Name: surgery and sample processing for correlative imaging of the murine pulmonary valve
Uploaded: 2021-08-05T14:00:00
Description: Watch this Scientific Journal Video about Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve at JoVE.com

This work is supported, in part, by R01HL139796 and R01HL128847 grants to CKB and RO1DE028297 and CBET1608058 for DWM.

The use of animals in this study was in accordance with Nationwide Children&#39;s Hospital institutional animal care and use committee under protocol AR13-00030.
1. Pulmonary valve excision
<ol>
	<li>Autoclave the necessary tools needed for the mouse dissection. This includes fine scissors, micro forceps, micro vascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors.</li>
	<li>Acclimate all mice for a minimum of 2 weeks before the operation. Remove C57BL...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aortic+valve+microstructure:+Effects+of+transvalvular+pressure.">The aortic valve microstructure: Effects of transvalvular pressure.</a> Journal of Biomedical Materials Research. 41 (1), 131-141 (1998).</li><li>Ayoub, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+biomechanics+and+underlying+mechanobiology.">Heart valve biomechanics and underlying mechanobiology.</a> Comprehensive Physiology. 6 (4), 1743-1780 (2016).</li><li>Stella, J. A., Liao, J., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+biaxial+mechanical+behavior+of+the+aortic+heart+valve+leaflet.">Time-dependent biaxial mechanical behavior of the aortic heart valve leaflet.</a> Journal of Biomechanics. 40 (14), 3169-3177 (2007).</li><li>Korn, E. D., Weisman, R. A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=loss+of+lipids+during+preparation+of+amoebae+for+electron+microscopy.">loss of lipids during preparation of amoebae for electron microscopy.</a> Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism. 116 (2), 309-316 (1966).</li><li>Tapia, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-contrast+en+bloc+staining+of+neuronal+tissue+for+field+emission+scanning+electron+microscopy.">High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy.</a> Nature Protocols. 7 (2), 193-206 (2012).</li><li>Hinton, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+heart+valve+structure+and+function:+Echocardiographic+and+morphometric+analyses+from+the+fetus+through+the+aged+adult.">Mouse heart valve structure and function: Echocardiographic and morphometric analyses from the fetus through the aged adult.</a> American Journal of Physiology - Heart and Circulatory Physiology. 294 (6), 2480-2488 (2008).</li><li>Denk, W., Horstmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+to+reconstruct+three-dimensional+tissue+nanostructure.">Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.</a> Plos Biology. 2 (11), 1900-1909 (2004).</li><li>Lincoln, J., Florer, J. B., Deutsch, G. H., Wenstrup, R. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ColVa1+and+ColXIa1+are+required+for+myocardial+morphogenesis+and+heart+valve+development.">ColVa1 and ColXIa1 are required for myocardial morphogenesis and heart valve development.</a> Developmental Dynamics. 235 (12), 3295-3305 (2006).</li><li>Hamatani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+investigation+of+congenital+bicuspid+aortic+valve+stenosis,+compared+with+atherosclerotic+tricuspid+aortic+valve+stenosis+and+congenital+bicuspid+aortic+valve+regurgitation.">Pathological investigation of congenital bicuspid aortic valve stenosis, compared with atherosclerotic tricuspid aortic valve stenosis and congenital bicuspid aortic valve regurgitation.</a> PLoS One. 11 (8), (2016).</li></ol>

Removal of the ventricles serves two purposes. First, exposing the ventricle side to the atmospheric pressure, thereby only needing to apply a transvalvular pressure from the arterial side of the pulmonary valve to close, and second, providing a stable base to prevent twisting of the pulmonary trunk. During pressurization, the pulmonary trunk distends radially and inferiorly, making it prone to twisting, causing the collapse of the pulmonary trunk. Preloading the pulmonary valve with a saline solution offers an additiona...

