Name: measuring the densities of aqueous glasses at cryogenic temperatures
Uploaded: 2017-06-28T14:00:00
Description: Watch this Scientific Journal Video about Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures at JoVE.com

This work was supported by the NSF under award MCB-1330685. DWM acknowledges partial support from Cornell University&#39;s Molecular Biophysics Training Grant (NIH T32GM0082567).

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Please use all appropriate safety practices when using compressed gases, including appropriate calibrated gas handling regulators and valves, and approved gas tubing. Contact with liquid cryogens can cause&#160; severe frostbite and necrosis. Use appropriate personal protective equipment (face shield, gloves, lab coat, full length pants, closed-toe shoes), all of which must be impermeable to liquid nitrogen. Remain standing and ensure an ...

<ol>
	<li>Fahy, G. M., Wowk, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+Cryopreservation+by+Vitrification.">Principles of Cryopreservation by Vitrification.</a> Cryopreservation and Freeze-Drying Protocols. , 21-82 (2015).</li><li>Nagy, Z. P., Nel-Themaat, L., Chang, C. -. C., Shapiro, D. B., Berna, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+eggs.">Cryopreservation of eggs.</a> Human Fertility: Methods and Protocols. , 439-454 (2014).</li><li>Kriminski, S., Caylor, C. L., Nonato, M. C., Finkelstein, K. D., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+cooling+and+annealing+of+protein+crystals.">Flash cooling and annealing of protein crystals.</a> Acta Cryst Sect D. 58 (3), 459-471 (2002).</li><li>Juers, D. H., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+lattice+repacking+illustrates+the+temperature+dependence+of+macromolecular+interactions.">Reversible lattice repacking illustrates the temperature dependence of macromolecular interactions.</a> J Mol Biol. 311 (4), 851-862 (2001).</li><li>Juers, D. H., Matthews, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-cooling+in+macromolecular+crystallography:+advantages,+disadvantages+and+optimization.">Cryo-cooling in macromolecular crystallography: advantages, disadvantages and optimization.</a> Q Rev Biophys. 37 (2), 105-119 (2004).</li><li>Alcorn, T., Juers, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+rational+methods+of+cryoprotection+in+macromolecular+crystallography.">Progress in rational methods of cryoprotection in macromolecular crystallography.</a> Acta Cryst Sect D. 66 (4), 366-373 (2010).</li><li>Warkentin, M., Sethna, J., Thorne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Droplet+Theory+Explains+the+Glass+Formability+of+Aqueous+Solutions.">Critical Droplet Theory Explains the Glass Formability of Aqueous Solutions.</a> Phys Rev Lett. 110 (1), 15703 (2013).</li><li>Kriminski, S., Kazmierczak, M., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+transfer+from+protein+crystals:+implications+for+flash-cooling+and+X-ray+beam+heating.">Heat transfer from protein crystals: implications for flash-cooling and X-ray beam heating.</a> Acta Cryst Sect D. 59 (4), 697-708 (2003).</li><li>Loerting, T., Bauer, M., Kohl, I., Watschinger, K., Winkel, K., Mayer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoflotation:+Densities+of+amorphous+and+crystalline+ices.">Cryoflotation: Densities of amorphous and crystalline ices.</a> J Phys Chem B. 115 (48), 14167-14175 (2011).</li><li>Shen, C., Julius, E. F., Tyree, T. J., Moreau, D. W., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+contraction+of+aqueous+glycerol+and+ethylene+glycol+solutions+for+optimized+protein-crystal+cryoprotection+Thermal+contraction+of+aqueous+glycerol+and+ethylene+glycol+solutions+for+optimized+protein-crystal+cryoprotection.">Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection.</a> Acta Cryst Sect D. 72 (6), 742-752 (2016).</li><li>Warkentin, M., Berejnov, V., Husseini, N. S., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperquenching+for+protein+cryocrystallography.">Hyperquenching for protein cryocrystallography.</a> J Appl Cryst. 39 (6), 805-811 (2006).</li><li>McFerrin, M. B., Snell, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+application+of+a+method+to+quantify+the+quality+of+cryoprotectant+solutions+using+standard+area-detector+X-ray+images.">The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images.</a> J Appl Cryst. 35 (5), 538-545 (2002).</li><li>Chinte, U., Shah, B., DeWitt, K., Kirschbaum, K., Pinkerton, A. A., Schall, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size:+An+important+parameter+in+flash-cooling+macromolecular+crystallization+solutions.">Sample size: An important parameter in flash-cooling macromolecular crystallization solutions.</a> J. Appl. Cryst. 38 (3), 412-419 (2005).</li><li>Bosart, L. W., Snoddy, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+gravity+of+glycerol.">Specific gravity of glycerol.</a> Ind Eng Chem. 20 (12), 1377-1379 (1928).</li><li>Rodrigues, M., Francesconi, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+the+excess+molar+volumes+of+binary+and+ternary+mixtures+containing+water+++(1,2-ethanediol,+or+1,2-propanediol,+or+1,3-propanediol,+or+1,2-butanediol)+++(1-n-butyl-3-methylimidazolium+bromide)+at+298.15+K+and+atmospheric+pressure.">Experimental study of the excess molar volumes of binary and ternary mixtures containing water + (1,2-ethanediol, or 1,2-propanediol, or 1,3-propanediol, or 1,2-butanediol) + (1-n-butyl-3-methylimidazolium bromide) at 298.15 K and atmospheric pressure.</a> J Solution Chem. 40 (11), 1863-1873 (2011).</li><li>Berejnov, V., Husseini, N. S., Alsaied, O. A., Thorne, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryoprotectant+concentration+and+cooling+rate+on+vitrification+of+aqueous+solutions.">Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions.</a> J Appl Cryst. 39 (2), 244-251 (2006).</li><li>Meisburger, S. P., Warkentin, M., 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=Breaking+the+Radiation+Damage+Limit+with+Cryo-SAXS.">Breaking the Radiation Damage Limit with Cryo-SAXS.</a> Biophys J. 104 (1), 227-236 (2013).</li></ol>

