1000 mm, 1500 mm, and 2000 mm) were fabricated and tested for pre-failure and post-failure stages. The dimensions of the reference specimen considered were 3 mm in thickness, 101.6 mm in diameter, and 1500 mm in span. The results have shown that on increasing the section thickness by 200%, ductility and bearing strength were enhanced by 58.04% and 81.75%, respectively. Meanwhile, decreasing the section thickness by 67%, ductility and bearing strength were reduced by 64.86% and 38.87%, respectively. Moreover, on increasing the specimen diameter and on decreasing span, a significant increase in bearing strength and stiffness was observed; however, ductility was reduced. Meanwhile, on increasing the span of the specimen, all the parameters observed, i.e., bearing strength, stiffness, and ductility, decreased. On observing the ultimate strength of each specimen with square opening, the ultimate strength was reduced by 17.88%, 19.71%, and 14.23% for one, two-, and three-square openings, respectively. Moreover, the ductility was significantly reduced by 72.40%, 67.71%, and 60.88% for one, two-, and three-square openings/apertures, respectively, and led to the sudden failure of these specimens. The local buckling failure dominated for specimens having a D/t ratio more than 50 and showed very negligible levels of ovalization of the cross-section. Local buckling failure was observed to be prevented after providing the circular rings in the specimen, since bearing strength increased compared with the specimen without rings.

The Conjugate Beam Effective Thickness Method

The structural performance of laminated glass is strongly dependent on the shear coupling offered by the interlayer between the bounding layered and monolithic limits of the glass plies. The most common simplified design approach consists of defining the effective thickness, i.e., the thickness of a monolithic section with equivalent flexural section properties. The Enhanced Effective Thickness (EET) method has been verified to estimate deflection in laminated glass for a range of load and boundary conditions for two-, three-, and multi-ply beams; however, for some static schemes, the EET method is less accurate for predicting stress. The recently proposed Conjugate Beam Effective Thickness (CBET) method, initially developed for cantilevered laminated glass balustrade applications, accounts for the relative displacement of glass plies across the interlayer for a range of loads and statically determinate boundary conditions. In this paper, the CBET method is extended to the evaluation of two-ply simply supported beams under concentrated, uniformly, and tapered distributed out-of-plane loads. Predicted deflection- and stress-effective thickness obtained from effective thickness methods is compared with finite element model results in illustrative examples, demonstrating improved accuracy. Closed-form formulas are summarized in tables to facilitate the practical application of the CBET method in the design practice.

Fracture mechanics solutions and operative formulae for isotropic bi-material layers with large elastic mismatch

Following previous works in the literature on the fracture of layered composites, semi-analytical expressions for energy release rate and mode-mixity phase angle in isotropic bi-material layers subjected to arbitrary end loadings are extended to materials characterized by a large elastic mismatch described by Dundurs’ parameters |α| ≤ 0.99 and |β| ≤ 0.4. The solutions depend on elementary bending, axial and shear loads acting at the crack tip cross section, elastic constants and relative thickness of the layers, and dimensionless coefficients describing the fracture parameters of single elementary loads. The coefficients associated to the shear forces are presented in the literature only for combinations with β = 0; here they are determined numerically for different values β ≠ 0 to describe materials used in current applications. Simplified fitting formulae having general validity, as polynomials, which highlight the dependence on Dundurs’ parameters and are practical to support laboratory tests, are derived for layers with mid-thickness delaminations and thin layers on semi-infinite substrates. Applications to some mixed mode fracture specimens highlight the effects of β ≠ 0 on the fracture parameters and the limitations of estimates based on the commonly adopted assumption β = 0. As expected, the results for mid-thickness delaminations show that such effects are stronger on mode-mixity phase angle than on energy release rate.

Effect of web perforations on end-two-flange web crippling behaviour of roll-formed aluminium alloy unlipped channels through experimental test, numerical simulation and deep learning

Aluminium alloy has recently become popular in New Zealand’s construction sector as a sustainable building material. However, in roll-formed aluminium alloy (RFA) channel beams, particularly those having web holes, cripples at points of concentrated or localised loading. In this paper, end-two-flange (ETF) web crippling behaviour of RFA unlipped channels having web holes was investigated using experimental tests, numerical simulations and a deep learning model referred as deep belief network (DBN). A total of 1080 data points was generated to train the DBN, using a finite element (FE) model that was validated using 30 new experimental results reported in this paper. Compared to the test data, the DBN predictions were conservative by around 6%. Using the DBN predictions, a comprehensive parametric study was undertaken which used the validated FE model in order to explore the effects of hole size, hole location, section thickness, and bearing plate on the web crippling strength of RFA channels having web holes. Web crippling strengths obtained from the DBN, tests and finite element analysis (FEA) were utilised to evaluate the performance of current design specifications from the American Iron and Steel Institute (AISI), Australian/New Zealand Standards (AS/NZS 1664; AS/NZS 4600:2018) and Eurocode (CEN 2006, 2007). It is shown that the current design rules are not reliable to predict the web crippling strength of RFA unlipped channels with web holes. As a consequence of the parametric analysis, new web crippling strength and web crippling strength reduction factor formulae for perforated RFA unlipped channels were proposed. A reliability analysis was then conducted, which confirmed that the proposed equations are capable of predicting the ETF web crippling strength of perforated RFA unlipped channels.

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (≤200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.