Entire Plate Research Articles

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

Performance Analysis of Double Pass Solar Air Heater with Bottom Extended Surface

Abstract A model of solar air heater with selective absorber coating with longitudinal fins at the bottom of absorber has been prepared to produce hot gases by consuming solar energy in day time. It is kept in position in such a way that air will flow due to forced convection and the entire setup placed at an angle of 15° to 17° inclination as per the considered global position (20.2961° N, 85.8245° E). Air flow rate is increased by applying a DC suction fan at outlet which sucks the air to flow over extended surfaces which are present at bottom inlet channel. Special selective coating applied over the entire Aluminum absorber plate to increase the heat absorbing capacity. Average ambient, inlet and plate temperatures are recorded daily along with solar flux. It is found that the instantaneous efficiency and air outlet temperature increased up to 69% and 94 °C respectively. Theoretical analysis has been made as well for the present set up. The difference between the theoretical and experimental values lies within 1.23-9.75%.

A Dual Image Sensor Approach for Automated, High Resolution, Region-of-Interest Imaging in a 96-Well Plate

Phenotypic screens are performed on model organisms to understand how chemical compounds affect biological systems. High-throughput devices are needed to efficiently gather large volumes of data for these screens. Recently we developed a low cost high-throughput imaging solution to capture the effects of chemicals on the egg-laying behaviour of C. elegans. We present a second-generation prototype capable of high-resolution (0.2 µm/px) imaging of individual worms and their internal structures. It features an automatic real time segmentation of C. elegans worms and is compatible with 96-well plates. In order to maximize efficiency in data collection, we developed an approach for simultaneous dual imaging through a single objective. A low-resolution (15 µm/px) arm continuously collects images of an entire well using a CCD camera (1280 px by 1024 px). The images are continuously fed into a Python image algorithm that tracks the movement of the worms in regions of interest (ROIs). Using this information, high resolution images of the worms are captured using a linescan camera (8000 px). This solution provides automated high-resolution imaging minimizing acquisition time by eliminating the need for multiple objectives, allowing image collection with a continuously moving system (high speed linescan camera) and minimizing the amount of stitching (large linescan sensor). The scan time of a 96-well plate is proportional to the number of ROIs, which is significantly less than imaging an entire plate. The high-resolution images can be fed into an image processing pipeline that will be able to classify the effects of chemicals on the internal structure of the worms. Simultaneous acquisition of high and low resolution data can be used to correlate deformations of internal structure with changes in mobility. This low cost solution provides users with an automated approach to obtain high-resolution images and mobility statistics of C. elegans.

Magnetic Resonance Imaging Evaluation of Physeal Violation in Adolescents After Transphyseal Anterior Cruciate Ligament Reconstruction

To quantify and compare the amount and location of physis violation of the distal femur and proximal tibia after transphyseal anterior cruciate ligament (ACL) reconstruction in skeletally immature patients. This study included 19 patients with open physes of the distal femur and proximal tibia who underwent ACL reconstruction with tibialis anterior allografts. Physeal tunnel volume and location on the growth plate, as well as obliquity to the growth plate, were measured by 3-dimensional postoperative magnetic resonance imaging of the distal femur and proximal tibia. The percentage of physeal violation (ratio of the tunnel to the entire growth plate area) was similar for the distal femur and proximal tibia (3.95% vs 3.65%, P= .582). There were no differences in tunnel obliquity to the growth plate in the coronal (56.1° vs 71.6°, P= .061) and sagittal (85.9° vs 74.9°, P= .092) planes. The distal femoral tunnel was located 6.2% (17.2% vs 23.4%, P= .001) more peripherally in the anteroposterior direction and 9.7% (27.1% vs 36.8%, P < .001) more peripherally in the mediolateral direction than was the tibial tunnel. The mean percentages of physeal violation of tunnel creation during ACL reconstruction in adolescent patients were 3.95% for the distal femur and 3.65% for the proximal tibia. Moreover, femoral tunnels were located more peripherally on the growth plate than were tibial tunnels, in both the anteroposterior and mediolateral directions. Level IV, case series.