The present apparatus and methods, developed primarily by undergraduates with limited access to instrument-building tools and machinery, nevertheless delivers highly accurate density measurements for individual liquid drops as small as 50 pL. In the concentration range near and above 50% w/w, where small cooling rates are sufficient to obtain vitrified samples, the densities agree with those obtained in previous measurements on bulk samples. Extrapolations of the present densities to 0% concentration &#8211; pure water &...

The physical properties of water and aqueous mixtures in their various phases are of fundamental interest, and are important to in vivo and in vitro understanding of biological systems. In contemporary cryobiology and biological cryopreservation, the vitreous or amorphous phases of aqueous cryoprotectant mixtures are of particular interest1,2. Nucleation and growth of ice crystals can disrupt cells and tissues, and promote protein denaturation and aggregation, so cryopreservation protocols that vitrify the solvent have become increasingly popular. In biomolecular crystallography, the crystallization of solvent in the channels between biomolecules disrupts crystal lattices and degrades diffraction properties. Vitrification is achieved through a combination of rapid cooling, dehydration, and addition of cryoprotective solutes such as glycerol, ethylene glycol, polyethylene glycols (PEGs), alcohols, and salts.
Vitrification limits ice crystallization and growth, but does not eliminate all cooling-related sample damage. For example, crystal mosaicity (a measure of the distribution of crystal plane orientations) routinely increases by a factor of 10 to 100 when protein crystals are cooled into a vitrified state3, and post-thaw survival rates of vitrified sperm cells and oocytes vary widely.
One damage mechanism is differential contraction of solvent and surrounding material during cooling3,4,5. The equilibrium solvent and solute concentrations within a crystal, cell or tissue are temperature dependent, and the solvent plus the solute and surrounding material may contract by different amounts. Rapid cooling may prevent solvent and solute redistribution prior to vitrification, and differential contraction may lead to large, inhomogeneous, nonequilibrium stresses that cause sample damage.
Rational approaches to reducing cooling-induced damage could thus benefit from the knowledge of temperature-dependent densities of liquid and vitrified aqueous mixtures. At solute concentrations above 50% weight of solute to weight of solution (w/w), most aqueous cryoprotectant mixtures can be vitrified with modest cooling rates of 10 K/s or less, allowing production of and density measurements using large vitreous samples6. Density can then be determined using Archimedes&#39; principle, by measuring the apparent weight of the sample when suspended in a liquid cryogen like nitrogen. However, as solute concentration decreases, cooling rates required for vitrification increase rapidly: Cooling rates for aqueous glycerol mixtures increase from &#60;10 K/s at 50% weight of solute in g to volume of solution in mL (w/v) to &#62;1,000 K/s at 25% w/v7. Heat transfer becomes boundary-layer limited, so that achieving larger cooling rates requires smaller and smaller samples8.
Measurements of the density of pure vitreous water and ice have been achieved by depositing micrometer-diameter (femtoliter volume) drops in a vacuum onto a cryogenically-cooled surface so as to build up a macroscopic (gram mass) sample. The density of this sample was determined by cryoflotation in a liquid nitrogen-argon mixture, in which the density of the cryogenic liquid was adjusted until the sample became neutrally buoyant9. However, generating large samples from a large number of small drops in a way that minimizes void volumes &#8211; an important source of error in previous vitreous phase density measurements &#8211; is non-trivial. For aqueous mixtures, differential evaporation of solution components during aerosolization and deposition in a vacuum can lead to substantial uncertainties in deposited concentrations.
We have developed a method, based on cryoflotation, that allows accurate density determination of aqueous mixtures using individual drops as small as 50 pL10. These drops can be rapidly cooled while retaining their original concentrations, and their cryogenic temperature state (vitrified or crystalline) can be assessed using a simple visual assay that correlates with X-ray diffraction measurements. This method is broadly applicable to aqueous and non-aqueous mixtures, and can be extended to a variety of biological samples including cells (e.g., stem and egg), tissue samples, and protein crystals having low-temperature densities between 0.8 and 1.4 g/mL.