The pulmonary valve (PV) serves to ensure unidirectional blood flow between the right ventricle and the pulmonary artery. Pulmonary valve malformations are associated with several forms of congenital heart disease. The current treatment for congenital heart valve disease (HVD) is valvular repair or valve replacement, which can necessitate multiple invasive surgeries throughout a patient&#39;s lifetime1. It has been widely accepted that the function of the heart valve is derived from its structure, often referred to as the structure-function correlate. More specifically, the geometric and biomechanical properties of the heart dictate its function. The mechanical properties, in turn, are determined by the composition and organization of the ECM. By developing a method for determining the biomechanical properties of murine heart valves, transgenic animal models can be used to interrogate the role of the ECM on heart valve function and dysfunction2,3,4,5.
The murine animal model has long been regarded as the standard for molecular studies because transgenic models are more readily available in mice compared to other species. Murine transgenic models provide a versatile platform for researching heart valve-related diseases6. However, the surgical expertise and instrumentation requirements to characterize both the geometry and ECM organization have been a major hurdle in progressing HVD research. Hstological data in literature provides a picture into murine heart valve extracellular matrix content, but only in the form of 2D images, and are unable to describe its 3D architecture7,8. Additionally, the heart valve is both spatially and temporally heterogeneous, making it difficult to draw conclusions across experiments regarding ECM organization if the sampling and conformation are not fixed. Conventional 3D characterization methods, such as MRI or 3D echocardiography, do not provide the resolution necessary to resolve ECM components9,10.
This work details a fully correlative workflow where the temporal heterogeneity due to the cardiac cycle was addressed by fixing the conformation of the murine PV with hydrostatic transvalvular pressure. The spatial heterogeneity was controlled precisely by sampling regions of interest and registering data sets from different imaging modalities, specifically &#181;CT and serial block face scanning electron microscopy, across different length scales. This method of scouting with &#181;CT for guiding downstream sampling has been proposed previously, but because the pulmonary valve exhibits temporal variation, an additional level of control was needed on the surgical level11.
In vivo studies describing murine heart valve biomechanics are sparse and, instead, rely on computational models when describing the deformation behavior. It is of critical importance that local extracellular data on the nanometer length scale be related to the geometry and location of the heart valve. This, in turn, provides quantifiable, spatially mapped distributions of mechanically contributing ECM proteins, which can be used to reinforce existing biomechanical heart valve models12,13,14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25% glutaraldehyde (aq)</td>
			<td>EMS</td>
			<td>16210</td>
			<td>Primary fixative component</td>
		</tr>
		<tr>
			<td>0.9% sodium chloride injection</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-4888-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>EMS</td>
			<td>15055</td>
			<td></td>
		</tr>
		<tr>
			<td>22G needle</td>
			<td>BD</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Smiths Medical ASD, Inc.</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr>
			<td>4% osmium tetroxide</td>
			<td>EMS</td>
			<td>19150</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde (aq)</td>
			<td>EMS</td>
			<td>157-4-100</td>
			<td>Primary fixative component</td>
		</tr>
		<tr>
			<td>Absorbable hemostat</td>
			<td>Ethicon</td>
			<td>1961</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMS</td>
			<td>10012</td>
			<td></td>
		</tr>
		<tr>
			<td>Black polyamide monofilament suture, 10-0</td>
			<td>AROSurgical instruments Corporation</td>
			<td>TI38402</td>
			<td></td>
		</tr>
		<tr>
			<td>Black polyamide monofilament suture, 6-0</td>
			<td>AROSurgical instruments Corporation</td>
			<td>SN-1956</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td>Approximately 1 yo</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp applying forcep</td>
			<td>FST</td>
			<td>00072-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tip applicators</td>
			<td>Fisher Scientific</td>
			<td>23-400-118</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forcep</td>
			<td>FST</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 forceps</td>
			<td>FST</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 fine forcep</td>
			<td>FST</td>
			<td>11274-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Durcupan ACM resin</td>
			<td>EMS</td>
			<td>14040</td>
			<td>For embedding</td>
		</tr>
		<tr>
			<td>Fine scissor</td>
			<td>FST</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heliscan microCT</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Micro-CT</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-2053</td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Sigma-Aldrich</td>
			<td>56-84-8</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>EMS</td>
			<td>17900</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>low-vacuum backscatter detector</td>
			<td>Thermo Fisher Scientific</td>
			<td>VSDBS</td>
			<td>SEM backscatter detector</td>
		</tr>
		<tr>
			<td>Micro-adson forcep</td>
			<td>FST</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP filter, 0.22 um, PES 33mm, non-sterile</td>
			<td>EMD Millipore</td>
			<td>SLGP033NS</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-woven songes</td>
			<td>McKesson Corp.</td>
			<td>94442000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate(II) trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>14459-95-1</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>1310-58-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure monitor line</td>
			<td>Smiths Medical ASD, Inc.</td>
			<td>MX562</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution (sterile 0.9% sodium chloride)</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-0138-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Size 3 BEEM capsule</td>
			<td>EMS</td>
			<td>69910-01</td>
			<td>Embedding container</td>
		</tr>
		<tr>
			<td>Sodium cacodylate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>6131-99-3</td>
			<td>Buffer</td>
		</tr>
		<tr>
			<td>Solibri retractors</td>
			<td>FST</td>
			<td>17000-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter, carbon and e-beam coater</td>
			<td>Leica</td>
			<td>EM ACE600</td>
			<td>Gold coater</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide (TCH)</td>
			<td>EMS</td>
			<td>21900</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Tish needle holder/forcep</td>
			<td>Micrins</td>
			<td>MI1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Trimmer</td>
			<td>Wahl</td>
			<td>9854-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>EMS</td>
			<td>22400</td>
			<td>Staining component</td>
		</tr>
		<tr>
			<td>Volumescope scanning electron microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td>VOLUMESCOPESEM</td>
			<td>Serial Block Face Scanning Electron Microscope</td>
		</tr>
		<tr>
			<td>Xylazine sterile solution</td>
			<td>Akorn Inc.</td>
			<td>NADA# 139-236</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgery and sample processing for correlative imaging of the murine pulmonary valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Anastomosis of the pulmonary artery to the pressurization tubing is shown in Figure 1A. Following the application of hydrostatic pressure, the pulmonary trunk distends radially (Figure 1B) indicating that the pulmonary valve leaflets are in a closed configuration. Pulmonary valve conformation was confirmed by &#181;CT. In this case, the leaflets were coapt (closed) and the annulus was circular (Figure 2A). Figur...

