The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.
20 Related JoVE Articles!
A Dual Task Procedure Combined with Rapid Serial Visual Presentation to Test Attentional Blink for Nontargets
Institutions: Dartmouth College.
When viewers search for targets in a rapid serial visual presentation (RSVP) stream, if two targets are presented within about 500 msec of each other, the first target may be easy to spot but the second is likely to be missed. This phenomenon of attentional blink (AB) has been widely studied to probe the temporal capacity of attention for detecting visual targets. However, with the typical procedure of AB experiments, it is not possible to examine how the processing of non-target items in RSVP may be affected by attention. This paper describes a novel dual task procedure combined with RSVP to test effects of AB for nontargets at varied stimulus onset asynchronies (SOAs). In an exemplar experiment, a target category was first displayed, followed by a sequence of 8 nouns. If one of the nouns belonged to the target category, participants would respond ‘yes’ at the end of the sequence, otherwise participants would respond ‘no’. Two 2-alternative forced choice memory tasks followed the response to determine if participants remembered the words immediately before or after the target, as well as a random word from another part of the sequence. In a second exemplar experiment, the same design was used, except that 1) the memory task was counterbalanced into two groups with SOAs of either 120 or 240 msec and 2) three memory tasks followed the sequence and tested remembrance for nontarget nouns in the sequence that could be anywhere from 3 items prior the target noun position to 3 items following the target noun position. Representative results from a previously published study demonstrate that our procedure can be used to examine divergent effects of attention that not only enhance targets but also suppress nontargets. Here we show results from a representative participant that replicated the previous finding.
Behavior, Issue 94, Dual task, attentional blink, RSVP, target detection, recognition, visual psychophysics
Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy
Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.
optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.
Bioengineering, Issue 82, biophysics, protein unfolding, atomic force microscopy, surface immobilization
Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
Institutions: University of Utah School of Medicine, University of Utah School of Medicine, University of Utah School of Medicine, University of Utah School of Medicine, University of Utah School of Medicine.
Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.
Basic Protocol, Issue 84, genotyping, high-resolution melting analysis (HRMA), PCR, zebrafish, mutation, transgenes
Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior
Institutions: Rutgers University, Rutgers University, Rutgers University, Rutgers University, Rutgers University.
Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.
Behavior, Issue 86, vision for action, vision for perception, motor control, reach, grasp, visuomotor, ventral stream, dorsal stream, illusion, space perception, depth inversion
Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism
Institutions: Washington University in St. Louis, Washington University in St. Louis, Washington University in St. Louis, Washington University in St. Louis, Washington University in St. Louis.
Quantitative cardiac function assessment remains a challenge for physiologists and clinicians. Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself. The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion. This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k
), viscoelasticity/relaxation (c
), and load (xo
). The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data. As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2
, maximum A-V pressure gradient kxo
, load independent index of diastolic function, etc
.) and select the aspect of physiology or pathophysiology to be quantified.
Bioengineering, Issue 91, cardiovascular physiology, ventricular mechanics, diastolic function, mathematical modeling, Doppler echocardiography, hemodynamics, biomechanics
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm
Institutions: Trier University, Trier University.
Selection tasks in which simple stimuli (e.g.
letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.
Behavior, Issue 87, stimulus-response binding, distractor-response binding, response retrieval, distractor inhibition, event file, action control, selection task
Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example
Institutions: The Hebrew University of Jerusalem, Université catholique de Louvain, Université catholique de Louvain.
Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf
) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield Pf
Plant Biology, Issue 92, Osmotic water permeability coefficient, aquaporins, protoplasts, curve fitting, non-instantaneous osmolarity change, volume change time course
Dependence of Laser-induced Breakdown Spectroscopy Results on Pulse Energies and Timing Parameters Using Soil Simulants
Institutions: Alvernia University, Applied Research Associates (ARA), Inc..
The dependence of some LIBS detection capabilities on lower pulse energies (<100 mJ) and timing parameters were examined using synthetic silicate samples. These samples were used as simulants for soil and contained minor and trace elements commonly found in soil at a wide range of concentrations. For this study, over 100 calibration curves were prepared using different pulse energies and timing parameters; detection limits and sensitivities were determined from the calibration curves. Plasma temperatures were also measured using Boltzmann plots for the various energies and the timing parameters tested. The electron density of the plasma was calculated using the full-width half maximum (FWHM) of the hydrogen line at 656.5 nm over the energies tested. Overall, the results indicate that the use of lower pulse energies and non-gated detection do not seriously compromise the analytical results. These results are very relevant to the design of field- and person-portable LIBS instruments.