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			<td height="21" style="height:21px;width:216px;">centrifuge tube</td>
			<td style="width:71px;">Falcon</td>
			<td style="width:119px;">6029236</td>
			<td style="width:167px;">15 mL conical centrifuge tube</td>
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			<td height="21" style="height:21px;width:216px;">glycerol, &#62;99.5%</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:119px;">G9012-100 mL</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">ethylene glycol, &#62;99.8%</td>
			<td style="width:71px;">Sigma</td>
			<td>324558-100 mL</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">analytical microbalance</td>
			<td style="width:71px;">Mettler</td>
			<td style="width:119px;">AE240</td>
			<td>Analytical balance, 0.01 mg resolution, has hook on bottom for weighing below the balance&#160;</td>
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			<td height="42" style="height:42px;width:216px;">vortexer</td>
			<td style="width:71px;">Scientific Industries</td>
			<td style="width:119px;">SI-0236</td>
			<td>Vortex-Genie 2</td>
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			<td height="42" style="height:42px;width:216px;">Apparatus enclosure framing</td>
			<td style="width:71px;">Unistrut&#160;</td>
			<td style="width:119px;">1-5/8&#34; metal framing</td>
			<td>48&#34; wide x 24&#34; deep x 40&#34; tall&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Apparatus enclosure air barrier</td>
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			<td style="width:119px;"></td>
			<td>any clear plastic sheeting</td>
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			<td height="21" style="height:21px;width:216px;">neoprene rubber disk</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>4&#34; diameter, 1/8&#34; thick</td>
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			<td height="42" style="height:42px;width:216px;">dewar flask</td>
			<td style="width:71px;">Scilogix Dilvac</td>
			<td style="width:119px;">SS333</td>
			<td>4.5 liter dewar flask with steel case and clamp lid</td>
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			<td height="21" style="height:21px;width:216px;">copper chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>This fabricated part is comprised of a 1.43&#34; diameter, 0.017&#34; wall thickness copper tube with a solid cylindrical copper base soldered to seal one end.&#160; The copper base is 0.87&#34; tall and the overall chamber height is 7&#34;.</td>
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			<td height="21" style="height:21px;width:216px;">nitrogen gas</td>
			<td style="width:71px;">Airgas</td>
			<td style="width:119px;">NI HP300</td>
			<td>99.998% pure N2 gas</td>
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			<td height="21" style="height:21px;width:216px;">argon gas</td>
			<td style="width:71px;">Airgas</td>
			<td style="width:119px;">AR HP300</td>
			<td>99.998% pure Ar gas</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rotameter</td>
			<td style="width:71px;">Omega</td>
			<td style="width:119px;">FL3692ST</td>
			<td>2.52 l/min max flow rate</td>
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			<td height="24" style="height:24px;width:216px;">foam insulating lid</td>
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			<td>This part is fabricated from 4 lb/ft3 crosslinked polyethylene foam (supplied by Technifab, 1355 Chester Industrial Parkway, Avon, OH), and has an OD of 2.42&#34;, and ID of 1.52&#34;, and a thickness of 0.79&#34;.&#160;&#160;&#160;&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PTFE test mass</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>This fabricated part is a 0.246&#34; diameter, 0.580&#34; tall cylinder with a 0.060&#34; diameter hole running perpendicular to and intersecting the cylinder axis ~0.10&#34; from one end.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">microbalance platform</td>
			<td style="width:71px;">Unistrut</td>
			<td style="width:119px;">1-5/8&#34; metal framing</td>
			<td>11&#34; wide x 24&#34; long x 24&#34; high rectangular frame with an top aluminum sheet containing a hole for the monofilament and hanging test mass</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 mil (50 um) monofilament line</td>
			<td style="width:71px;">Berkley</td>
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			<td>Nanofil fishing line</td>
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			<td style="width:71px;">VWR</td>
			<td style="width:119px;">60985-512</td>
			<td>1/8&#34; ID x 1/4&#34; OD PVC tubing</td>
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			<td height="24" style="height:24px;width:216px;">perforated copper foil mixer</td>
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			<td>1.4&#34; diameter,&#160; 35 micron thick copper disk, cut from 1 ounce/ft2 copper sheet and perforated with holes using an awl or other sharp pointed tool.&#160; Insert 1-2 mm diameter rigid thermally insulating (plastic or wood) rod into the center and fix using epoxy as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">syringe</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">309628</td>
			<td>1 ml Luer-Lok tip syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vacuum generator</td>
			<td style="width:71px;">Gast</td>
			<td style="width:119px;">VG-015-00-00</td>
			<td>compressed air venturi single stage vacuum generator</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hydrophobic coating spray</td>
			<td style="width:71px;">RainX</td>
			<td style="width:119px;">620036</td>
			<td>plastic water repellent</td>
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			<td height="84" style="height:84px;width:216px;">long focal length stereo microscope</td>
			<td style="width:71px;">Bausch and Lomb Stereozoom 6</td>
			<td style="width:119px;"></td>
			<td>0.67-4 x zoom pod with 20x eyepieces, 10 cm working distance&#160;</td>
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			<td height="21" style="height:21px;width:216px;">LED ring illuminator</td>
			<td style="width:71px;">Amscope</td>
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			<td height="42" style="height:42px;width:216px;">LED spot illuminator</td>
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			<td style="width:119px;">NHCLP-LED</td>
			<td>3W LED gooseneck clamp lamp</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.8 ml cryo vial</td>
			<td style="width:71px;">Nunc</td>
			<td>V7634-500EA</td>
			<td>Any 1.8 or 2 ml cryovial is adequate</td>
		</tr>
	</tbody>
</table>

