Bead Position Research Articles

3D fluorescence imaging through scattering medium using transport of intensity equation and iterative phase retrieval.

In this paper, we have proposed a method of three-dimensional (3D) fluorescence imaging through a scattering medium. The proposed method combines the numerical digital phase conjugation propagation after measurement of the complex amplitude distribution of scattered light waves by the transport of intensity equation (TIE) with followed iterative phase retrieval to achieve 3D fluorescence imaging through a scattering medium. In the experiment, we present the quantitative evaluation of the depth position of fluorescent beads. In addition, for time-lapse measurement, cell division of tobacco-cultured cells was observed. Numerical results presented the effective range of the phase amount in the scattering medium. From these results, the proposed method is capable of recovering images degraded by a thin scattering phase object beyond a small phase change approximation.

Solution of steady state in the model polymer system with rupture and rebinding

In this paper, we study the steady state attained in our model polymer system that attempts to explain the relative motion between soft rubbing surfaces at the single polymer level. We generalize our one-dimensional model [Borah et al, 2016 Soft Matter 12 4406] by including the rebinding of interconnecting bonds between a flexible transducer (bead spring polymer) and a rigid fixed plate. The interconnecting bonds described as harmonic springs rupture and rebind stochastically when a constant force pulls the flexible transducer. We obtain a distinct steady state in stochastic simulations of the model when the bead positions and the bond states (closed or open) are independent of time, analogous to creep states in frictional systems and rupture termination states in earthquakes. The simulation results of the stochastic model for specific parameter sets agree with the numerical solution to the mean-field equations developed for analytical tractability. We develop an analytical solution for the steady state within the homotopy analysis method, which converges and agrees well with the numerical results.

A combined experimental and finite element analysis of the human elbow under loads of daily living

Elbow trauma is often accompanied by a loss of independence in daily self-care activities, negatively affecting patients’ quality of life. Finite element models can help gaining profound knowledge about native human joint mechanics, which is crucial to adequately restore joint functionality after severe injuries. Therefore, a finite element model of the elbow is required that includes both the radio-capitellar and ulno-trochlear joint and is subjected to loads realistic for activities of daily living. Since no such model has been published, we aim to fill this gap.For comparison, 8 intact cadaveric elbows were subjected to loads of up to 1000 N, after they were placed in an extended position. At each load step, the displacement of the proximal humerus relative to the distal base plate was measured with optical tracking markers and the joint pressure was measured with a pressure mapping sensor.Analogously, eight finite element models were created based on subject-specific CT scans of the corresponding elbow specimens. The CT scans were registered to the positions of tantalum beads in the experiment. The optically measured displacements were applied as boundary conditions.We demonstrated that the workflow can predict the experimental contact pressure distribution with a moderate correlation, the experimental peak pressures in the correct joints and the experimental stiffness with moderate to excellent correlation. The predictions of peak pressure magnitude, contact area and load share on the radius require improvement by precise representation of the cartilage geometry and soft tissues in the model, and proper initial contact in the experiment.

Inspection Algorithm of Welding Bead Based on Image Projection

The shear reinforcement of dual-anchorage (SRD) is used to enhance the safety of reinforced concrete structures in construction sites. In SRD, welding is used to create shear reinforcement, and after production, a quality inspection of the welding bead is required. Since the welding bead of SRD is inspected for quality by measuring both horizontal and vertical lengths, it is necessary to obtain this information for quality inspection. However, it is difficult to inspect the quality of welding beads using existing methods based on segmentation, due to the similarity in texture between the welding bead and the base material, as well as discoloration around the welded area after welding. In this paper, we propose an algorithm that detects the welding bead using an image projection algorithm for pixels and classifies the quality of the welding bead. This algorithm detects the position of welding beads using the brightness values of an image. The proposed algorithm reduces the amount of computation time by first specifying the region of interest and then performing the analysis. Results from experiments reveal that the algorithm accurately classifies welding beads into good or bad classes by obtaining all brightness values in the vertical and horizontal directions in the SRD image. Furthermore, comparison tests with conventional algorithms demonstrate that the classification accuracy of the proposed algorithm is the highest. The proposed algorithm will be helpful in the real-time welding bead inspection field where fast and accurate inspection is crucial.