Watch this Scientific Journal Video about Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve at JoVE.com

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

This protocol is significant because it's designed to answer questions about the structure function correlate of the murine pulmonary valve, which is generally tricky because of its inherent heterogeneity. The controlled fixation and correlative imaging were used to capture the structure on multiple length scales. Local high-resolution images can then be mapped back to the precise anatomical location on the pulmonary valve.Demonstrating the procedure will be Dr.Tai Yi, the Director of Microsurgery in the Center of Regenerative Medicine at Nationwide Children's Hospital. Begin by autoclaving the tools needed for the mouse dissection, including fine scissors, microforceps, microvascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors. After euthanizing an adult C57BL/6 mouse, place it in a dorsal recumbent position on a tray, secure its limbs with tape and perform the thoracotomy.Expose the heart by removing any excess adipose tissue and fascia, then remove the right atrium and perfuse the left ventricle with room temperature saline solution. Remove the entire heart by severing the superior vena cava, inferior vena cava, pulmonary artery, and aorta, and cut approximately two millimeters above the ventriculoatrial junction, which will serve as the conduit for pressurization. Remove the left and right ventricles to expose the chambers to atmospheric pressure, ensuring that the pulmonary trunk structure is unaffected by removing the ventricles.Anastomose pressurization tubing with the pulmonary artery, leaving approximately one millimeter distance between the sinotubular junction and the end of the tubing to accommodate for large movements of the leaflets and pulmonary trunk. Elevate the reservoir to an analogous physiological pressure and fill it with the saline solution. Test the flow-through system to ensure there are no blockages or air bubbles.Attach a stopcock to the anastomosed pulmonary valve and ensure adequate flow through the tubing by switching the outflow tract. Once the flow is sufficient, switch the outflow to the anastomosed pulmonary valve and ensure pressurization of the pulmonary trunk identified by trunk distension. After confirming the pressurization of the pulmonary trunk, gradually incorporate primary fixative solution until 25%of the reservoir capacity of the saline solution is purged.Place a fixative-soaked gauze over the tissue sample to prevent drying. Perfuse the fixative for three hours, refilling the reservoir to maintain a constant pressure. After perfusion, store the heart valve in the fixative solution at four degrees Celsius until use for up to one week.For staining, wash the fixed heart valve sample three times with cold 0.15 molar cacodylate buffer for five minutes and completely submerge the heart valve in a solution of 1.5 potassium ferrocyanide, 0.15 molar cacodylate, two millimolar calcium chloride, and 2%osmium tetroxide on ice for one hour. Wash the samples with room temperature double distilled water by placing them in a tube for five minutes with slight agitation. After three more washes, place the sample in filtered thiocarbohydrazide solution at room temperature.After 20 minutes, wash the sample three times with water as demonstrated earlier. Next, place the sample in 2%osmium tetroxide at room temperature. After 30 minutes, wash it with water three times for five minutes each and incubate the sample in 1%uranyl acetate overnight at four degrees Celsius.Meanwhile, prepare a solution of lead nitrate. On the following day, perform the washing step three times as demonstrated earlier, then incubate the heart valve tissue in lead aspartate solution at 60 degrees Celsius for 30 minutes. Wash the samples three more times, then perform a serial dehydration treatment on the heart valve tissues with freshly prepared 20, 50, 70, and 90%ethanol on ice for five minutes each, followed by two subsequent treatments with 100%ethanol.After dehydration, move the tissues to ice cold acetone, then place it in fresh acetone at room temperature for 10 minutes. For embedding, make the resin mixture according to the manufacturer's specifications. Place tissues in subsequent treatments of 25 to 75, 50 to 50 and 75 to 25 resin to acetone mixture for two hours each.Finally, place the tissues in 100%resin overnight. On the following day, place tissues in fresh 100%resin. After two hours, transfer the tissues to an embedding capsule, add fresh 100%resin and place it in a 60 degree Celsius oven for 48 hours to cure.The excised anastomosed pulmonary artery before and following the application of hydrostatic pressure is shown here. The pulmonary trunk distends radially indicating that the pulmonary valve leaflets are in a closed configuration. Microcomputed tomography confirmed the pulmonary valve confirmation.The leaflets were closed and the annulus was circular while varying degrees of inadequate pulmonary valve pressurization by either fixation or arterial collapse were observed. The micro CT volume rendering virtual cross-sections was correlated with optical images to confirm the slicing direction and location. High-resolution SBF SEM images were taken at a local region within a leaflet when the specimen block was at the desired location and orientation.Correlated images between the micro CT volume rendering virtual slice, low-resolution, and high-resolution SBF SEM images are shown here. In 3D representation of a segmented region of the pulmonary valve, extracellular components like endothelial cells, valvular interstitial cells, and extracellular fibers can be identified. When attempting this protocol, please keep in mind that performing the pressurization is critical and will ensure the yield to be as high as possible.