measuring the densities of aqueous glasses at cryogenic temperatures

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid cooling, to prepare the desired cryogenic temperature phase. Microliter to picoliter size drops are cooled by projection into a liquid nitrogen-argon (N2-Ar) mixture. The cryogenic temperature phase of the drop is evaluated using a visual assay that correlates with X-ray diffraction measurements. The density of the liquid N2-Ar mixture is adjusted by adding N2 or Ar until the drop becomes neutrally buoyant. The density of this mixture and thus of the drop is determined using a test mass and Archimedes principle. With appropriate care in drop preparation, management of gas above the liquid cryogen mixture to minimize icing, and regular mixing of the cryogenic mixture to prevent density stratification and phase separation, densities accurate to &#60;0.5% of drops as small as 50 pL can readily be determined. Measurements on aqueous cryoprotectant mixtures provide insight into cryoprotectant action, and provide quantitative data to facilitate thermal contraction matching in biological cryopreservation.

Density measurements at T = 77 K for vitrified drops of aqueous glycerol and ethylene glycol versus cryoprotectant concentration are shown in Figure 1A and Figure 1B respectively, and the corresponding change in specific volume between T = 298 K and 77 K, calculated using previously determined T = 298 K densities, is shown in Figure 2. At h...

Watch this Scientific Journal Video about Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures at JoVE.com

Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures

A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid ...

The overall goal of this experiment is to determine the glass or vitreous state densities of aqueous solutions at cryogenic temperatures. This method can help answer key questions in the fields of Cryobiology and Cryocrystallography, such as, how much do aqueous cryo protected mixtures contract or expand when cooled into a vitreous state at cryogenic temperatures. The main advantage of this technique is that both cryogenic temperature sample density and phase, can be determined using small samples that can be cooled rapidly.Though this method was developed for cryoprotecting solutions, it can also be used to determine cryogenic temperature densities of tissues, cell aggregates and other biological samples. I first had the idea for this method when I learned from Thomas Lutin, how to make a cryogenic liquid with a tunable density and the range between 0.9 and 1.3 grams per milliliter. To begin this procedure, place a disk of neoprene rubber on the bottom of a 4.5 liter glass dewar flask, to protect the dewar flask from damage.Carefully insert a high thermal conductivity copper chamber into the flask, until it rests on the rubber disk. Adjust struts projecting outward from the chamber to the dewar flask walls, so that the chamber centered and has no tendency to rock. Insert the outlet of a gas tube with dry nitrogen gas flowing at approximately two liters per minute, down to the bottom of the copper chamber and purge the chamber of moist air.Now, slowly pour liquid nitrogen into the dewar flask, outside the copper chamber, allowing time for nitrogen boil off. Remove the dry nitrogen gas tube from the copper chamber and insert it into a matching opening in a foam insulating lid. Then cover the outer portion of the dewar flask with the lid.Secure the lid in place with elastic straps. Slowly pour liquid nitrogen into the copper chamber, so that the final fill level after boiling has ceased, is within approximately five centimeters of the top of the copper chamber. Following this, place a thin optically transparent plastic sheet over the central opening in the lid.Reduce the nitrogen gas flow rate to approximately 0.2 liters per minute, leaving a slight overpressure of nitrogen gas within the gas spaces above the cryogenic liquids. Determine the apparent mass of a one gram 0.4 milliliter PTFE test mass, in the air, at 298 Kelvin, by placing it on the pan of a calibrated analytical micro balance. Next, suspend the test mass using a 50 micron monofilament line strung from the hook on the underside of the micro balance and through a hole in the test mass.Then, determine the apparent mass in air and compare to the mass measurement, correcting as needed for the mass of the line. To determine the volume of the test mass at 77 Kelvin, lower the test mass into the copper chamber containing liquid nitrogen until it is fully submerged. When boiling has ceased, measure the apparent mass.Flow Argon gas at a flow rate of