Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.
28 Related JoVE Articles!
A Novel Capsulorhexis Technique Using Shearing Forces with Cystotome
Institutions: Hairmyres Hospital, NHS Lanarkshire, Department of Ophthalmology, South Devon Healthcare NHS Trust.
To demonstrate a capsulorhexis technique using predominantly shearing forces with a cystotome on a virtual reality simulator and on a human eye.
Our technique involves creating the initial anterior capsular tear with a cystotome to raise a flap. The flap left unfolded on the lens surface. The cystotome tip is tilted horizontally and is engaged on the flap near the leading edge of the tear. The cystotome is moved in a circular fashion to direct the vector forces. The loose flap is constantly swept towards the centre so that it does not obscure the view on the tearing edge.
Our technique has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. The capsulorhexis flap is moved away from the tear leading edge allowing better visualisation of the direction of tear. This technique offers superior control of the capsulorhexis by allowing the surgeon to change the direction of the tear to achieve the desired capsulorhexis size.
The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis techniques. The shearing forces technique is a suitable alternative and in some cases a far better technique in achieving the desired capsulorhexis.
JoVE Medicine, Issue 39, Phacoemulsification surgery, cataract surgery, capsulorhexis, capsulotomy, technique, Continuous curvilinear capsulorhexis, cystotome
Making Gynogenetic Diploid Zebrafish by Early Pressure
Institutions: University of Oregon, Fred Hutchinson Cancer Research Center - FHCRC.
Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2
described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3
Developmental Biology, Issue 28, Zebrafish, Early Pressure, Homozygous Diploid, Haploid, Gynogenesis
A Novel Technique of Rescuing Capsulorhexis Radial Tear-out using a Cystotome
Institutions: Hairmyres Hospital, NHS Lanarkshire, Royal Devon and Exeter NHS Foundation Trust, National Institute of Ophthalmology, South Devon Healthcare NHS Trust.
Part 1 : Purpose: To demonstrate a capsulorhexis radial tear out rescue technique using a cystotome on a virtual reality cataract surgery simulator and in a human eye. Part 2 : Method: Steps: When a capsulorhexis begins to veer radially towards the periphery beyond the pupillary margin the following steps should be applied without delay. 2.1) Stop further capsulorhexis manoeuvre and reassess the situation. 2.2) Fill the anterior chamber with ophthalmic viscosurgical device (OVD). We recommend mounting the cystotome to a syringe containing OVD so that the anterior chamber can be reinflated rapidly. 2.3) The capsulorhexis flap is then left unfolded on the lens surface. 2.4) The cystotome tip is tilted horizontally to avoid cutting or puncturing the flap and is engaged on the flap near the leading edge of the tear but not too close to the point of tear. 2.5) Gently push or pull the leading edge of tear opposite to the direction of tear. 2.6) The leading tearing edge will start to do a 'U-Turn'. Maintain the tension on the flap until the tearing edge returns to the desired trajectory. Part 3 : Results: Using our technique, a surgeon can respond instantly to radial tear out without having to change surgical instruments. Changing surgical instruments at this critical stage runs a risk of further radial tear due to sudden shallowing of anterior chamber as a result of forward pressure from the vitreous. Our technique also has the advantage of reducing corneal wound distortion and subsequent anterior chamber collapse. Part 4 : Discussion The EYESI Surgical Simulator is a realistic training platform for surgeons to practice complex capsulorhexis tear-out techniques. Capsulorhexis is the most important and complex part of phacoemulsification and endocapsular intraocular lens implantation procedure. A successful cataract surgery depends on achieving a good capsulorhexis. During capsulorhexis, surgeons may face a challenging situation like a capsulorhexis radial tear-out. A surgeon must learn to tackle the problem promptly without making the situation worse. Some other methods of rescuing the situation have been described using a capsulorhexis forceps. However, we believe our method is quicker, more effective and easier to manipulate as demonstrated on the EYESi surgical simulator and on a human eye. Acknowledgments: List acknowledgements and funding sources. We would like to thank Dr. Wael El Gendy, for video clip. Disclosures: describe potential conflicting interests or state We have nothing to disclose. References: 1. Brian C. Little, Jennifer H. Smith, Mark Packer. J Cataract Refract Surg 2006; 32:1420 1422, Issue-9. 2. Neuhann T. Theorie und Operationstechnik der Kapsulorhexis. Klin Monatsbl Augenheilkd. 1987; 1990: 542-545. 3. Gimbel HV, Neuhann T. Development, advantages and methods of the continuous circular capsulorhexis technique. J Cataract Refract Surg. 1990; 16: 31-37. 4. Gimbel HV, Neuhann T. Continuous curvilinear capsulorhexis. (letter) J Cataract Refract Sur. 1991; 17: 110-111.