surgery-sample-processing-for-correlative-imaging-murine-pulmonary

Research

JoVE Journal

Engineering

Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 &deg;C), flammable, and volatile organic electrolytes. These organic based electrolyte systems are viable at ambient temperatures, but require a cooling system to ensure that temperatures do not exceed 80 &deg;C. These cooling systems tend to increase battery costs and can malfunction which can lead to battery malfunction and explosions, thus endangering human life. Increases in petroleum prices lead to a huge demand for safe, electric hybrid vehicles that are more economically viable to operate as oil prices continue to rise. Existing organic based electrolytes used in lithium ion batteries are not applicable to high temperature automotive applications. A safer alternative to organic electrolytes is solid polymer electrolytes. This work will highlight the synthesis for a graft copolymer electrolyte (GCE) poly(oxyethylene) methacrylate (POEM) to a block with a lower glass transition temperature (Tg) poly(oxyethylene) acrylate (POEA). The conduction mechanism has been discussed and it has been demonstrated the relationship between polymer segmental motion and ionic conductivity indeed has a Vogel-Tammann-Fulcher (VTF) dependence. Batteries containing commercially available LP30 organic (LiPF6 in ethylene carbonate (EC):dimethyl carbonate (DMC) at a 1:1 ratio) and GCE were cycled at ambient temperature. It was found that at ambient temperature, the batteries containing GCE showed a greater overpotential when compared to LP30 electrolyte. However at temperatures greater than 60 &deg;C, the GCE cell exhibited much lower overpotential due to fast polymer electrolyte conductivity and nearly the full theoretical specific capacity of 170 mAh/g was accessed.

Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (&gt;120 &deg;C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes.

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 ...

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Fast Imaging Technique to Study Drop Impact Dynamics of Non-Newtonian Fluids

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for detailed observation, scenarios that make it challenging to observe with conventional imaging systems. One of these is the drop impact of liquids, which usually happens within one tenth of millisecond. To tackle this challenge, a fast imaging technique is introduced that combines a high-speed camera (capable of up to one million frames per second) with a macro lens with long working distance to bring the spatial resolution of the image down to 10 &#181;m/pixel. The imaging technique enables precise measurement of relevant fluid dynamic quantities, such as the flow field, the spreading distance and the splashing speed, from analysis of the recorded video. To demonstrate the capabilities of this visualization system, the impact dynamics when droplets of non-Newtonian fluids impinge on a flat hard surface are characterized. Two situations are considered: for oxidized liquid metal droplets we focus on the spreading behavior, and for densely packed suspensions we determine the onset of splashing. More generally, the combination of high temporal and spatial imaging resolution introduced here offers advantages for studying fast dynamics across a wide range of microscale phenomena.