Abstract 4303: High-throughput 3D tumoroid models for immunotherapy and drug discovery

Abstract The tumor microenvironment (TME) is a complex 3D cellular system comprised of a diverse amalgam of cells. To characterize the TME, we developed a 3D cell culture platform which facilitates imaging-based analysis and cell viability for extended durations. We used this platform to collect in-situ spatiotemporal measurements of cytokine concentrations in the tumor vicinity, track cellular activity, and model local tumor invasion of glioblastoma (GBM) using fast-scanning confocal microscopy. The mechanism by which we achieve long-term 3D culture is perfusion, modeled as flow through a porous medium. Perfusion facilitates regulated transport of nutrients and waste within the soft microgel medium: Liquid-Like Solids (LLS). The interstitial space is tuned to mimic a capillary bed, and surface bioconjugation of the microgels promotes cellular adhesion and migration. To measure cytokine concentrations, GBM tumoroids were grown in LLS and printed into a dispersion of ELISA beads in LLS. The mechanical stability of LLS ensures the tumor and ELISA beads remain stationary without impeding cellular activity. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. Co-culture of patient-derived tumor explants and autologous PBMCs printed into type I collagen-bioconjugated LLS enabled studies of cancer-immune interactions. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml−1 after 10 hours. A cellular production rate was estimated at 2 molecules cell−1 s−1. Invasive behavior into the proximal space was determined to be super-diffusive; off-lattice agent-based simulations indicated this behavior is a result of the confinement of invasive fronts to the microgel interstitium. The invading glioblastoma cells used anchorage-dependent migration and were guided by geometric cues to traverse the porous bioconjugated LLS network. Cancer-immune interaction studies revealed average CD8+ speeds greater than 2.8 µm min−1 and both chemotaxis and chemokinetic behavior. CD8+ T-cell killing rates were estimated at approximately 3 cancer cells hr−1 initially, monotonically falling over 12 hours to roughly 1 cell hr−1. The development of the in-situ 3D ELISA assay and imaging-based analysis techniques have enabled the tracking of tumor-immune cell interactions, observation of dynamic tumor progression, and local cytokine concentration profiling at appreciable spatiotemporal resolutions. The combination of a physiologically relevant 3D culture platform with the capacity for in-situ qualitative and quantitative observation may lead to new, powerful, preclinical models that allow for interrogation of the TME and decrease the rate of false discovery. Citation Format: Duy Nguyen, Alexander McGhee, Diego Pedro, Alfonso Pepe, Matthew Schaller, Ryan Smolchek, Jack Famiglietti, Stephanie Warrington, W. Gregory Sawyer. High-throughput 3D tumoroid models for immunotherapy and drug discovery. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4303.

Hoverfly (Eristalis tenax) pursuit of artificial targets.

The ability to visualize small moving objects is vital for the survival of many animals, as these could represent predators or prey. For example, predatory insects, including dragonflies, robber flies and killer flies, perform elegant, high-speed pursuits of both biological and artificial targets. Many non-predatory insects, including male hoverflies and blowflies, also pursue targets during territorial or courtship interactions. To date, most hoverfly pursuits have been studied outdoors. To investigate hoverfly (Eristalis tenax) pursuits under more controlled settings, we constructed an indoor arena that was large enough to encourage naturalistic behavior. We presented artificial beads of different sizes, moving at different speeds, and filmed pursuits with two cameras, allowing subsequent 3D reconstruction of the hoverfly and bead position as a function of time. We show that male E. tenax hoverflies are unlikely to use strict heuristic rules based on angular size or speed to determine when to start pursuit, at least in our indoor setting. We found that hoverflies pursued faster beads when the trajectory involved flying downwards towards the bead. Furthermore, we show that target pursuit behavior can be broken down into two stages. In the first stage, the hoverfly attempts to rapidly decreases the distance to the target by intercepting it at high speed. During the second stage, the hoverfly's forward speed is correlated with the speed of the bead, so that the hoverfly remains close, but without catching it. This may be similar to dragonfly shadowing behavior, previously coined 'motion camouflage'.

Single-spot spiral phase plate for tracking 3D particle motion.

We have imaged and tracked submicron fluorescent particles in three dimensions by inserting a spiral phase plate (SPP) of our design into a conventional wide-field optical microscope. Our novel SPP consists of concentric annular zones that each impose a unique quantized orbital angular momentum (OAM) on the ring of fluorescent light it intercepts. Each ring of the SPP consists of a vortex surface that produces one additional unit of OAM than the smaller ring just inside it, with the seven successive rings each contributing n = 1 to 7 quantum units of OAM respectively. The light rings interfere to produce a single-spot rotating point spread function (SS-RPSF). This image ‘spot’ rotates with the axial depth of a particle, permitting 3D mapping of the particle position. The SPP is easily incorporated into existing microscopes. We used the DIC slider here for the SPP. 3D positions of fluorescent beads were retrieved using template matching. Bead positions could be inferred over a 6 µm depth range. Best results were obtained over a depth range of 2.3 μm, with a mean absolute error of 20 nm in this range. As proof-of-concept for live-cell imaging we tracked DNA loci and determined their diffusion coefficients in the 3D environment of the nucleus of U2OS cells. We are pursuing chromatin tracking experiments to better understand how DNA damage affects chromatin motions and, reciprocally, how chromatin dynamics impacts the DNA damage response. Beyond this specific application, our SPP-based imaging approach should enable researchers to track dynamic fluorescent objects in samples with high temporal resolution because x, y, and z are determined at exactly the same time.

Measuring tension states of hiPSC-cardiomyocytes via traction force microscopy.

Cardiomyocytes (CMs), the cells responsible for the contraction of the heart, experience mechanical forces such as active and resting tension as the cell contracts and relaxes. Active tension is generated by the contractile cytoskeleton, which is composed of actin-myosin arrays called sarcomeres. Resting tension is the tension that results when spring-like elements are stretched beyond their resting length, and this resting tension determines how forceful a contraction will be. The ability to investigate how resting tension is regulated has been a challenge due to the lack of a robust method to measure resting tension at the cell level. Previous methods involve stretching an individual cell and measuring the resulting resting tension; however, this method is low throughput and invasive. Single-cell Traction Force Microscopy (TFM) serves as a promising alternative method to measure resting tension due to the ability to measure the tension states of individual CMs with high throughput. We culture human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) on polyacrylamide (PA) gels with stiffness of healthy myocardium (10 kPa) patterned with islands of adhesive protein (Matrigel). We use TFM video analysis of beating cells to measure the traction forces when the hiPSC-CM is contracted and relaxed. We measure bead positions again after treatment with blebbistatin, and finally after the cell is dissociated from the gel. We calculate resting tension by calculating the TFM bead displacements from their positions under a relaxed cell to their positions after blebbistatin and after the cell is dissociated. To probe how resting tension is regulated by increased loading, we culture hiPSC-CMs on PA gels of increasing stiffness to mimic fibrotic tissue. These results provide insight into how the resting tension of a CM is regulated and how the CM adapts in response to pathophysiological conditions.

Dean-Flow Affected Lateral Focusing and Separation of Particles and Cells in Periodically Inhomogeneous Microfluidic Channels.

The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli (E. coli), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae, and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.

Bayesian Information Engine that Optimally Exploits Noisy Measurements.

We have experimentally realized an information engine consisting of an optically trapped, heavy bead in water. The device raises the trap center after a favorable "up" thermal fluctuation, thereby increasing the bead's average gravitational potential energy. In the presence of measurement noise, poor feedback decisions degrade its performance; below a critical signal-to-noise ratio, the engine shows a phase transition and cannot store any gravitational energy. However, using Bayesian estimates of the bead's position to make feedback decisions can extract gravitational energy at all measurement noise strengths and has maximum performance benefit at the critical signal-to-noise ratio.

Pulling, failing, and adaptive mechanotransduction of macrophage filopodia

Macrophages use filopodia to withdraw particles toward the cell body for phagocytosis. This can require substantial forces, which the cell generates after bio-mechanical stimuli are transmitted to the filopodium. Adaptation mechanisms to mechanical stimuli are essential for cells, but can a cell iteratively improve filopodia pulling? If so, the underlying mechanic adaptation principles organized on the protein level are unclear. Here, we tackle this problem using optically trapped 1 μm beads, which we tracked interferometrically at 1 MHz during connection to the tips of dorsal filopodia of macrophages. We observe repetitive failures while the filopodium tries to pull the bead out of the optical trap. Analyses of mean bead motions and position fluctuations on the nano-meter and microsecond scale indicate mechanical ruptures caused by a force-dependent actin-membrane connection. We found that beads are retracted three times slower under any load between 5 and 40 pN relative to the no-load transport, which has the same speed as the actin retrograde flow obtained from fluorescent speckle tracking. From this duty ratio of pulling velocities, we estimated a continuous on/off binding with τoff = 2⋅τon, with measured off times τoff = 0.1–0.5 s. Remarkably, we see a gradual increase of filopodia pulling forces from 10 to 30 pN over time and after failures, which points toward an unknown adaptation mechanism. Additionally, we see that the attachment strength and friction between the bead and filopodium tip increases under load and over time. All observations are typical for catch-bond proteins such as integrin-talin complexes. We present a mechanistic picture of adaptive mechanotransduction, which formed by the help of mathematical models for repetitive tip ruptures and reconnections. The analytic mathematical model and the stochastic computer simulations, both based on catch-bond lifetimes, confirmed our measurements. Such catch-bond characteristics could also be important for other immune cells taking up counteracting pathogens.

Tiling with \U0001d579 c _class

This work is devoted to apply classes of nested chain abacus of tiling in finite regions. a mapping notations for generating an algorithms are presented. The mapping notations are used to move subsets of bead positions as well as empty bead positions after embedding the class of nested chain abacus in finite grid for tiling a rectangle.

Hydrodynamic interaction and complex viscosity of multi-bead rods

One good way to explain the elasticity of a polymeric liquid is to just consider the orientation distribution of the macromolecules. When exploring how macromolecular architecture affects the elasticity of a polymeric liquid, we find the general rigid bead–rod theory to be both versatile and accurate. This theory sculpts macromolecules using beads and rods. Whereas beads represent points of Stokes flow resistances, the rods represent rigid separations. In this way, how the shape of the macromolecule affects its rheological behavior in suspension is determined. Until recently, general rigid bead–rod theory has neglected interferences of the Stokes flow velocity profiles between nearby beads. We call these hydrodynamic interactions, and we here employ our new method for exploring how these interactions affect the complex viscosity of suspensions of multi-bead rods. These multi-bead rods are also called shish-kebabs. We use the center-to-center distance between adjacent beads as the characteristic length. We proceed analytically, beginning with a geometric expression for the shish-kebab bead positions. Our analytical solution for the complex viscosity presents as one for N=3,5,6,7,8,…, one for N=4, and another for the rigid dumbbell, N=2. We find that for shish-kebabs, hydrodynamic interactions (i) increase zero-shear viscosity, (ii) increase zero-shear first normal stress coefficient, (iii) decrease the real part of the dimensionless complex viscosity, and (iv) increase minus the dimensionless imaginary part. We find that the combination of (iii) and (iv) explains crossovers of the parts of the complex viscosity. We further find that for a monodisperse polystyrene solution, the general rigid bead–rod theory with hydrodynamic interaction, for both parts of the complex viscosity, provides stunning improvement over without.

The impact of self-lacing technology on foot containment during dynamic cutting

The sport of basketball requires repeated dynamic side-to-side movements that can cause foot sliding and place excessive stress on the athlete. Footwear that provides containment to reduce the motion of the foot relative to the shoe may have performance and injury prevention advantages. Foot containment can vary significantly across different types of footwear due to its dependence on the interaction between the shoe lacing system of the upper and specific design features within the shoe. This study used high-speed stereo radiography to assess the impact of a self-lacing system with three conditions of increasing lace tension compared to a control basketball shoe on foot containment by measuring foot–shoe motion during cutting and jab step activities. Twenty healthy athletes performed dynamic cutting activities in view of a unique stereo radiography system positioned to measure motion of specific foot bones while motion capture and perception data were also collected. By tracking bead positions on the foot and shoe, relative motion of the foot inside the shoe was measured along shoe planes during activity. The self-lacing system showed statistically significant reductions in foot–shoe motion of as much as 75.2% while performing dynamic basketball cuts. Controlling tension with the lacing system provides athletes flexibility in containment to go from a loose fit to a tight fit and reduce in-shoe motion by up to 39.3%. Furthermore, perception data showed athletes had 20.1% greater confidence to perform cutting tasks with increases in lace tension. Reduction of in-shoe foot motion with a self-lacing system is an important consideration in the design of footwear for performance athletes.

