Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37°C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.
25 Related JoVE Articles!
The Generation of Higher-order Laguerre-Gauss Optical Beams for High-precision Interferometry
Institutions: University of Birmingham.
Thermal noise in high-reflectivity mirrors is a major impediment for several types of high-precision interferometric experiments that aim to reach the standard quantum limit or to cool mechanical systems to their quantum ground state. This is for example the case of future gravitational wave observatories, whose sensitivity to gravitational wave signals is expected to be limited in the most sensitive frequency band, by atomic vibration of their mirror masses. One promising approach being pursued to overcome this limitation is to employ higher-order Laguerre-Gauss (LG) optical beams in place of the conventionally used fundamental mode. Owing to their more homogeneous light intensity distribution these beams average more effectively over the thermally driven fluctuations of the mirror surface, which in turn reduces the uncertainty in the mirror position sensed by the laser light.
We demonstrate a promising method to generate higher-order LG beams by shaping a fundamental Gaussian beam with the help of diffractive optical elements. We show that with conventional sensing and control techniques that are known for stabilizing fundamental laser beams, higher-order LG modes can be purified and stabilized just as well at a comparably high level. A set of diagnostic tools allows us to control and tailor the properties of generated LG beams. This enabled us to produce an LG beam with the highest purity reported to date. The demonstrated compatibility of higher-order LG modes with standard interferometry techniques and with the use of standard spherical optics makes them an ideal candidate for application in a future generation of high-precision interferometry.
Physics, Issue 78, Optics, Astronomy, Astrophysics, Gravitational waves, Laser interferometry, Metrology, Thermal noise, Laguerre-Gauss modes, interferometry
Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive
Institutions: Lawrence Livermore National Laboratory, University of California, San Francisco.
Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro
test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (>3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo
recording data from the rat cortex.
Bioengineering, Issue 79, Nervous System Diseases, Surgical Procedures, Operative, Investigative Techniques, Nonmetallic Materials, Engineering (General), neural interfaces, polymer neural probes, surgical insertion, polyethylene glycol, microelectrode arrays, chronic implantation
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis
Institutions: North Carolina State University & University of North Carolina at Chapel Hill, University of North Carolina School of Medicine, Atlantic Prosthetics & Orthotics, LLC.
To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user's movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.
Biomedical Engineering, Issue 89, neural control, powered transfemoral prosthesis, electromyography (EMG), neural-machine interface, experimental setup and protocol
In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint
Institutions: University of California San Francisco, University of California San Francisco, Xradia Inc..
This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ
loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e.
from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo
conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.
Bioengineering, Issue 85, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent
Construction and Characterization of External Cavity Diode Lasers for Atomic Physics
Institutions: The Australian National University.
Since their development in the late 1980s, cheap, reliable external cavity diode lasers (ECDLs) have replaced complex and expensive traditional dye and Titanium Sapphire lasers as the workhorse laser of atomic physics labs1,2
. Their versatility and prolific use throughout atomic physics in applications such as absorption spectroscopy and laser cooling1,2
makes it imperative for incoming students to gain a firm practical understanding of these lasers. This publication builds upon the seminal work by Wieman3
, updating components, and providing a video tutorial. The setup, frequency locking and performance characterization of an ECDL will be described. Discussion of component selection and proper mounting of both diodes and gratings, the factors affecting mode selection within the cavity, proper alignment for optimal external feedback, optics setup for coarse and fine frequency sensitive measurements, a brief overview of laser locking techniques, and laser linewidth measurements are included.
Physics, Issue 86, External Cavity Diode Laser, atomic spectroscopy, laser cooling, Bose-Einstein condensation, Zeeman modulation
Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators
Institutions: University of California, Davis, Texas Instruments, Purdue University.