Drop impact of non-Newtonian fluids is a complex process since different physical parameters influence the dynamics over a very short time (less than one tenth of a millisecond). A fast imaging technique is introduced in order to characterize the impact behaviors of different non-Newtonian fluids.

In the field of fluid mechanics, many dynamical processes not only occur over a very short time interval but also require high spatial resolution for ...

Electron Channeling Contrast Imaging for Rapid III-V Heteroepitaxial Characterization

Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging (ECCI) in a scanning electron microscope (SEM). ECCI allows for imaging of defects and crystallographic features under specific diffraction conditions, similar to that possible via plan-view transmission electron microscopy (PV-TEM). A particular advantage of the ECCI technique is that it requires little to no sample preparation, and indeed can use large area, as-produced samples, making it a considerably higher throughput characterization method than TEM. Similar to TEM, different diffraction conditions can be obtained with ECCI by tilting and rotating the sample in the SEM. This capability enables the selective imaging of specific defects, such as misfit dislocations at the GaP/Si interface, with high contrast levels, which are determined by the standard invisibility criteria. An example application of this technique is described wherein ECCI imaging is used to determine the critical thickness for dislocation nucleation for GaP-on-Si by imaging a range of samples with various GaP epilayer thicknesses. Examples of ECCI micrographs of additional defect types, including threading dislocations and a stacking fault, are provided as demonstration of its broad, TEM-like applicability. Ultimately, the combination of TEM-like capabilities &#8211; high spatial resolution and richness of microstructural data &#8211; with the convenience and speed of SEM, position ECCI as a powerful tool for the rapid characterization of crystalline materials.

The use of electron channeling contrast imaging in a scanning electron microscope to characterize defects in III-V/Si heteroexpitaxial thin films is described. This method yields similar results to plan-view transmission electron microscopy, but in significantly less time due to lack of required sample preparation.

Misfit dislocations in heteroepitaxial layers of GaP grown on Si(001) substrates are characterized through use of electron channeling contrast imaging ...

Use of a Multi-compartment Dynamic Single Enzyme Phantom for Studies of Hyperpolarized Magnetic Resonance Agents

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due to fundamental constraints imposed by the hyperpolarized state, exotic imaging and reconstruction techniques are commonly used. A practical system for characterization of dynamic, multi-spectral imaging methods is critically needed. Such a system must reproducibly recapitulate the relevant chemical dynamics of normal and pathological tissues. The most widely utilized substrate to date is hyperpolarized [1-13C]-pyruvate for assessment of cancer metabolism. We describe an enzyme-based phantom system that mediates the conversion of pyruvate to lactate. The reaction is initiated by injection of the hyperpolarized agent into multiple chambers within the phantom, each of which contains varying concentrations of reagents that control the reaction rate. Multiple compartments are necessary to ensure that imaging sequences faithfully capture the spatial and metabolic heterogeneity of tissue. This system will aid the development and validation of advanced imaging strategies by providing chemical dynamics that are not available from conventional phantoms, as well as control and reproducibility that is not possible in vivo.

A multi-compartment dynamic phantom is used to simulate some biology of interest for metabolic studies using hyperpolarized magnet resonance agents.

Imaging of hyperpolarized substrates by magnetic resonance shows great clinical promise for assessment of critical biochemical processes in real time. Due ...

Wicking Tests for Unidirectional Fabrics: Measurements of Capillary Parameters to Evaluate Capillary Pressure in Liquid Composite Molding Processes

During impregnation of a fibrous reinforcement in liquid composite molding (LCM) processes, capillary effects have to be understood in order to identify their influence on void formation in composite parts. Wicking in a fibrous medium described by the Washburn equation was considered equivalent to a flow under the effect of capillary pressure according to the Darcy law. Experimental tests for the characterization of wicking were conducted with both carbon and flax fiber reinforcement. Quasi-unidirectional fabrics were then tested by means of a tensiometer to determine the morphological and wetting parameters along the fiber direction. The procedure was shown to be promising when the morphology of the fabric is unchanged during capillary wicking. In the case of carbon fabrics, the capillary pressure can be calculated. Flax fibers are sensitive to moisture sorption and swell in water. This phenomenon has to be taken into account to assess the wetting parameters. In order to make fibers less sensitive to water sorption, a thermal treatment was carried out on flax reinforcements. This treatment enhances fiber morphological stability and prevents swelling in water. It was shown that treated fabrics have a linear wicking trend similar to those found in carbon fabrics, allowing for the determination of capillary pressure.

