This paper is supported by grants from Vertex Pharmaceuticals (Ehre RIA Award) and CFF-supported Research EHRE20XX0.

In the present study, samples were collected during a clinically indicated bronchoscopy after obtaining informed consent under a protocol approved by the UNC Institutional Review Board.
1. Sputum/mucus collection and storage
<ol>
	<li>Collect airway mucus via sputum collection or bronchoscopy aspiration.
	<ol>
		<li>Collect sputum either via spontaneous expectoration or induce sputum by 3% hypertonic saline inhalation. Alternatively, directly aspirate mucus from the airways during a bronchoscopy procedure.</li>
		<li>Store collected airway sputum/mucus in sterile specimen cups. In the case of sputum, remove excess saliva from the sample immediately upon collection.</li>
		<li>Place the samples on ice for transport. Limit the transport time to less than 4 h.</li>
	</ol>
	</li>
	<li>Analyze the samples at the time of collection or store at -80 &#176;C until processed.
	<ol>
		<li>Before storage, homogenize the mucus by gently pipetting up and down three to five times with a positive displacement pipette or pipette directly into the microcentrifuge tubes.</li>
		<li>Aliquot the samples for storage in volumes &#8805;500 &#181;L to ensure sufficient volume for experiments. 
		NOTE: Freezing and thawing may affect the viscoelastic properties of the sample. Only compare samples that have undergone similar freeze/thaw cycles.</li>
	</ol>
	</li>
</ol>
2.&#160;Sample preparation
<ol>
	<li>Pipette fresh and frozen sputa/mucus directly or homogenize specimens using a positive displacement pipette by gently pipetting up and down three to five times before aliquoting. 
	NOTE: Homogenization is important for samples that contain thick plugs that can affect reproducibility.</li>
	<li>Aliquot 400-500 &#181;L of the sample into separate microcentrifuge tubes. Prepare as many aliquots as needed for repeat measurements and/or treatment with pharmacological reagents (e.g., rhDNase, N-acetyl cysteine). Incubate the aliquots to be tested at 37 &#176;C for a minimum of 5 min prior to measurement.</li>
	<li>For testing pharmacological agents (optional), use high concentrations of stock solutions to prevent sample dilution.
	<ol>
		<li>Add between 0.4% and 10% volume (to minimize specimen dilution) of the desired reagent (e.g., TCEP) directly onto the sample. Make sure no drop of the compound stays on the side of the tube.</li>
		<li>Incubate the samples at 37 &#176;C for the desired length of time to allow a chemical reaction (&#60;1 h to prevent the proteolytic degradation of the mucus).</li>
		<li>Mix the mucus sample and reagent by flicking the bottom of the microcentrifuge tube every 2 min to allow progressive penetration of the reagent into the mucus sample without compromising the mucin network (e.g., mimicking ciliary beating and mucociliary clearance). When comparing multiple drug reagents, ensure that the incubation time is similar.</li>
	</ol>
	</li>
</ol>
3. Instrument initialization and calibration
<ol>
	<li>Turn on the machine (Table of Materials) and initialize the software.</li>
	<li>Select New Measurement. Enter the sample identification number under Measure ID and the name of the operator under Operator to continue. Enter additional information or comments under Comments.</li>
	<li>Select a geometry set (i.e., rough, or smooth 25 mm parallel plates) and inspect large and small plates carefully to ensure that plates are clean and in perfect condition). 
	NOTE: Rough plates are designed for large volumes (350-500 &#181;L) and smooth plates are designed for smaller volumes (250-350 &#181;L). Using a lower or higher sample volume than recommended can cause inaccurate measurements.</li>
	<li>Insert the large plate firmly on the bottom pulpit.</li>
	<li>Insert the small plate gently on the upper pulpit and lock the plate by slightly rotating until hearing a &#34;click&#34;, which indicates that the plate is properly clamped. Note that free oscillation of the upper plate is normal.</li>
	<li>Wait until the temperature reaches the 37 &#176;C target value. Then, initiate automatic calibration as prompted by the software. 
	&#8203;NOTE: Do not disturb the machine or benchtop surface during this process.</li>
</ol>
4. Sample loading
<ol>
	<li>Using a positive displacement pipette, slowly pipette between 250 and 500 &#181;L of the sample on the center of the large bottom plate. Once deposited on the plate, viscous samples will adopt a dome shape whereas highly elastic samples may require physical severing (use dissecting scissors). 
	NOTE: Avoid introducing air bubbles. If needed, remove residual bubbles by pushing away with a pipette tip.</li>
	<li>Lower the measuring head carrying the small plate via the software and observe the sample. If properly loaded on the bottom plate, the sample will make contact and be centered between the two plates.</li>
	<li>To ensure that the sample fills the gap (i.e., by spreading to the edges of the plates), use the Reduce Gap function until the sample is no longer in a biconcave shape or is aligned with the edge of the plates. The Reduce Gap function lowers the measuring head in 0.1 mm increments and is limited to seven increments. 
	NOTE: Monitor the sample carefully and adjust the gap progressively to avoid overspill.
	<ol>
		<li>If a gap remains after seven increments, click on Redo Installation to return to the initial position and adjust the position and/or volume of the sample.</li>
		<li>If the gap is exceedingly reduced (e.g., biconvex shape), remove the excess sample with a spatula by a circular motion along the edge of the upper plate. Make sure to trim the excess sample gently to avoid shear stress. 
		&#8203;NOTE: At the end of this step, the edge of the sample should be aligned with the edge of the upper plate as shown in the user guidelines.</li>
	</ol>
	</li>
	<li>Lower the protective cover to avoid any accidental projection of contaminated fluids during oscillation.</li>
</ol>
5. Initiate biophysical measurement
<ol>
	<li>To initiate measurement, click on Start Analysis. A full cycle will take 4-7 min.
	<ol>
		<li>Avoid talking loudly and touching the device or the bench during the entire length of the cycle. A quiet environment is particularly important for the first 2 min. 
		NOTE: During the cycle, the instrument performs a standardized strain sweep test, which consists of successive oscillating steps. Each step is a series of 10 oscillations at constant amplitude and frequency (1 Hz), during which the corresponding torque is measured in real-time. The strain and torque signals allow computation of the complex (G*), elastic (G&#39;), and viscous (G&#34;) moduli, as well as the damping ratio (tan &#948;) at each step. Oscillations gradually increase in amplitude, which intensifies the deformation imposed on the sample.</li>
	</ol>
	</li>
</ol>
6. Sample removal
<ol>
	<li>Once the cycle is complete, click on Next to raise the measuring head and generate the sample analysis report. 
	NOTE: For the report, the software computes the recorded data and automatically graphs two curves showing the evolution of the viscous and the elastic moduli in relation to the deformation exerted to the sample and displays the linear viscoelastic regime (i.e., a plateau at low deformation) if present. If no linear regime is detected, the values of G&#39;, G&#34;, G*, and tan &#948; are extracted at 0.05 strain. In addition, the crossover strain and yield stress (&#947;c, and &#963;c) are calculated at tan &#948; = 1. