Name: force-clamp rheometry for characterizing protein-based hydrogels
Uploaded: 2018-08-21T15:00:00
Description: Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

We acknowledge financial support from Research Growth Initiative (Award No. 101X340), National Science Foundation, Major Research Instrumentation Program (Grant No. PHY-1626450), Greater Milwaukee Foundation (Shaw Award) and University of Wisconsin System (Applied Research Grant).

1. Reagents Solution Preparation
<ol>
	<li>Prepare a starting protein solution by dissolving/diluting the protein of interest to the desired concentration, using a Tris buffer [20 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl, pH 7.4]. 
	NOTE: The smallest protein concentration for which cross-linking leads to hydrogels depends on the protein used and is typically &#62; 1 mM.</li>
	<li>Prepare stocks of ammonium persulfate (APS) (1 M) and tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]2+</s...

<ol>
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P., 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=Probing+the+chemistry+of+thioredoxin+catalysis+with+force.">Probing the chemistry of thioredoxin catalysis with force.</a> Nature. 450 (7166), 124-127 (2007).</li><li>Lv, 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=Designed+biomaterials+to+mimic+the+mechanical+properties+of+muscles.">Designed biomaterials to mimic the mechanical properties of muscles.</a> Nature. 465 (7294), 69-73 (2010).</li><li>Plumere, N., 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=A+redox+hydrogel+protects+hydrogenase+from+high-potential+deactivation+and+oxygen+damage.">A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage.</a> Nature Chemistry. 6 (9), 822-827 (2014).</li><li>Kong, N., Peng, Q., Li, H. 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Herein, we describe a force-clamp rheometry technique to investigate the biomechanical response of low-volume protein-based hydrogels. Additionally, a protocol is provided to synthesize a uniform cylindrical low-volume protein hydrogel sample. A protocol is also presented which describes how to tie different types of protein-based hydrogels with various elasticities without causing any mechanical deformation or damage to the protein-based hydrogel samples or slippage of the gel on the hooks. The analog PID system, togeth...