Chemistry, Issue 79, analytical chemistry, laser research, atomic physics, [LIBS, Laser-induced breakdown spectroscopy, gated and non-gated detection, energy study]
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
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
Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
Institutions: University of Massachusetts Medical School, Broad Institute of Harvard and Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University , Harvard University , Massachusetts Institute of Technology, Harvard Medical School, Massachusetts Institute of Technology.
The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6
. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.
Cellular Biology, Issue 39, Chromosome conformation capture, chromatin structure, Illumina Paired End sequencing, polymer physics.
Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy
Institutions: University of Illinois at Chicago and Rehabilitation Institute of Chicago, Rehabilitation Institute of Chicago.
Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other forms of biofeedback. Previous human-robot interactive systems have required different software commands to be implemented for each robot leading to unnecessary developmental overhead time each time a new system becomes available. For example, when a haptic/graphic virtual reality environment has been coded for one specific robot to provide haptic feedback, that specific robot would not be able to be traded for another robot without recoding the program. However, recent efforts in the open source community have proposed a wrapper class approach that can elicit nearly identical responses regardless of the robot used. The result can lead researchers across the globe to perform similar experiments using shared code. Therefore modular "switching out"of one robot for another would not affect development time. In this paper, we outline the successful creation and implementation of a wrapper class for one robot into the open-source H3DAPI, which integrates the software commands most commonly used by all robots.
Bioengineering, Issue 54, robotics, haptics, virtual reality, wrapper class, rehabilitation robotics, neural engineering, H3DAPI, C++
Creating Objects and Object Categories for Studying Perception and Perceptual Learning
Institutions: Georgia Health Sciences University, Georgia Health Sciences University, Georgia Health Sciences University, Palo Alto Research Center, Palo Alto Research Center, University of Minnesota .
In order to quantitatively study object perception, be it perception by biological systems or by machines, one needs to create objects and object categories with precisely definable, preferably naturalistic, properties1
. Furthermore, for studies on perceptual learning, it is useful to create novel objects and object categories (or object classes
) with such properties2
Many innovative and useful methods currently exist for creating novel objects and object categories3-6
(also see refs. 7,8). However, generally speaking, the existing methods have three broad types of shortcomings.
First, shape variations are generally imposed by the experimenter5,9,10
, and may therefore be different from the variability in natural categories, and optimized for a particular recognition algorithm. It would be desirable to have the variations arise independently of the externally imposed constraints.
Second, the existing methods have difficulty capturing the shape complexity of natural objects11-13
. If the goal is to study natural object perception, it is desirable for objects and object categories to be naturalistic, so as to avoid possible confounds and special cases.
Third, it is generally hard to quantitatively measure the available information in the stimuli created by conventional methods. It would be desirable to create objects and object categories where the available information can be precisely measured and, where necessary, systematically manipulated (or 'tuned'). This allows one to formulate the underlying object recognition tasks in quantitative terms.
Here we describe a set of algorithms, or methods, that meet all three of the above criteria. Virtual morphogenesis (VM) creates novel, naturalistic virtual 3-D objects called 'digital embryos' by simulating the biological process of embryogenesis14
. Virtual phylogenesis (VP) creates novel, naturalistic object categories by simulating the evolutionary process of natural selection9,12,13
. Objects and object categories created by these simulations can be further manipulated by various morphing methods to generate systematic variations of shape characteristics15,16
. The VP and morphing methods can also be applied, in principle, to novel virtual objects other than digital embryos, or to virtual versions of real-world objects9,13
. Virtual objects created in this fashion can be rendered as visual images using a conventional graphical toolkit, with desired manipulations of surface texture, illumination, size, viewpoint and background. The virtual objects can also be 'printed' as haptic objects using a conventional 3-D prototyper.
We also describe some implementations of these computational algorithms to help illustrate the potential utility of the algorithms. It is important to distinguish the algorithms from their implementations. The implementations are demonstrations offered solely as a 'proof of principle' of the underlying algorithms. It is important to note that, in general, an implementation of a computational algorithm often has limitations that the algorithm itself does not have.