Medicine, Issue 47, Phacoemulsification surgery, cataract surgery, capsulorhexis, capsulotomy, technique, Continuous curvilinear capsulorhexis, cystotome, capsulorhexis radial tear, capulorhexis COMPLICATION
Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium
Institutions: Duke University , University of Rochester , University of Rochester .
The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity.
Microcontact printing (μ
CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9
Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 μ
In contrast to traditional printing, inkless μ
CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (<200 nm) features.17-23
However, up till now, inkless μ
CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many μ
CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.
Bioengineering, Issue 58, Soft lithography, microcontact printing, protein arrays, catalytic printing, oxide-free silicon
Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents
Institutions: University of Missouri.
In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5
These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7
While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12
The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13
Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2
Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16
Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.
Bioengineering, Issue 63, optical biosensors, microspheres, surface functionalization
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage
Institutions: SUNY Upstate Medical University, SUNY Upstate Medical University.
Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 μm ± 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 ± 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8
. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.
Medicine, Issue 71, Anatomy, Physiology, Neurobiology, Neuroscience, Immunology, Surgery, Aneurysm, cerebral, hemorrhage, model, mortality, rat, rodent, subarachnoid, vasospasm, animal model
Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip
Institutions: West Virginia University, University of California at Riverside.
Efforts have been focused on developing in vitro
assays for the study of microvessels because in vivo
animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro
microvessel assays have limitations when representing in vivo
microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo
microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.
Bioengineering, Issue 80, Bioengineering, Tissue Engineering, Miniaturization, Microtechnology, Microfluidics, Reflow photoresist, PDMS, Perfusion flow, Primary endothelial cells
Production and Targeting of Monovalent Quantum Dots
Institutions: University of California, San Francisco, University of California, Berkeley, Lawrence Berkeley National Laboratory, University of California, San Francisco, University of California, San Francisco, University of California, San Francisco, University of California, San Francisco.
The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.
Bioengineering, Issue 92, monovalent quantum dots, single particle tracking, SNAP tag, steric exclusion, phosphorothioate, DNA, nanoparticle bioconjugation, single molecule imaging
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro
Institutions: University of Central Florida.
The development of more predictive and biologically relevant in vitro
assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro,
leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.
Bioengineering, Issue 92, cantilever, in vitro, contraction, skeletal muscle, NMJ, cardiomyocytes, functional
Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
Institutions: Oregon Health & Science University, School of Medicine, Oregon Health & Science University, School of Medicine, Oregon Health & Science University, School of Medicine.
We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using “off-the-shelf” microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.
Bioengineering, Issue 86, Label-free optics, quantitative microscopy, cellular biophysics, cell mass, cell volume, cell density
Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g.
changes in redox states and bond lengths). In situ
experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ
on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ
and in situ
samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures
Institutions: La Paz University Hospital, Magnetecs Corp., Geffen School of Medicine at UCLA Los Angeles.
New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.
Medicine, Issue 74, Anatomy, Physiology, Biomedical Engineering, Surgery, Cardiology, catheter ablation, remote navigation, magnetic, robotic, catheter, positioning, electrophysiology, clinical techniques
Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements
Institutions: Joint Bioenergy Institute, Lawrence Berkeley National Laboratory.
is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40–50%), hemicellulose (25–30%), and lignin (20–30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas Mäule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana
model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.
Cellular Biology, Issue 87, Xylem, Fibers, Lignin, polysaccharides, Plant cell wall, Mäule staining, Phloroglucinol, Congo red, Toluidine blue O, Calcofluor white, Cell wall staining methods
A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis
Institutions: University of Nevada, Reno, St. Joseph's Medical Center, University of Rochester Medical Center .
Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e.
, medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroC
n of ischeM
sive to phaR
atment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.
Medicine, Issue 70, Anatomy, Physiology, Cardiology, Myocardial Ischemia, Cardiovascular Diseases, Health Occupations, Health Care, transient myocardial ischemia, Acute Coronary Syndrome, electrocardiogram, ST-segment monitoring, Holter monitoring, research methodology
Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation
Institutions: Piedmont Heart Institute, Medtronic Inc..
Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.