approximately two liters per minute through a coiled tube to its outlet. Place the coiled tube on top of the upper struts that stabilize the position of the copper chamber, just above the liquid nitrogen level and below top surface in the dewar flask. After allowing the coiled tube to cool for five minutes, place the outlet and the tube in the copper chamber at least 10 centimeters below the surface and the liquid nitrogen.Then, cover the dewar flask with the annular lid and transparent sheet. Following this, adjust the Argon flow rate until bubbles rise from the tube outlet to the top surface of the liquid nitrogen. Then, reduce the flow rate until the bubbles form at the outlet but dissolve or liquefy just before breaking the liquid nitrogen surface.Periodically, mix the liquid by inserting a thin circular sheet of copper foil attached to a thin insulating rod into the copper chamber and slowly moving it up and down like a piston. After measuring the apparent mass of the test mass in air, remove the insulating dewar cover and cool the mass to 77 Kelvin by lowering it into the liquid nitrogen outside the copper chamber. Raise the cool test mass into the cold layer of nitrogen gas above the liquid nitrogen and wait for residual liquid nitrogen to evaporate off the test mass.Then, lower the cool, dried test mass into the liquid nitrogen Argon mixture until it is fully submerged and within two centimeters of the liquid surface. After any boiling and surface waves have disappeared, measure the apparent mass of the test mass. Immediately prior to drop dispensing in cooling, mix the nitrogen argon cryogenic liquid, as previously described.After removing the sample tubes cap, extract up to one milliliter solution using a clean one milliliter syringe. Attach a 27 to 33 gauge needle onto the syringe and then push a small amount of sample through the needle to expel air and any residues from previous dispensing. For samples that require faster cooling for vitrification, place the outlet of a gas tube connected to a vacuum generator, in the gas space above the liquid nitrogen argon mixture and gently suction away the cold gas layer that forms.For samples with large non-aqueous component concentrations that can be vitrified with modest cooling rates, lightly press the syringe to displace a small diameter, 10 nanoliter to one microliter drop that hangs from the needle tip by surface tension. Gently tap the needle to detach and project the drop towards the liquid nitrogen argon mixture. Using a long working distance binocular microscope, and bright, cool illumination from an LED illuminator, carefully examine the drop while keeping it immersed in the liquid nitrogen argon mixture.Vitrified drops should appear clear. Reject drops are cloudy or show an optical imperfections including cracks. The drop floats, decrease the density of the liquid nitrogen argon mixture by adding liquid nitrogen using a 1.8 milliliter cryo vial.Gently mix the liquid nitrogen argon mixture, using a thin perforated copper foil sheet and moving it up and down in the copper chamber. After each liquid nitrogen addition, move a small prequel rod beneath the floating drop to gently displace it downward into the liquid and observe its speed as it returns to the surface. Density measurements at 77 Kelvin for vitrified drops of aqueous glycerol and ethylene glycol, versus cryo protecting concentration, are shown here.The corresponding change in specific body in between 298 and 77 Kelvin, calculated using previously determined 298 Kelvin densities is displayed here. Near 20%to 25%solutions of both cryo protectants are predicted to show no net expansion or contraction. The slope of the volume change versus concentration has the largest magnitude below 40%where the effects of additional cryo protectant on waters, tetrahedral low temperature structure are most pronounced.Once mastered, the initial high density liquid nitrogen argon cryogenic solution can be prepared in two to two and a half hours and the densities of individual drops can be determined in 10 to 15 minutes. While attempting this procedure, it's important to remember to watch for icing and remove as much of the cold gas layer that forms on the liquid nitrogen argon mixture surface as possible, using a vacuum generator. Following this procedure, drops can be collected, stored in liquid nitrogen and later measured using cryocrystallography to verify the phase of a drop.Don't forget, that working with liquid cryogens can be extremely hazardous and precautions such as, wearing appropriate gloves, face shields, clothing and footwear, should always be taken while performing this procedure.