High-Resolution Optical Tweezers Combined with Multicolor Single-Molecule Microscopy.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or "fleezers," combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest "random phase" trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.

Tweezepy: A Python package for calibrating forces in single-molecule video-tracking experiments.

Single-molecule force spectroscopy (SMFS) instruments (e.g., magnetic and optical tweezers) often use video tracking to measure the three-dimensional position of micron-scale beads under an applied force. The force in these experiments is calibrated by comparing the bead trajectory to a thermal motion-based model with the drag coefficient, γ, and trap spring constant, κ, as parameters. Estimating accurate parameters is complicated by systematic biases from spectral distortions, the camera exposure time, parasitic noise, and least-squares fitting methods. However, while robust calibration methods exist that correct for these biases, they are not always used because they can be complex to implement computationally. To address this barrier, we present Tweezepy: a Python package for calibrating forces in SMFS video-tracking experiments. Tweezepy uses maximum likelihood estimation (MLE) to estimate parameters and their uncertainties from a single bead trajectory via the power spectral density (PSD) and Allan variance (AV). It is well-documented, fast, easy to use, and accounts for most common sources of biases in SMFS video-tracking experiments. Here, we provide a comprehensive overview of Tweezepy's calibration scheme, including a review of the theory underlying thermal motion-based parameter estimates, a discussion of the PSD, AV, and MLE, and an explanation of their implementation.

On-the-Fly Ring-Polymer Molecular Dynamics Calculations of the Dissociative Photodetachment Process of the Oxalate Anion.

The dissociative photodetachment dynamics of the oxalate anion, C2O4H− + hν → CO2 + HOCO + e−, were theoretically studied using the on-the-fly path-integral and ring-polymer molecular dynamics methods, which can account for nuclear quantum effects at the density-functional theory level in order to compare with the recent experimental study using photoelectron–photofragment coincidence spectroscopy. To reduce computational time, the force acting on each bead of ring-polymer was approximately calculated from the first and second derivatives of the potential energy at the centroid position of the nuclei beads. We find that the calculated photoelectron spectrum qualitatively reproduces the experimental spectrum and that nuclear quantum effects are playing a role in determining spectral widths. The calculated coincidence spectrum is found to reasonably reproduce the experimental spectrum, indicating that a relatively large energy is partitioned into the relative kinetic energy between the CO2 and HOCO fragments. This is because photodetachment of the parent anion leads to Franck–Condon transition to the repulsive region of the neutral potential energy surface. We also find that the dissociation dynamics are slightly different between the two isomers of the C2O4H− anion with closed- and open-form structures.

Radiopaque drug-eluting embolisation beads as fiducial markers for stereotactic liver radiotherapy.

Objective:To determine the feasibility of using radiopaque (RO) beads as direct tumour surrogates for image-guided radiotherapy (IGRT) in patients with liver tumours after transarterial chemoembolisation (TACE).Methods:A novel vandetanib-eluting RO bead was delivered via TACE as part of a first-in-human clinical trial in patients with either hepatocellular carcinoma or liver metastases from colorectal cancer. Following TACE, patients underwent simulated radiotherapy imaging with four-dimensional computed tomography (4D-CT) and cone-beam CT (CBCT) imaging. RO beads were contoured using automated thresholding, and feasibility of matching between the simulated radiotherapy planning dataset (AVE-IP image from 4D data) and CBCT scans assessed. Additional kV, MV, helical CT and CBCT images of RO beads were obtained using an in-house phantom. Stability of RO bead position was assessed by comparing 4D-CT imaging to CT scans taken 6–20 days following TACE.Results:Eight patients were treated and 4D-CT and CBCT images acquired. RO beads were visible on 4D-CT and CBCT images in all cases and matching successfully performed. Differences in centre of mass of RO beads between CBCT and simulated radiotherapy planning scans (AVE-IP dataset) were 2.0 mm mediolaterally, 1.7 mm anteroposteriorally and 3.5 mm craniocaudally. RO beads in the phantom were visible on all imaging modalities assessed. RO bead position remained stable up to 29 days post TACE.Conclusion:RO beads are visible on IGRT imaging modalities, showing minimal artefact. They can be used for on-set matching with CBCT and remain stable over time.Advances in knowledge:The role of RO beads as fiducial markers for stereotactic liver radiotherapy is feasible and warrants further exploration as a combination therapy approach.