Bootstrapped total least squares orocline test: A robust method to quantify vertical-axis rotation patterns in orogens, with examples from the Cantabrian and Aegean oroclines

Most mountain belts on Earth show some degree of curvature in plan view, from a slight bend to horseshoe shapes. Such curvatures may occur on different scales, from individual thrust sheets to entire plate boundaries. Curvature may be acquired by vertical-axis rotation during or after orogenesis, or reflect primary lateral variations in shortening directions or physiographical features. Quantifying the amount of vertical-axis rotations of plan-view curvature is therefore helpful to our understanding of orogenesis, geodynamics, and paleogeography. The orocline test assesses to what extent vertical-axis rotations have played a role in the acquisition of an orogen’s curvature. The test quantifies through linear regression the relationships between changes in structural trends and the orientations of a geologic fabric. However, the current mathematical approaches to the orocline test show potential biases. In this paper we aim to overcome such biases by developing a novel orocline test that applies total least squares (TLS) regression combined with a novel approach to bootstrapping. This bootstrap TLS orocline test can be used with all types of directional data acquired from structural geology, paleomagnetism, or sedimentology. It quantifies, for the first time, secondary curvature with confidence bands. We also provide several graphical and analytical tests to evaluate the statistical significance of the result. An open source online application implementing this method is available for use on www.paleomagnetism.org. We illustrate the use of the methodology by reanalyzing published data sets from two well-known oroclines in the Cantrabrian (northwest Iberia) and Aegean (Greece) regions.

Gaskinetic Modeling on Dilute Gaseous Plume Impingement Flows

This paper briefly reviews recent work on gaseous plume impingement flows. As the major part of this paper, also included are new comprehensive studies on high-speed, collisionless, gaseous, circular jet impinging on a three-dimensional, inclined, diffuse or specular flat plate. Gaskinetic theories are adopted to study the problems, and several crucial geometry-location and velocity-direction relations are used. The final complete results include impingement surface properties such as pressure, shear stress, and heat flux. From these surface properties, averaged coefficients of pressure, friction, heat flux, moment over the entire flat plate, and the distance from the moment center to the flat plate center are obtained. The final results include accurate integrations involving the geometry and specific speed ratios, inclination angle, and the temperature ratio. Several numerical simulations with the direct simulation Monte Carlo method validate these analytical results, and the results are essentially identical. The gaskinetic method and processes are heuristic and can be used to investigate other external high Knudsen (Kn) number impingement flow problems, including the flow field and surface properties for a high Knudsen number jet from an exit and flat plate of arbitrary shapes. The results are expected to find many engineering applications, especially in aerospace and space engineering.

On thermal stability of plates with functionally graded coefficient of thermal expansion

In this article, a four-variable refined plate theory is presented for buckling analysis of functionally graded plates subjected to uniform, linear and non-linear temperature rises across the thickness direction. The theory accounts for parabolic distribution of the transverse shear strains, and satisfies the zero traction boundary conditions on the surfaces of the plate without using shear correction factor. Young\'s modulus and Poisson ratio of the FGM plates are assumed to remain constant throughout the entire plate. However, the coefficient of thermal expansion of the FGM plate varies according to a power law form through the thickness coordinate. Equilibrium and stability equations are derived based on the present theory. The influences of many plate parameters on buckling temperature difference such ratio of thermal expansion, aspect ratio, side-to-thickness ratio and gradient index will be investigated.

An analytic model employing an elliptical surface area to determine the gaseous thermal conductance of uncooled VOx microbolometers

This work presents a detailed overview of the analytic methods for calculating the beam and gaseous thermal conductance components associated with uncooled VOx microbolometers. The conventional method to calculate the gaseous component relies on the assumption that the entire plate is maintained at a uniform temperature, thus the surface area of the plate is used for the calculation. We have observed using an industry leading multiphysics simulator that this assumption is not strictly true for VOx microbolometers as the conduction pattern exhibits an elliptical shape. Based on this, we have developed and propose an analytic method that employs an elliptical surface area scaled appropriately with the device dimensions to obtain an estimate of the average temperature conduction pattern. Prototype devices were manufactured and experimentally characterised. The devices exhibit thermal conduction characteristics comparable to those in literature and industry, and we could achieve 0.5μW/K under vacuum conditions and 15μW/K at atmospheric pressure with a TCR of −1%/K. However, both simulated and experimental result sets of the gaseous thermal conductance exhibit large deviations from the conventional analytic method, on average approximately 40%. The proposed method reduces this average error significantly to less than 10% when compared to the simulated results.