Medicine, Issue 72, Anatomy, Physiology, Cardiology, Biomedical Engineering, Surgery, Cardiovascular System, Cardiovascular Diseases, Surgical Procedures, Operative, Investigative Techniques, Atrial fibrillation, Cryoballoon Ablation, Pulmonary Vein Occlusion, Pulmonary Vein Isolation, electrophysiology, catheterizatoin, heart, vein, clinical, surgical device, surgical techniques
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
In Vivo Microinjection and Electroporation of Mouse Testis
Institutions: Leibniz Institute for Farm Animal Biology (FBN).
This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo
. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo
gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.
Molecular Biology, Issue 90, electroporation, transfection, microinjection, testis, sperm, spermatogenesis, reproduction
Prehospital Thrombolysis: A Manual from Berlin
Institutions: Charité - Universitätsmedizin Berlin, Charité - Universitätsmedizin Berlin, Universitätsklinikum Hamburg - Eppendorf, Berliner Feuerwehr, STEMO-Consortium.
In acute ischemic stroke, time from symptom onset to intervention is a decisive prognostic factor. In order to reduce this time, prehospital thrombolysis at the emergency site would be preferable. However, apart from neurological expertise and laboratory investigations a computed tomography (CT) scan is necessary to exclude hemorrhagic stroke prior to thrombolysis. Therefore, a specialized ambulance equipped with a CT scanner and point-of-care laboratory was designed and constructed. Further, a new stroke identifying interview algorithm was developed and implemented in the Berlin emergency medical services. Since February 2011 the identification of suspected stroke in the dispatch center of the Berlin Fire Brigade prompts the deployment of this ambulance, a stroke emergency mobile (STEMO). On arrival, a neurologist, experienced in stroke care and with additional training in emergency medicine, takes a neurological examination. If stroke is suspected a CT scan excludes intracranial hemorrhage. The CT-scans are telemetrically transmitted to the neuroradiologist on-call. If coagulation status of the patient is normal and patient's medical history reveals no contraindication, prehospital thrombolysis is applied according to current guidelines (intravenous recombinant tissue plasminogen activator, iv rtPA, alteplase, Actilyse).
Thereafter patients are transported to the nearest hospital with a certified stroke unit for further treatment and assessment of strokeaetiology. After a pilot-phase, weeks were randomized into blocks either with or without STEMO care. Primary end-point of this study is time from alarm to the initiation of thrombolysis. We hypothesized that alarm-to-treatment time can be reduced by at least 20 min compared to regular care.
Medicine, Issue 81, Telemedicine, Emergency Medical Services, Stroke, Tomography, X-Ray Computed, Emergency Treatment,[stroke, thrombolysis, prehospital, emergency medical services, ambulance
Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis
Institutions: Bionics Institute, St Vincent's Hospital Melbourne, University of Melbourne, University of Melbourne.
With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.
Medicine, Issue 78, Anatomy, Physiology, Biomedical Engineering, Bioengineering, Surgery, Ophthalmology, Pathology, Tissue Engineering, Prosthesis Implantation, Implantable Neurostimulators, Implants, Experimental, Histology, bionics, Retina, Prosthesis, Bionic Eye, Retinal, Implant, Suprachoroidal, Fixation, Localization, Safety, Preclinical, dissection, embedding, staining, tissue, surgical techniques, clinical techniques
PDMS Device Fabrication and Surface Modification
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, Bioengineering
Title Cell Encapsulation by Droplets
Institutions: Harvard Medical School, Brigham and Women's Hospital, Harvard Medical School, Brigham and Women's Hospital.
Cellular Biology, Issue 8, tissue engineering, microfluidics, ejection, imaging, bioengineering
Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies
Institutions: Ottawa Health Research Institute, Ottawa Health Research Institute.
In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.
Cellular Biology, Issue 11, Springer Protocols, Immunohistochemistry, epidermis, differentiation, keratins, antibody
Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools
Institutions: Brigham and Women's Hospital.
Cellular Biology, Issue 8, microfluidics, diagnostics, capture, blood, HIV, bioengineering
Development of New Therapeutic Applications Using Microfluidics
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 8, Microfluidics, Translational Research, Diagnostics, Bioengineering
Experimental Approaches to Tissue Engineering
Institutions: Brigham and Women's Hospital.
Issue 7, Cell Biology, tissue engineering, microfluidics, stem cells
Cell Capture Using a Microfluidic Device
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 8, microfluidics, cell isolation, diagnostics, Bioengineering