Together, these methods represent a set of powerful and flexible tools for studying object recognition and perceptual learning by biological and computational systems alike. With appropriate extensions, these methods may also prove useful in the study of morphogenesis and phylogenesis.
Neuroscience, Issue 69, machine learning, brain, classification, category learning, cross-modal perception, 3-D prototyping, inference
Adaptation of a Haptic Robot in a 3T fMRI
Institutions: University of California, University of California, University of California.
Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1
with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2
, and nearly real-time analyses 3
. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4
allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6
, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of ˜380 to ˜330, and ˜250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (˜2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6
Bioengineering, Issue 56, neuroscience, haptic robot, fMRI, MRI, pointing
Morphometric Analyses of Retinal Sections
Institutions: The University of Hong Kong, The University of Hong Kong, The University of Hong Kong.
Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)–induced excitotoxicity1
, retinal ischemia-reperfusion injury2
. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4
. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6
. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience — MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.
Neuroscience, Issue 60, morphometric analysis, retina, thickness, cell size, Stereo Investigator, neuroscience
Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers
Institutions: University of Michigan , University of Michigan .
Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2
, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4
. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs.
The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6,
(see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension.
Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces.
We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8
. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.
Bioengineering, Issue 56, Genetics, DNA stretching, DNA, Axial Optical Tweezers, Single-Molecule Biophysics, Biophysics
Echo Particle Image Velocimetry
Institutions: University of New Hampshire.
The transport of mass, momentum, and energy in fluid flows is ultimately determined by spatiotemporal distributions of the fluid velocity field.1
Consequently, a prerequisite for understanding, predicting, and controlling fluid flows is the capability to measure the velocity field with adequate spatial and temporal resolution.2
For velocity measurements in optically opaque fluids or through optically opaque geometries, echo particle image velocimetry (EPIV) is an attractive diagnostic technique to generate "instantaneous" two-dimensional fields of velocity.3,4,5,6
In this paper, the operating protocol for an EPIV system built by integrating a commercial medical ultrasound machine7
with a PC running commercial particle image velocimetry (PIV) software8
is described, and validation measurements in Hagen-Poiseuille (i.e.
, laminar pipe) flow are reported.
For the EPIV measurements, a phased array probe connected to the medical ultrasound machine is used to generate a two-dimensional ultrasound image by pulsing the piezoelectric probe elements at different times. Each probe element transmits an ultrasound pulse into the fluid, and tracer particles in the fluid (either naturally occurring or seeded) reflect ultrasound echoes back to the probe where they are recorded. The amplitude of the reflected ultrasound waves and their time delay relative to transmission are used to create what is known as B-mode (brightness mode) two-dimensional ultrasound images. Specifically, the time delay is used to determine the position of the scatterer in the fluid and the amplitude is used to assign intensity to the scatterer. The time required to obtain a single B-mode image, t
, is determined by the time it take to pulse all the elements of the phased array probe. For acquiring multiple B-mode images, the frame rate of the system in frames per second (fps) = 1/δt
. (See 9 for a review of ultrasound imaging.)
For a typical EPIV experiment, the frame rate is between 20-60 fps, depending on flow conditions, and 100-1000 B-mode images of the spatial distribution of the tracer particles in the flow are acquired. Once acquired, the B-mode ultrasound images are transmitted via an ethernet connection to the PC running the PIV commercial software. Using the PIV software, tracer particle displacement fields, D(x,y)
[pixels], (where x and y denote horizontal and vertical spatial position in the ultrasound image, respectively) are acquired by applying cross correlation algorithms to successive ultrasound B-mode images.10
The velocity fields, u(x,y)
[m/s], are determined from the displacements fields, knowing the time step between image pairs, ΔT
[s], and the image magnification, M
. The time step between images ΔT
= 1/fps + D(x,y)/B
, where B
[pixels/s] is the time it takes for the ultrasound probe to sweep across the image width. In the present study, M = 77[μm/pixel], fps
= 49.5[1/s], and B
= 25,047[pixels/s]. Once acquired, the velocity fields can be analyzed to compute flow quantities of interest.
Mechanical Engineering, Issue 70, Physics, Engineering, Physical Sciences, Ultrasound, cross correlation, velocimetry, opaque fluids, particle, flow, fluid, EPIV
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
Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Institutions: University of California, Los Angeles.
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Cellular Biology, Issue 5, mosquito, malaria, popuulation, replacement, modeling, infectious disease