Mechanically underdamped electrostatic fringing-field MEMS actuators are well known for their fast switching operation in response to a unit step input bias voltage. However, the tradeoff for the improved switching performance is a relatively long settling time to reach each gap height in response to various applied voltages. Transient applied bias waveforms are employed to facilitate reduced switching times for electrostatic fringing-field MEMS actuators with high mechanical quality factors. Removing the underlying substrate of the fringing-field actuator creates the low mechanical damping environment necessary to effectively test the concept. The removal of the underlying substrate also a has substantial improvement on the reliability performance of the device in regards to failure due to stiction. Although DC-dynamic biasing is useful in improving settling time, the required slew rates for typical MEMS devices may place aggressive requirements on the charge pumps for fully-integrated on-chip designs. Additionally, there may be challenges integrating the substrate removal step into the back-end-of-line commercial CMOS processing steps. Experimental validation of fabricated actuators demonstrates an improvement of 50x in switching time when compared to conventional step biasing results. Compared to theoretical calculations, the experimental results are in good agreement.
Physics, Issue 90, microelectromechanical systems, actuators, switching time, settling time, electrostatic devices, micromachining, thin film devices
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond
Institutions: University of Antwerp.
Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1
and in zebrafish2
, to control of seizures following cortical stroke using optogenetics3
. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4
and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5
was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.
Neuroscience, Issue 100, Electrophysiology, cellular neurobiology, dynamic clamp, Active Electrode Compensation, command-line interface, real-time computing, closed-loop, scripted electrophysiology.
Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks
Institutions: Istituto Italiano di Tecnologia.
Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e.
, a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.
Neuroscience, Issue 97, Micro Electrode Arrays (MEA), in vitro cultures, coding, decoding, tetanic stimulation, spike, burst
Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice
Institutions: Brigham and Women's Hospital, Harvard Medical School, Chi-Mei Medical Center, Tainan.
Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy.
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS).
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography.
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.
Medicine, Issue 98, Coronary flow reserve, Doppler echocardiography, non-invasive methodology, use of animals in research, pressure overload, aortic banding
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo
. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls.
DTI data analysis is performed in a variate fashion, i.e.
voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e.
differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale
Institutions: University of Lethbridge.
Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.
Neuroscience, Issue 18, rat skilled reaching, rat reaching scale, rat, rat movement element rating scale, reaching elements
A Tactile Automated Passive-Finger Stimulator (TAPS)
Institutions: Duquesne University, McMaster University.
Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich
Medicine, Neuroscience, Issue 28, tactile, somatosensory, touch, cutaneous, acuity, psychophysics, Bayesian, grating orientation, sensory neuroscience, spatial discrimination
An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces
Institutions: Imperial College London.
The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.
Neuroscience, Issue 49, Stabilization reflexes, Sensorimotor control, Adaptive control, Insect vision
Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Institutions: School of Medicine, New York University.
The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant β-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2
used the ab initio
accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio
folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5
. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.
Infection, Issue 43, HIV-1, structure-activity relationships, ab initio simulations, antibody-mediated neutralization, vaccine design
Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans
Institutions: University of Utah.
Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1
. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3
. However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4
). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.
Neuroscience, Issue 57, laser axotomy, regeneration, growth cone, time lapse, C. elegans, neuroscience, Nd:Yag laser
Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
Institutions: University of Nebraska-Lincoln, University of Nebraska-Lincoln.
Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1
. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2
. The method proposed in the following protocol uses microscopic magnetic resonance elastography (μMRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3
. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4
. Recently, μMRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5
. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6
. The resulting measurements at each voxel have been shown to strongly correlate (R2
>0.9914) with data collected using dynamic mechanical analysis7
. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (h
MSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.
Bioengineering, Issue 60, mesenchymal stem cells, tissue engineering (TE), regenerative medicine, adipose TE, magnetic resonance elastography (MRE), biomechanics, elasticity
Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images
Institutions: Virginia Commonwealth University, Virginia Commonwealth University Reanimation Engineering Science (VCURES) Center, Virginia Commonwealth University, Virginia Commonwealth University, Virginia Commonwealth University.