Self-propulsion of a flapping flexible plate near the ground.

The self-propulsion of a three-dimensional flapping flexible plate near the ground is studied using an immersed boundary-lattice Boltzmann method for fluid flow and a finite-element method for plate motion. When the leading edge of the flexible plate is forced into a vertical oscillation near the ground, the entire plate moves freely due to the fluid-structure interaction. The mechanisms underlying the dynamics of the plate near the ground are elucidated. Based on the propulsive behaviors of the flapping plate, three distinct regimes due to the ground effect can be qualitatively identified. These regimes can be described briefly as the expensive, benefited, and uninfluenced propulsion regimes. The analysis of unsteady dynamics and plate deformation indicates that the ground effect becomes weaker for a more flexible plate. We have found that a suitable degree of flexibility can improve propulsion near the ground. The vortical structure around the plate and the pressure distribution on the plate are analyzed to understand propulsive behaviors. The results obtained in this study can provide some physical insights into the propulsive mechanisms of a flapping flexible plate near the ground.

Deformation and faulting of subduction overriding plate caused by a subducted seamount

AbstractWe conducted numerical experiments to simulate elastoplastic deformation of the overriding plate caused by a subducted seamount. Calculations revealed development of a distinct pair of fault‐like shear zones, including a landward dipping forethrust fault initiated from the seamount top and a seaward dipping backthrust fault from the landward base of the seamount. Significant dome‐shaped surface uplift was predicted above the thrust faults. Lesser‐developed seaward dipping backthrust faults were calculated to develop under certain conditions. The overriding plate was calculated to deform in two stages: In Stage I, elastic deformation leads to the formation of fault‐like shear zones. After major faults have cut through the entire plate, plastic deformation on faults dominates Stage II. On the subduction interface, compressional normal stress was calculated to increase on the landward leading flank of the seamount and decrease on the seaward trailing flank. These changes, together with associated stress singularities at seamount edges, could affect earthquake processes.

High resolution local heat transfer and pressure drop infrared measurements of two-phase flow of R245fa within a compact plate heat exchanger

An experimental study is presented to characterize two-phase thermal and hydraulic performance of a prototype corrugated plate heat exchanger with small pressing depth. In particular, upward flow boiling of low pressure refrigerant R245fa was investigated for a prototype fabricated with two plates of only 1mm pressing depth with a chevron angle of 65°. High spatial and temporal resolution infrared measurements were obtained by self-calibration of the IR camera to measure local (pixel-by-pixel) heat transfer coefficients during diabatic tests and local frictional pressure drops over the entire plate during adiabatic tests. Two-phase experiments were carried out for mass fluxes from 10 to 85kgm−2s−1, heat fluxes from 225 to 4100Wm−2, saturation temperatures from 19 to 35°C and vapor qualities from 0.05 to 0.90. The two-phase frictional pressure drops increased with mass flux and vapor quality while they decreased with increasing saturation temperature. A new correlation for predicting the local frictional pressure gradient through the test section was proposed. The present model captured the entire frictional pressure drop database within a bandwidth of ±30%, providing a mean absolute error of 10% and mean error of 0.2%. The local flow boiling heat transfer coefficients were found to increase with mass flux, heat flux and saturation temperature while rising, leveling off and then decreasing with increasing vapor quality.

Rapid guided wave delamination detection and quantification in composites using global-local sensing

This paper presents a rapid guided ultrasonic wave inspection approach through global inspection by phased array beamforming and local damage evaluation via wavenumber analysis. The global-local approach uses a hybrid system consisting of a PZT wafer and a non-contact laser vibrometer. The overall inspection is performed in two steps. First, a phased array configured by a small number of measurements performs beamforming and beamsteering over the entire plate in order to detect and locate the presence of the damage. A local area is identified as target damage area for the second step. Then a high density wavefield measurement is taken over the target damage area and a spatial wavenumber imaging is performed to quantitatively evaluate the damage. The two-step inspection has been applied to locate and quantify impact-induced delamination damage in a carbon fiber reinforced polymer composite plate. The detected delamination location, size and shape agree well with those of an ultrasonic C-scan. For the test case studied in this work the global-local approach reduced the total composite inspection (damage detection and characterization) time by ∼97% compared to using a full scan approach.

Source Separations and Identification by Structural Holography

The source field reconstruction aims at identifying the excitation field measuring the response of the system. In Near-field Acoustic Holography, the response of the system (the radiated acoustic pressure) is measured on a hologram using a microphones array and the source field (the acoustic velocity field) is reconstructed with a back-propagation technique performed in the wave number domain. The objective of the present works is to use such a technique to reconstruct displacement field on the whole surface of a plate by measuring vibrations on a one-dimensional holograms. This task is much more difficult in the vibratory domain because of the complexity of the equation of motion of the structure. The method presented here and called is particularly interesting when a direct measurement of the velocity field is not possible. Moreover, Structural Holography decreases the number of measurements required to reconstruct the displacement field of the entire plate. This method permits to separate the sources in the case of multi-sources excitations by considering them as direct or back waves. It's possible to compute the structural intensity of one particular source without the contributions of others sources. The purpose of this paper is to introduce the Structural Holography method. The first part presents the theoretical background of the method. A numerical simulation of displacement fields generate by few sources for an infinite plate is presented in a second part. The structural intensity for each source is computed by removing the contribution of others source. Finally, some results are presented for a simply supported plate.

Analytical solution of forced convective heat transfer in parallel-plate channel partially filled with metallic foams

The forced convection flow and heat transfer characteristics in parallel-plate channel partially filled with metallic foams have been studied analytically. To predict fluid and thermal transport in the bottom heated partially filled channel, the Brinkman-extended Darcy momentum model and Non-equilibrium heat transfer model for porous media were employed for the foam filled region coupling with the momentum and energy conservation equations for foam free region. The interface between foam filled region and foam free region are subject to continuous temperature and energy balance to derive the analytical solutions for fully developed flow and convection heat transfer in entire plate channel including foam filled region and non-foam region. Then, the velocity and temperature distributions were predicted for the whole region of the parallel-plate channel based on the analytical solutions. The effects of key parameters on flow resistance and heat transfer performance has been investigated. Unlike the completely filled channel, the influences of pore density and porosity on flow and heat transport are dependent on the height of the foam.

Proteobacteria, extremophiles and unassigned species dominate in a tape-like showerhead biofilm.

The development of showerhead biofilms exposes the user to repeated contact with potentially pathogenic microbes, yet we know relatively little about the content of these aggregates. The aim of the present study was to examine the microbial content of tape-like films found protruding from a domestic showerhead. Culturing showed that the films were dominated by aerobic α- and β-proteobacteria. Three isolates made up almost the entire plate count. These were a Brevundimonas species, a metalophilic Cupriavidus species and a thermophile, Geobacillus species. Furthermore, it was shown that the Cupriavidus isolate alone had a high capacity for biofilm formation and thus might be the initiator of biofilm production. A clone library revealed the same general composition. However, half of the 70 clones analyzed could not be assigned to a particular bacterial phylum and of these 29 differed from one another by only 1–2 base pairs, indicating a single species. Thus both the culture dependent and culture independent characterizations suggest a simple yet novel composition. The work is important as the biofilm is fundamentally different in form (tape-like) and content from that of all previously reported ones, where variously Mycobacterium, Methylobacterium and Xanthomonas species have dominated, and extremophiles were not reported.