Refocusing and locating effect of fluorescence scattering field* *Project supported by the National Key Research and Development Program of China (Grant No. 2019YFC0119800), the Youth Talent Support Program of Universities of Hebei Province, China (Grant No. BJ2021038), the National Natural Science Foundation of China (Grant No. 12004265), the Natural Science Foundation of Hebei Province, China (Grant No. A2020210001), and the

Optical imaging deep inside scattering medium has always been one of the challenges in the field of bioimaging, which significantly drawbacks the employment of con-focal microscopy system. Although a variety of feedback techniques, such as acoustic or nonlinear fluorescence-based schemes have realized the refocusing of the coherent light, the problems of non-invasively refocusing and locating of linearly-excited fluorescent beads inside the scattering medium have not been thoroughly explored. In this paper, we linearly excited the fluorescent beads inside a scattering medium by using our homemade optical con-focal system, collected the fluorescence scattering light as the optimized target, and established a theoretical model of target contrast enhancement, which is consistent with the experimental data. By improving both the cost function and variation rate within the genetic algorithm, we could refocus the fluorescence scattering field while improving the contrast enhancement factor to 12.8 dB. Then, the positions of the fluorescent beads are reconstructed by subpixel accuracy centroid localization algorithm, and the corresponding error is no more than 4.2 μm with several fluorescent beads within the field of view. Finally, the main factors such as the number of fluorescent beads, the thickness of the scattering medium, the modulating parameter, the experimental noise and the system long-term stability are analyzed and discussed in detail. This study proves the feasibility of reconstructing fluorescent labeled cells inside biological tissues, which provides certain reference value for deep imaging of biological tissues.

Bead geometry modeling on uneven base metal surface by fuzzy systems for multi-pass welding

This paper presents a modeling method of weld bead profiles deposited on uneven base metal surfaces and its application in multi-pass welding. The robotized multi-pass tungsten inert gas welding requires precise positioning of the weld beads to avoid welding defects and achieve the desirable welding join since the weld bead shapes depend on the surface of the previously deposited beads. The proposed model consists of fuzzy systems to estimate the coefficients of the profile function. The characteristic points of the trapezoidal membership functions in the rule bases are tuned by the Bacterial Memetic Algorithm during supervised training. The fuzzy systems are structured as multiple-input-single-output systems, where the inputs are the welding process variables and the coefficients of the shape functions of the segments underlying the modeled bead; the outputs are the coefficients of the bead shape function. Each segment surface is approximated by a second-order polynomial function defined in the weld bead’s local coordinate system. The model is developed from empirical data collected from single and multi-pass welding. The performance of the proposed model is compared with a multiple linear regression model. During the experimental validation, first, the individual beads are evaluated by comparing the estimated coefficients of the profile function and other bead characteristics (bead area, width, contact angles, and position of the toe points) with the measurements, and the estimations of a multiple linear regression model. Second, the sequential placement of the weld beads is evaluated while filling a straight V-groove by comparing the estimated bead characteristics with the measurements and calculating the accumulated error of the filled groove cross-section. The results show that the proposed model provides a good estimation of the bead shapes during deposition on uneven base metal surfaces and outperforms the regression model with low error in both validation cases. Furthermore, it is experimentally validated that the derived bead characteristics provide a suitable measure to identify locations sensitive to welding defects.