measuring-the-densities-of-aqueous-glasses-at-cryogenic-temperatures

Research

JoVE Journal

Engineering

Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures

The physical properties of a material are defined by its electronic structure. Electrons in solids are characterized by energy (&omega;) and momentum (k) and the probability to find them in a particular state with given &omega; and k is described by the spectral function A(k, &omega;). This function can be directly measured in an experiment based on the well-known photoelectric effect, for the explanation of which Albert Einstein received the Nobel Prize back in 1921. In the photoelectric effect the light shone on a surface ejects electrons from the material. According to Einstein, energy conservation allows one to determine the energy of an electron inside the sample, provided the energy of the light photon and kinetic energy of the outgoing photoelectron are known. Momentum conservation makes it also possible to estimate k relating it to the momentum of the photoelectron by measuring the angle at which the photoelectron left the surface. The modern version of this technique is called Angle-Resolved Photoemission Spectroscopy (ARPES) and exploits both conservation laws in order to determine the electronic structure, i.e. energy and momentum of electrons inside the solid. In order to resolve the details crucial for understanding the topical problems of condensed matter physics, three quantities need to be minimized: uncertainty* in photon energy, uncertainty in kinetic energy of photoelectrons and temperature of the sample.
In our approach we combine three recent achievements in the field of synchrotron radiation, surface science and cryogenics. We use synchrotron radiation with tunable photon energy contributing an uncertainty of the order of 1 meV, an electron energy analyzer which detects the kinetic energies with a precision of the order of 1 meV and a He3 cryostat which allows us to keep the temperature of the sample below 1 K. We discuss the exemplary results obtained on single crystals of Sr2RuO4 and some other materials. The electronic structure of this material can be determined with an unprecedented clarity.

The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.

The physical properties of a material are defined by its  electronic structure. Electrons in solids are characterized by energy (&omega;)  and momentum ...