Nonlocal Effect on Buckling of Triangular Nano-composite Plates

In the present study, small scale effect on critical buckling loads of triangular nano- composite plates under uniform in-plane compression is studied. Since at nano-scale the structure of the plate is discrete, the size dependent nonlocal elasticity theory is employed to develop an equivalent continuum plate model for this nanostructure incorporating the changes in its mechanical behavior. The two-parameter, Winkler-Pasternak, elastic medium is used for precisely modelling the behavior of matrix surrounding the nano-plate. The governing stability equations are then derived using the classical plate theory and the principle of virtual work for a perfect uniform triangular nano-plate composite. The well-known numerical Galerkin method in conjunction with the areal coordinates system is used as the basis for the solution. The solution procedure views the entire nano-composite plate as a single super-continuum element. Effects of nonlocal parameter, length, aspect ratio, mode number, anisotropy, edge supports and elastic medium on the buckling loads are rigorously investigated. All of these parameters are seen to have significant effect on the stability of nano-composite plate. It is shown that the results depend obviously on the non-locality of buckled nano-composite plate, especially at very small dimensions, small aspect ratios, higher mode numbers, higher anisotropy degree, stiffer edge supports and softer elastic mediums. Also, it is seen that the classical continuum mechanics overestimates the buckling results which can lead to deficient design and analysis of these widely used nanostructures.

Self-propulsion of a three-dimensional flapping flexible plate

The self-propulsion of a 3-D flapping flexible plate in a stationary fluid is numerically studied by an immersed boundary-lattice Boltzmann method for the fluid flow and a finite element method for the plate motion. When the leading-edge of the plate is forced to heave sinusoidally, the entire plate starts to move freely as a result of the fluid-structure interaction. Based on our simulation and analysis on the dynamical behaviors of the flapping flexible plate, we have found that the effect of plate aspect ratio on its propulsive properties can be divided into three typical regimes which are related to the plate flexibility, i.e. stiff, medium flexible, and more flexible regime. It is also identified that a suitable structure flexibility, corresponding to the medium flexible regime, can improve the propulsive speed and efficiency. The wake behind the flapping plate is investigated for several aspect ratios to demon- strate some typical vortical structures. The results obtained in this study can provide some physical insights into the understanding of the propulsive mechanisms in the flapping-based locomotion.

SUPR: Spectral Unmixinig Plate Reader for Live-Cell FRET Biosensor Drug Screening

We have developed a high-throughput Spectral Unmixing Plate Reader (SUPR) which can acquire a fluorescence emission spectrum across 384 wells, with short (< 4 min) read time for the entire plate. The entire spectrum is recorded at once with a compact spectrometer equipped with an uncooled one-dimensional array detector. For application to FRET in living cells, FRET protein biosensors, with genetically encoded GFP (donor) and RFP (acceptor) tags, are expressed in HEK cells, and single-component spectra are acquired from water (Raman), untransfected cells (autofluorescence), cells expressing donor only, and cells expressing acceptor only. Each observed spectrum is decomposed into a linear combination of component spectra to determine FRET efficiency. The method enables unprecedented speed, precision, and accuracy. It rigorously and painlessly accounts for phenomena such as bleed-through of the donor emission into the acceptor channel, cross-talk associated with direct excitation of the acceptor, and cell autofluorescence. Excitation and detection are both from the top, so low-cost polypropylene plates typically used in qPCR work very well in this application. This novel technology has been combined with our previously developed fluorescence lifetime technology (Petersen et al, Rev Sci Inst, 2014) allowing for high-throughput quantitation of FRET efficiency from independent measurements. We have screened small-molecule libraries with an intramolecular cardiac calcium pump FRET biosensor, expressed in living cells (Gruber et al., J. Biomol Screening, 2014) using this multiplexed fluorescence detection platform and identified several previously unknown small-molecule effectors. The information obtained from both fluorescence lifetime and spectrum measurements reduced the number of false-positives and increased confidence in our hit selection methods. The development of this technology and screening assay will prove useful for drug discovery but also answering basic biological questions with fluorescence based assays.