Apart from having unique physical properties, protein-based hydrogels hold the promise of revolutionizing force spectroscopy by enabling the measurement of several billion molecules in one &#39;pull&#39;, thus enabling the study of proteins in crowded environments, similar to those encountered in skin and other tissues. Protein domains remain folded inside hydrogels, allowing the study of their biomechanical response to force, binding partners, and chemical conditions. Additionally, the biomechanical response of protein domains inside hydrogels resembles the response seen with single-molecule force spectroscopy techniques. For example, chemical denaturants and oxidizing agents decrease the stability of the folded state, both at the single protein domain level1,2,3 and at the macroscopic level4,5,6,7. Similarly, osmolytes increase the stability of single proteins8,9, leading to a decrease in the viscoelastic response&#160;of hydrogels, for the same force conditions7,10.
Several approaches have been implemented to synthesize protein-based hydrogels, by either using physical interactions11,12 or covalent cross-linking4,13. Covalent reactions allow for fixed cross-linking locations and these hydrogels&#160;can recover the initial state upon a removal of the mechanical or chemical perturbations. A successful approach for covalent cross-linking relies on forming covalent carbon-carbon bonds between exposed tyrosine amino acids using ammonium persulfate (APS) as an oxidant and a ruthenium (II) salt as an initiator (Figure 1)14. Upon exposure to white light, a solution of concentrated proteins can be turned into a hydrogel. By controlling when the reaction starts, the protein-APS mix can be injected into any casting form, such as polytetrafluoroethylene (PFTE) tubes (Figure 1B and 1C), allowing the use of an extremely small solution volume15. Furthermore, the use of white light to trigger the cross-linking reaction results in a limited bleaching of fluorescent proteins and allows the formulation of composite hydrogels with fluorescent markers (Figure 1). Other protein-based hydrogel formation methods use cross-linking based on the SpyTag-SpyCatcher covalent interaction16, amine cross-linking via glutaraldehyde13, or biotin-streptavidin interactions17.
Dynamic mechanical analysis (DMA) is currently a technique extensively used to study polymer-based hydrogels13,18. While DMA can apply constant force protocols to biomaterials, it requires Young&#39;s moduli over 10 kPa, and large sample volumes of more than 200 &#181;L19. Due to these limitations, protein hydrogels are generally too soft to be investigated by this technique. As engineered polyproteins are harder to synthesize than polymers, since they require a living system to produce, such high volumes are inefficient, at best4,15. Furthermore, most biological tissues are softer than 10 kPa. Several approaches were developed for biological samples, especially in the study of muscle elasticity20,21. These techniques can also operate under feedback to apply constant force but are optimized for samples with small diameters (in the micron range) exposed to force for very short times (typically less than 1 s).
Protein-based hydrogels were successfully studied with modified rheometry techniques. For example, casting the hydrogel in a ring shape allows the use of extensional rheometry to measure the change in the experienced force as a function of extension4,22. Other approaches for studying the rheological properties of protein-based hydrogels use controlled shear-stress rheometry. These techniques can also achieve low sample volume and tolerate soft materials. However, these methods lack the ability to mimic the pulling forces that cause protein unfolding in vivo, and Young&#39;s modulus is calculated based on complex theories that require various assumptions and corrections23.
We have recently reported a new approach that utilizes a small volume of proteins, polymerized inside tubes with&#160;diameters &#60; 1 mm. Our first implementation of this technique was operating in length-clamp mode, where the gel was extended following the desired protocol15. In this method, the proteins experience a continuous change in both extension and force while the domains unfold, making the data interpretation cumbersome. Recently, we have reported a new force-clamp rheometry technique, where a feedback loop can expose low-volume protein hydrogels to a predefined force protocol7 (Figure 2). An analog PID system compares the force measured by the force sensor with the set point sent from the computer and adjusts the gel extension by moving the voice coil to minimize the difference between the two inputs. This &#39;clamping&#39; of the force now allows for new types of experiments to measure the biomechanics of protein hydrogels.
In the force-ramp mode, a tethered protein hydrogel experiences a constant increase and decrease of force with time. The PID compensates for any viscoelastic deformation by changing the extension in a non-linear way, depending on the type of protein and hydrogel formulation. The main advantage of force ramp is that it allows the quantification of standard parameters, such as Young&#39;s modulus and energy dissipation, due to an unfolding and refolding of protein domains.
In constant-force mode, the applied force changes in a step-like fashion. In this mode, the gel extends and contracts elastically when the force is increased or decreased, respectively, followed by a time-dependent deformation. This viscoelastic deformation, taking place while the gel experiences a constant force, is directly related to domain unfolding/refolding. In a simplified way, this extension can be seen as the equivalent of several billion single molecule traces averaged together and measured all at once. Constant-force protocols can be used to study the creep and relaxation of protein hydrogels as a function of force and time. As a function of force, for BSA-based protein hydrogels, we have recently shown that there is a linear dependency between the elastic and viscoelastic extension and recoil with the applied strain7.
Here we detail the operation of a force-clamp rheometer using composite gels made from a mixture of protein L (8 domains24, depicted as L8) and a protein L-eGFP construct (L-eGFP), which makes the overall hydrogel fluorescent and easy to demonstrate.

<table>
	<tbody>
		<tr>
			<td>SI-KG4A force transducer</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SI-KG4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Voice Coil Motor</td>
			<td>Equipement Solutions</td>
			<td>LFA2010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Rocky Mountain Biologicals (RMBIO)</td>
			<td>BSA-AAF-1XG / 100 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma</td>
			<td>Sigma-Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>248614-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(bipyridine)ruthenium(II) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>544981-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>EXPRESS MEDICAL SUPPLIES&#160;6-0 NYLON SUTURE 12/PK</td>
			<td>Fisher Scientific</td>
			<td>NC0395626</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe Only, Luer-Lok Tip</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Silane, Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbore PTFE Tubing, 0.022&#34;ID x 0.042&#34;OD, 100 ft/roll</td>
			<td>Cole-Parmer</td>
			<td>EW-06417-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 23 Gauge</td>
			<td>Healthcare Supply Pros</td>
			<td>305194</td>
			<td></td>
		</tr>
		<tr>
			<td>Jensen Global JG24-1.5X Red IT Dispensing Tips - 24 gauge</td>
			<td>KIMCO</td>
			<td>JG24-1.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>USH-103D USHIO 100W Short Arc Mercury Lamp</td>
			<td>ALB</td>
			<td>USH-103D USHIO</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The computer code and CAD design of the custom parts can be made available on request to the corresponding author.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

force-clamp rheometry for characterizing protein-based hydrogels

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels&#160;enables the tethering of extremely low-volume hydrogel samples (&#60; 5 &#181;L) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

Figure 1A shows the scheme of the photoactive reaction used to synthesize the L-EGP/L8 hydrogel. Figure 1B shows the hydrogel mixture in the PTFE tube before and after the photoactivation. Figure 1C presents the extruded L-eGFP-L8 hydrogel inside a Tris solution. The hydrogel sample has no structural defects such as notches. Hydrogels with clearly visible damage should be discar...

Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the &#39;clamping&#39; of the force experienced to the desired protocol.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog ...

The main advantages of this technique are that it utilizes a PID, which is a proportional integral derivative system to apply controlled force protocols to protein hydrogel samples, and it utilizes small sample volume. The protocols for the forces clamp allow for straight-forward interpretation of the data while low volumes are critical when working with hard to produce proteins that are available only in small amounts. The implications of this technique extend toward developing and characterizing new biometric materials with durable elasticity, using down-folding, refolding transition characteristic for proteins.This method can answer questions in tissue and biomaterial mechanics through enabling the measurements of billions of protein molecules in one pull and simulating crowded environments specific to biological tissues. Begin this procedure with reagent solutions preparation as described in the text protocol. To synthesize the protein-based hydrogel, first fix a 23-gauge needle on a one milliliter syringe with a pressed plunger.Then, cut a 10 centimeter polytetrafluoroethylene, or PTFE, tube using a razor blade. Attach the needle and syringe to one end of the PTFE tube. Insert the second end of the tube into a silane solution and fill the tube by retracting the syringe plunger.Leave the tube for approximately 30 minutes. Then, remove the silane solution and dry the tube with compressed air. Now, mix the protein solution with APS and Tris(bipyridine)ruthenium(II)chloride in a 1.5 milliliter tube using a constant volume ratio.Vortex the photoactive solution until it is mixed completely. Then, centrifuge the mix at maximum speed to remove any bubbles from the solution. Bubbles can form while loading the hydrogel photoactive mix into the Teflon tube, leading to sample damage.To prevent bubble formation, keep the Teflon tube end in the solution mix during the loading process and retract the syringe plunger slowly. Insert the open end of the treated PTFE tube into the photoactive mixture and draw the solution into the tube by retracting the syringe plunger. Now, place the loaded tube approximately 10 centimeters away from a 100 watt mercury lamp to prevent heating it and keep it there for up to 30 minutes at room temperature.Remove the tube from the needle and cut the edges of the tube near the hydrogel ends with a razor blade. Then, use a blunted 24-gauge needle to extrude the hydrogel into the Tris solution. Visually inspect the gels for any defects that might form during the extrusion or due to bubbles and discard any defective gels.Start the instrument control program and turn on the voice coil motor. Then, set the coil position to a value toward the end of the range. Displace the hooks in the Z direction and align them at the bend in the X direction.Then, record the values of the micrometer screws for the X direction. Now, tie a loose double-overhand knot at the end of the suture strand, such that the diameter of the loop is about four millimeters. Then, cut the loop off the strand.Repeat to form a second loop. Then, place the two loops on the hook connected to the force sensor. Fill the experimental chamber with Tris buffer and transfer the hydrogel sample into the filled chamber using medical tweezers.Place the voice coil and force sensor hooks close to the solution surface and align the hooks in all directions with the XYZ positioning manipulators. Using medical tweezers, hang both sides of the protein hydrogel sample on the hooks connected to the voice coil and force sensor. A typical error is an over-tightening of the suture loops around the hydrogel samples during the attachment process.This may lead to a notch formation and cutting of the hydrogel sample. Carefully tighten one suture loop around the hydrogel sample on the voice coil hook by holding both ends of the suture loop with medical tweezers and pulling them simultaneously. Repeat this step for the loop connected to the force sensor.Tighten the suture loops on the bends of each hook to prevent any slippage. Use these bends as reference points to find the zero separation between the hooks. Cut the excess lengths of the sutures using medical scissors.Move the attached hydrogel using Z manipulators along the Z axis towards the experimental chamber to immerse the hydrogel in the experimental solution. Align the hydrogel sample in YZ using the manipulators such that the gel is not under any stress. Zero the force sensor and separate the two hooks using the X micrometer stages until the gel starts to experience force.Once this happens, slightly turn back the micrometer screw in the X direction. Record the position of both manipulators for the voice coil motor and the sensor. Then, use the difference between these values and the ones previously measured to calculate the exact separation between the tethering hooks at the start of the experiment.To perform a force-ramp cycle by increasing the force at the desired loading rate, input the starting and final force values, as well as the duration of the protocol, which appears as a flipped V, followed by a constant low force for about 200 seconds to allow the protein domains to refold before the next cycle. Perform a constant-force protocol by applying a low force for 30 seconds. Then, increase the force to a constant force for a defined amount of time, followed by quenching the force back to the same low value for greater than 300 seconds to allow the protein domains to refold and the gel elasticity to recover.Finally, perform data analysis as described in the text protocol. Each measurement starts with a slack curve. By fitting two lines, the zero force on the sensor and the true gel length is determined.The true gel length is calculated from the intersection of the fits and the position of the micrometer screws. The force-clamp rheometry system can apply two different types of protocols. In the force ramp mode, the hydrogel sample experiences a force changing protocol with time that resembles an inverted V.In constant force mode, the applied stress changes in a step-like pattern.During measurements, the PID system adjusts the hydrogel extension by changing the coil position to follow the pre-defined set point from the force protocol. The strain is then calculated by dividing the measured extension to the true gel length. The stress is determined by dividing the applied force to the cross-sectional area of the hydrogel sample.Force Ramp traces are best represented as stress versus strain. The Young's modulus can be calculated from the slope measure during the loading phase. The hysteresis gives the energy dissipation coming from unfolding and refolding the protein.Constant force traces are best represented as a function of time. The change in strain can be used to measure the unfolding and refolding rates by fitting a double exponential. While attempting this procedure, it's important to remember to dry any silane from the tube before adding the protein, to centrifuge the protein mix to remove bubbles, to check the bubbles after injecting the protein mix inside the tube and to discard any mechanically damaged hydrogels after extrusion from the tube.Generally, individuals new to this method will struggle at first with attaching the gels to the hooks using the surgical sutures without causing damage to the hydrogel sample. Extruding the gel from the tube can damage the hydrogel as well. This method enables the applications of constant force protocols on low volume hydrogel samples.These experiments allow the decoupling of the elastic and the viscoelastic behaviors and the study of protein unfolding and folding mechanics in a bulk approach. After its development, this technique allows researchers in the field of material sciences to explore new soft biomaterials such as engineered polyproteins-based hydrogels that have an excellent potential for serving as tissue engineering scaffolds, drug delivery systems and biological ink for 3-D printing. Not only does this method provide insight into the mechanics of protein-based hydrogels, it can also be applied to other systems such as measuring the isometric response of muscle fibers or the elasticity of tissues such as skin.

force-clamp-rheometry-for-characterizing-protein-based-hydrogels

Research

JoVE Journal

Engineering

Bringing the Visible Universe into Focus with Robo-AO

The angular resolution of ground-based optical telescopes is limited by the degrading effects of the turbulent atmosphere. In the absence of an atmosphere, the angular resolution of a typical telescope is limited only by diffraction, i.e., the wavelength of interest, &lambda;, divided by the size of its primary mirror's aperture, D. For example, the Hubble Space Telescope (HST), with a 2.4-m primary mirror, has an angular resolution at visible wavelengths of ~0.04 arc seconds. The atmosphere is composed of air at slightly different temperatures, and therefore different indices of refraction, constantly mixing. Light waves are bent as they pass through the inhomogeneous atmosphere. When a telescope on the ground focuses these light waves, instantaneous images appear fragmented, changing as a function of time. As a result, long-exposure images acquired using ground-based telescopes - even telescopes with four times the diameter of HST - appear blurry and have an angular resolution of roughly 0.5 to 1.5 arc seconds at best.
Astronomical adaptive-optics systems compensate for the effects of atmospheric turbulence. First, the shape of the incoming non-planar wave is determined using measurements of a nearby bright star by a wavefront sensor. Next, an element in the optical system, such as a deformable mirror, is commanded to correct the shape of the incoming light wave. Additional corrections are made at a rate sufficient to keep up with the dynamically changing atmosphere through which the telescope looks, ultimately producing diffraction-limited images.
The fidelity of the wavefront sensor measurement is based upon how well the incoming light is spatially and temporally sampled1. Finer sampling requires brighter reference objects. While the brightest stars can serve as reference objects for imaging targets from several to tens of arc seconds away in the best conditions, most interesting astronomical targets do not have sufficiently bright stars nearby. One solution is to focus a high-power laser beam in the direction of the astronomical target to create an artificial reference of known shape, also known as a 'laser guide star'. The Robo-AO laser adaptive optics system2,3 employs a 10-W ultraviolet laser focused at a distance of 10 km to generate a laser guide star. Wavefront sensor measurements of the laser guide star drive the adaptive optics correction resulting in diffraction-limited images that have an angular resolution of ~0.1 arc seconds on a 1.5-m telescope.

Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used.

The angular  resolution of ground-based optical telescopes is limited by the degrading  effects of the turbulent atmosphere. In the absence of an ...

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2 In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61BM system correspond well with literature data. The P3HT:PC61BM blend separates onto PC61BM-rich and P3HT-rich domains. Mechanical properties of PC61BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.

Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution.

Organic photovoltaic  (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale  inhomogeneity of OPV materials affects performance ...

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O2 pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (&gt;80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO2, Al2O3, WO3 and Ag4O4.

We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.

Nanosecond Pulsed  Laser Deposition (PLD) in the presence of a background gas allows the deposition  of metal oxides with tunable morphology, structure, ...

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 ...

The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and polar dielectric liquids. These bridges are unique from capillary bridges in that they exhibit extensibility beyond a few millimeters, have complex bi-directional mass transfer patterns, and emit non-Planck infrared radiation. A number of common solvents can form such bridges as well as low conductivity solutions and colloidal suspensions. The macroscopic behavior is governed by electrohydrodynamics and provides a means of studying fluid flow phenomena without the presence of rigid walls. Prior to the onset of a liquid bridge several important phenomena can be observed including advancing meniscus height (electrowetting), bulk fluid circulation (the Sumoto effect), and the ejection of charged droplets (electrospray). The interaction between surface, polarization, and displacement forces can be directly examined by varying applied voltage and bridge length. The electric field, assisted by gravity, stabilizes the liquid bridge against Rayleigh-Plateau instabilities. Construction of basic apparatus for both vertical and horizontal orientation along with operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

Horizontal and vertical electrohydrodynamic liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields and polar dielectric liquids. The construction of basic apparatus and operational examples, including thermographic images, for three liquids (e.g., water, DMSO, and glycerol) is presented.

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and ...

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of biosensing devices. We report on a protocol of using KrF and ArF lasers irradiating Si (001) samples immersed in a liquid environment with low number of pulses and operating at moderately low pulse fluences to induce Si wettability modification. Wafers immersed for up to 4 hr in a 0.01% H2O2/H2O solution did not show measurable change in their initial contact angle (CA) ~75&#176;. However, the 500-pulse KrF and ArF lasers irradiation of such wafers in a microchamber filled with 0.01% H2O2/H2O solution at 250 and 65 mJ/cm2, respectively, has decreased the CA to near 15&#176;, indicating the formation of a superhydrophilic surface. The formation of OH-terminated Si (001), with no measurable change of the wafer&#8217;s surface morphology, has been confirmed by X-ray photoelectron spectroscopy and atomic force microscopy measurements. The selective area irradiated samples were then immersed in a biotin-conjugated fluorescein-stained nanospheres solution for 2 hr, resulting in a successful immobilization of the nanospheres in the non-irradiated area. This illustrates the potential of&#160;the method for selective area biofunctionalization and fabrication of advanced Si-based biosensing architectures. We also describe a similar protocol of irradiation of wafers immersed in methanol (CH3OH) using ArF laser operating at pulse fluence of 65 mJ/cm2 and in situ formation of a strongly hydrophobic surface of Si (001) with the CA of 103&#176;. The XPS results indicate ArF laser induced formation of Si&#8211;(OCH3)x compounds responsible for the observed hydrophobicity. However, no such compounds were found by XPS on the Si surface irradiated by KrF laser in methanol, demonstrating the inability of the KrF laser to photodissociate methanol and create -OCH3 radicals.

We report on a process of in situ alteration of HF treated Si (001) surface into a hydrophilic or hydrophobic state by irradiating samples in microfluidic chambers filled with&#160;H2O2/H2O solution (0.01%-0.5%) or methanol solutions using pulsed UV laser of a relative low pulse fluence.

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Writing Bragg Gratings in Multicore Fibers

Fiber Bragg gratings in multicore fibers can be used as compact and robust filters in astronomical and other research and commercial applications. Strong suppression at a single wavelength requires that all cores have matching transmission profiles. These gratings cannot be inscribed using the same method as for single-core fibers because the curved surface of the cladding acts as a lens, focusing the incoming UV laser beam and causing variations in exposure between cores. Therefore we use an additional optical element to ensure that the beam shape does not change while passing through the cross-section of the multicore fiber. This consists of a glass capillary tube which has been polished flat on one side, which is then placed over the section of the fiber to be inscribed. The laser beam enters the fiber through the flat surface of the capillary tube and hence maintains its original dimensions. This paper demonstrates the improvements in core-to-core uniformity for a 7-core fiber using this method. The technique can be generalized to larger multicore fibers.

We describe a technique for inscribing identical fiber Bragg gratings into each core of a multicore fiber. This is achieved by introducing an additional surface into the optical path to mitigate lensing by the curved surface of the fiber cladding.

Fiber Bragg gratings in multicore fibers can be used as compact and robust filters in astronomical and other research and commercial applications. Strong ...

Characterizing Far-infrared Laser Emissions and the Measurement of Their Frequencies

The generation and subsequent measurement of far-infrared radiation has found numerous applications in high-resolution spectroscopy, radio astronomy, and Terahertz imaging. For about 45 years, the generation of coherent, far-infrared radiation has been accomplished using the optically pumped molecular laser. Once far-infrared laser radiation is detected, the frequencies of these laser emissions are measured using a three-laser heterodyne technique. With this technique, the unknown frequency from the optically pumped molecular laser is mixed with the difference frequency between two stabilized, infrared reference frequencies. These reference frequencies are generated by independent carbon dioxide lasers, each stabilized using the fluorescence signal from an external, low pressure reference cell. The resulting beat between the known and unknown laser frequencies is monitored by a metal-insulator-metal point contact diode detector whose output is observed on a spectrum analyzer. The beat frequency between these laser emissions is subsequently measured and combined with the known reference frequencies to extrapolate the unknown far-infrared laser frequency. The resulting one-sigma fractional uncertainty for laser frequencies measured with this technique is &#177; 5 parts in 107. Accurately determining the frequency of far-infrared laser emissions is critical as they are often used as a reference for other measurements, as in the high-resolution spectroscopic investigations of free radicals using laser magnetic resonance. As part of this investigation, difluoromethane, CH2F2, was used as the far-infrared laser medium. In all, eight far-infrared laser frequencies were measured for the first time with frequencies ranging from 0.359 to 1.273 THz. Three of these laser emissions were discovered during this investigation and are reported with their optimal operating pressure, polarization with respect to the CO2 pump laser, and strength.

We describe the generation of far-infrared radiation using an optically pumped molecular laser along with the measurement of their frequencies with heterodyne techniques. The experimental system and techniques are demonstrated using difluoromethane (CH2F2) as the laser medium whose results include three new laser emissions and eight measured laser frequencies.

The generation and subsequent measurement of far-infrared radiation has found numerous applications in high-resolution spectroscopy, radio astronomy, and ...

Experimental Methods for Investigation of Shape Memory Based Elastocaloric Cooling Processes and Model Validation

Shape Memory Alloys (SMA) using elastocaloric cooling processes have the potential to be an environmentally friendly alternative to the conventional vapor compression based cooling process. Nickel-Titanium (Ni-Ti) based alloy systems, especially, show large elastocaloric effects. Furthermore, exhibit large latent heats which is a necessary material property for the development of an efficient solid-state based cooling process. A scientific test rig has been designed to investigate these processes and the elastocaloric effects in SMAs. The realized test rig enables independent control of an SMA&#39;s mechanical loading and unloading cycles, as well as conductive heat transfer between SMA cooling elements and a heat source/sink. The test rig is equipped with a comprehensive monitoring system capable of synchronized measurements of mechanical and thermal parameters. In addition to determining the process-dependent mechanical work, the system also enables measurement of thermal caloric aspects of the elastocaloric cooling effect through use of a high-performance infrared camera. This combination is of particular interest, because it allows illustrations of localization and rate effects &#8212; both important for efficient heat transfer from the medium to be cooled.
The work presented describes an experimental method to identify elastocaloric material properties in different materials and sample geometries. Furthermore, the test rig is used to investigate different cooling process variations. The introduced analysis methods enable a differentiated consideration of material, process and related boundary condition influences on the process efficiency. The comparison of the experimental data with the simulation results (of a thermomechanically coupled finite element model) allows for better understanding of the underlying physics of the elastocaloric effect. In addition, the experimental results, as well as the findings based on the simulation results, are used to improve the material properties.

Experimental methods for investigation of solid state cooling processes and characterization of elastocaloric material properties of Shape Memory Alloys (SMA) are presented. A custom-built test rig has been designed for controlling and comprehensive monitoring of elastocaloric cooling processes. Furthermore, it provides a validation platform for thermomechanically coupled modeling approaches.

Shape Memory Alloys (SMA) using elastocaloric cooling processes have the potential to be an environmentally friendly alternative to the conventional vapor ...