Synthesis and Microdiffraction at Extreme Pressures and Temperatures

High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary interiors, design materials with novel properties, understand the mechanical behavior of materials exposed to very high stresses as in explosions or impacts. Synthesis and structural analysis of materials at extreme conditions of pressure and temperature entails remarkable technical challenges. In the laser heated diamond anvil cell (LH-DAC), very high pressure is generated between the tips of two opposing diamond anvils forced against each other; focused infrared laser beams, shined through the diamonds, allow to reach very high temperatures on samples absorbing the laser radiation. When the LH-DAC is installed in a synchrotron beamline that provides extremely brilliant x-ray radiation, the structure of materials under extreme conditions can be probed in situ. LH-DAC samples, although very small, can show highly variable grain size, phase and chemical composition. In order to obtain the high resolution structural analysis and the most comprehensive characterization of a sample, we collect diffraction data in 2D grids and combine powder, single crystal and multigrain diffraction techniques. Representative results obtained in the synthesis of a new iron oxide, Fe4O5 1 will be shown.

The laser heated diamond anvil cell combined with synchrotron micro-diffraction techniques allows researchers to explore the nature and properties of new phases of matter at extreme pressure and temperature (PT) conditions. Heterogeneous samples can be characterized in situ under high pressure by 2D mapping and combined powder, single-crystal and multigrain diffraction approaches.

High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary ...

Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids&#160;and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.
The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.1&#160;Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.

This paper demonstrates the experimental procedure to measure terminal settling velocities of spherical particles in surfactant-based shear thinning viscoelastic fluids. Fluids over a wide range of rheological properties are prepared and settling velocities are measured for a range of particle sizes in unbounded fluids and fluids between parallel walls.

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic ...

Ambient Method for the Production of an Ionically Gated Carbon Nanotube Common Cathode in Tandem Organic Solar Cells

A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two solution-processed polymeric cells connected in parallel by a transparent carbon nanotubes (CNT) interlayer. This structure includes improvements in fabrication techniques for tandem OPV devices. First the need for ambient-processed cathodes is considered. The CNT anode in the tandem device is tuned via ionic gating to become a common cathode. Ionic gating employs electric double layer charging to lower the work function of the CNT electrode. Secondly, the difficulty of sequentially stacking tandem layers by solution-processing is addressed. The devices are fabricated via solution and dry-lamination in ambient conditions with parallel processing steps. The method of fabricating the individual polymeric cells, the steps needed to laminate them together with a common CNT cathode, and then provide some representative results are described. These results demonstrate ionic gating of the CNT electrode to create a common cathode and addition of current and efficiency as a result of the lamination procedure.

A method of fabricating, in ambient conditions, organic photovoltaic tandem devices in a parallel configuration is presented. These devices feature an air-processed, semi-transparent, carbon nanotube common cathode.

A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two ...

A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and a CCD spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, fast sample freezing, fast alignment of the sample, accurate and stable sample temperatures, and small size and portability of the Dewar/cryostat cryogenic unit. When coupled with the fast data acquisition rates of the CCD spectrograph, this Dewar/cryostat is capable of supporting cryogenic luminescence spectroscopic measurements on luminescent samples at a series of known, stable temperatures in the 77-300 K range. A temperature-dependent study of the oxygen quenching of luminescence in a rhodium(III) transition metal complex is presented as an example of the type of investigation possible with this Dewar/cryostat. In the context of this apparatus, a stable temperature for cryogenic spectroscopy means a luminescent sample that is thermally equilibrated with either liquid nitrogen or gaseous nitrogen at a known measureable temperature that does not vary (&#916;T &#60; 0.1 K) during the short time scale (~1-10 sec) of the spectroscopic measurement by the CCD. The Dewar/cryostat works by taking advantage of the positive thermal gradient dT/dh that develops above liquid nitrogen level in the Dewar where h is the height of the sample above the liquid nitrogen level. The slow evaporation of the liquid nitrogen results in a slow increase in h over several hours and a consequent slow increase in the sample temperature T over this time period. A quickly acquired luminescence spectrum effectively catches the sample at a constant, thermally equilibrated temperature.

A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and ...