In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift estimation and intracranial pressure (ICP) pre-screening system. To estimate the midline shift, first an estimation of the ideal midline is performed based on the symmetry of the skull and anatomical features in the brain CT scan. Then, segmentation of the ventricles from the CT scan is performed and used as a guide for the identification of the actual midline through shape matching. These processes mimic the measuring process by physicians and have shown promising results in the evaluation. In the second component, more features are extracted related to ICP, such as the texture information, blood amount from CT scans and other recorded features, such as age, injury severity score to estimate the ICP are also incorporated. Machine learning techniques including feature selection and classification, such as Support Vector Machines (SVMs), are employed to build the prediction model using RapidMiner. The evaluation of the prediction shows potential usefulness of the model. The estimated ideal midline shift and predicted ICP levels may be used as a fast pre-screening step for physicians to make decisions, so as to recommend for or against invasive ICP monitoring.
Medicine, Issue 74, Biomedical Engineering, Molecular Biology, Neurobiology, Biophysics, Physiology, Anatomy, Brain CT Image Processing, CT, Midline Shift, Intracranial Pressure Pre-screening, Gaussian Mixture Model, Shape Matching, Machine Learning, traumatic brain injury, TBI, imaging, clinical techniques
Origami Inspired Self-assembly of Patterned and Reconfigurable Particles
Institutions: The Johns Hopkins University , The Johns Hopkins University .
There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20
wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23
and (b) grippers that self-fold due to residual stress powered hinges24,25
. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26
and with a range of materials including metals21
. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 μm to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7
to possibly create even smaller nanoscale grasping devices.
Chemistry, Issue 72, Chemical Engineering, Biomolecular Engineering, Materials Science, Physics, Nanotechnology, Molecular Self-assembly, Electrochemistry, Folding, three dimensional, lithography, colloid, patchy particles, particles, nanoparticles, robotics, drug delivery, microfabrication, nanofabrication, nano, assembly, synthesis, reaction, origami
Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations
Institutions: Johns Hopkins University.
Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo
imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo
images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via
semiautomatic segmentation, from an in vivo
computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 °. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.
Bioengineering, Issue 71, Biomedical Engineering, Medicine, Anatomy, Physiology, Cardiology, Myocytes, Cardiac, Image Processing, Computer-Assisted, Magnetic Resonance Imaging, MRI, Diffusion Magnetic Resonance Imaging, Cardiac Electrophysiology, computerized simulation (general), mathematical modeling (systems analysis), Cardiomyocyte, biomedical image processing, patient-specific modeling, Electrophysiology, simulation
Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Institutions: University of Calgary , University of Calgary .
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via
quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering
Institutions: The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, VA Medical Center, Providence, RI, University of Texas Southwestern Medical Center .
We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.
Bioengineering, Issue 74, Biomedical Engineering, Biophysics, Cellular Biology, Medicine, Anatomy, Physiology, Cell Engineering, Bioreactors, Culture Techniques, Cell Engineering, Tissue Engineering, compression loads, shear loads, Tissues, bioreactor, mechanical loading, compression, shear, musculoskeletal, cartilage, bone, transplantation, cell culture
Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization
Institutions: The Ohio State University, The Ohio State University, The Ohio State University.
Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging (ECCI) in a scanning electron microscope (SEM). ECCI allows for imaging of defects and crystallographic features under specific diffraction conditions, similar to that possible via plan-view transmission electron microscopy (PV-TEM). A particular advantage of the ECCI technique is that it requires little to no sample preparation, and indeed can use large area, as-produced samples, making it a considerably higher throughput characterization method than TEM. Similar to TEM, different diffraction conditions can be obtained with ECCI by tilting and rotating the sample in the SEM. This capability enables the selective imaging of specific defects, such as misfit dislocations at the GaP/Si interface, with high contrast levels, which are determined by the standard invisibility criteria. An example application of this technique is described wherein ECCI imaging is used to determine the critical thickness for dislocation nucleation for GaP-on-Si by imaging a range of samples with various GaP epilayer thicknesses. Examples of ECCI micrographs of additional defect types, including threading dislocations and a stacking fault, are provided as demonstration of its broad, TEM-like applicability. Ultimately, the combination of TEM-like capabilities – high spatial resolution and richness of microstructural data – with the convenience and speed of SEM, position ECCI as a powerful tool for the rapid characterization of crystalline materials.
Engineering, Issue 101, Electron channeling contrast imaging, ECCI, electron microscopy, lattice-mismatch, misfit dislocations, semiconductors, heterostructures, rapid characterization