An experimental method to measure geometrical parameters and the apparent advancing contact angles describing capillary wicking in unidirectional synthetic and natural fabrics is proposed. These parameters are mandatory for the determination of the capillary pressures that must be taken into account for liquid composite molding (LCM) applications.

During impregnation of a fibrous reinforcement in liquid composite molding (LCM) processes, capillary effects have to be understood in order to identify ...

Processing of Bulk Nanocrystalline Metals at the US Army Research Laboratory

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued development of nanocrystalline metals. Despite these efforts, the transition of these materials from the lab bench to actual applications has been blocked by the inability to produce large scale parts that retain the desired nanocrystalline microstructures. Following the development of a method proven to stabilize the nanosized grain structure to temperatures approaching that of the melting point for the given metal, the US Army Research Laboratory (ARL) has progressed to the next stage in the development of these materials - namely the production of large scale parts suitable for testing and evaluation in a range of relevant test environments. This report provides a broad overview of the ongoing efforts in the processing, characterization, and consolidation of these materials at ARL. In particular, focus is placed on the methodology used for producing the nanocrystalline metal powders, in both small and large-scale amounts, that are at the center of ongoing research efforts.

This paper provides a brief overview of the ongoing efforts at the Army Research Laboratory on the processing of bulk nanocrystalline metals with an emphasis on the methodologies used for the production of the novel metal powders.

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued ...

Guidelines and Experience Using Imaging Biomarker Explorer (IBEX) for Radiomics

Imaging Biomarker Explorer (IBEX) is an open-source tool for medical imaging radiomics work. The purpose of this paper is to describe how to use IBEX's graphical user interface (GUI) and to demonstrate how IBEX calculated features have been used in clinical studies. IBEX allows for the import of DICOM images with DICOM radiation therapy structure files or Pinnacle files. Once the images are imported, IBEX has tools within the Data Selection GUI to manipulate the viewing of the images, measure voxel values and distances, and create and edit contours. IBEX comes with 27 preprocessing and 132 feature choices to design feature sets. Each preprocessing and feature category has parameters that can be altered. The output from IBEX is a spreadsheet that contains: 1) each feature from the feature set calculated for each contour in a data set, 2) image information about each contour in a data set, and 3) a summary of the preprocessing and features used with their selected parameters. Features calculated from IBEX have been used in studies to test the variability of features under different imaging conditions and in survival models to improve current clinical models.

Guidelines and Experience Using Imaging Biomarker Explorer IBEX for Radiomics

We describe IBEX, an open-source tool designed for medical imaging radiomics studies, and how to use this tool. In addition, some published works that have used IBEX for uncertainty analysis and model building are showcased.

Imaging Biomarker Explorer (IBEX) is an open-source tool for medical imaging radiomics work. The purpose of this paper is to describe how to use IBEX's ...

Fused Filament Fabrication (FFF) of Metal-Ceramic Components

Technical ceramics are widely used for industrial and research applications, as well as for consumer goods. Today, the demand for complex geometries with diverse customization options and favorable production methods is increasing continuously. With fused filament fabrication (FFF), it is possible to produce large and complex components quickly with high material efficiency. In FFF, a continuous thermoplastic filament is melted in a heated nozzle and deposited below. The computer-controlled print head is moved in order to build up the desired shape layer by layer. Investigations regarding printing of metals or ceramics are increasing more and more in research and industry. This study focuses on additive manufacturing (AM) with a multi-material approach to combine a metal (stainless steel) with a technical ceramic (zirconia: ZrO2). Combining these materials offers a broad variety of applications due to their different electrical and mechanical properties. The paper shows the main issues in preparation of the material and feedstock, device development, and printing of these composites.

Fused Filament Fabrication FFF of Metal-Ceramic Components

This study shows multi-material additive manufacturing (AM) using fused filament fabrication (FFF) of stainless steel and zirconia.

Technical ceramics are widely used for industrial and research applications, as well as for consumer goods. Today, the demand for complex geometries with ...

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

We present a protocol for the real-time optical detection of single unlabeled proteins as they are secreted from living cells. This is based on interferometric scattering (iSCAT) microscopy, which can be applied to a variety of different biological systems and configurations.

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living ...