Design and Evaluation of Smart Glasses for Food Intake and Physical Activity Classification

This study presents a series of protocols of designing and manufacturing a glasses-type wearable device that detects the patterns of temporalis muscle activities during food intake and other physical activities. We fabricated a 3D-printed frame of the glasses and a load cell-integrated printed circuit board (PCB) module inserted in both hinges of the frame. The module was used to acquire the force signals, and transmit them wirelessly. These procedures provide the system with higher mobility, which can be evaluated in practical wearing conditions such as walking and waggling. A performance of the classification is also evaluated by distinguishing the patterns of food intake from those physical activities. A series of algorithms were used to preprocess the signals, generate feature vectors, and recognize the patterns of several featured activities (chewing and winking), and other physical activities (sedentary rest, talking, and walking). The results showed that the average F1 score of the classification among the featured activities was 91.4%. We believe this approach can be potentially useful for automatic and objective monitoring of ingestive behaviors with higher accuracy as practical means to treat ingestive problems.

This study presents a protocol of designing and manufacturing a glasses-type wearable device that detects the patterns of food intake and other featured physical activities using load cells inserted in both hinges of the glasses.

This study presents a series of protocols of designing and manufacturing a glasses-type wearable device that detects the patterns of temporalis muscle ...

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 ...

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely M&#246;ssbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. M&#246;ssbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

Methods of Ex Situ and In Situ Investigations of Structural Transformations: The Case of Crystallization of Metallic Glasses

Here, we present a protocol to describe ex situ and in situ investigations of structural transformations in metallic glasses. We employed nuclear-based analytical methods which inspect hyperfine interactions. We demonstrate the applicability of M&#246;ssbauer spectrometry and nuclear forward scattering of synchrotron radiation during temperature-driven experiments.

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic ...

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed electric body force has to be induced by ionic motions in liquid. On the other hand, positive and negative charges attract each other, and electroneutrality is maintained everywhere in equilibrium conditions. Furthermore, an increase in an applied voltage has to be suppressed to avoid water electrolysis, which causes the solutions to become unstable. Usually, EHD flows can be induced in non-aqueous solutions by applying extremely high voltages, such as tens of kV, to inject electrical charges. In this study, two methods are introduced to generate EHD flows induced by electrical charge separations in aqueous solutions, where two liquid phases are separated by an ion-exchange membrane. Due to a difference in the ionic mobility in the membrane, ion concentration polarization is induced between both sides of the membrane. In this study, we demonstrate two methods. (i) The relaxation of ion concentration gradients occurs via a flow channel that penetrates an ion-exchange membrane, where the transport of the slower species in the membrane selectively becomes dominant in the flow channel. This is a driving force to generate an EHD flow in the liquid. (ii) A long waiting time for the diffusion of ions passing through the ion-exchange membrane enables the generation of an ion-dragged flow by externally applying an electric field. Ions concentrated in a flow channel of a 1 x 1 mm2 cross-section determine the direction of the liquid flow, corresponding to the electrophoretic transport pathways. In both methods, the electric voltage difference required for an EHD flow generation is drastically reduced to near 2 V by rectifying the ion transport pathways.

The rectification of ion transport pathways is an effective method to generate one-directional ion-dragged electrohydrodynamic flows. By setting an ion-exchange membrane in a flow channel, an electrically polarized condition is generated and causes a liquid flow to be driven when an electric field is externally applied.

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed ...

Measurement of Dynamic Force Acted on Water Strider Leg Jumping Upward by the PVDF Film Sensor

This study aimed to make an explanation for the phenomenon in nature that water strider usually jumps or glides on the water surface easily but quickly, with its peak locomotion speed reaching 150 cm/s. First of all, we observed the microstructure and hierarchy of water strider legs using the scanning electron microscope. On the basis of the observed morphology of the legs, a theoretical model of the detachment from water surface was established, which explained water striders' capability to slide on water surface effortlessly in terms of energy reduction. Secondly, a dynamic force measurement system was devised using the PVDF film sensor with excellent sensitivity, which could detect the whole interaction process. Subsequently, a single leg in contact with water was pulled upward at different speeds, and the adhesion force was measured at the same time. The results of the departing experiment suggested a deep understanding of the fast jumping of water striders.

The protocol here is dedicated to investigating the free and quick maneuvering of water strider on water surface. The protocol includes observing the microstructure of legs and measuring the adhesion force when departing from water surface at different speeds.

This study aimed to make an explanation for the phenomenon in nature that water strider usually jumps or glides on the water surface easily but quickly, ...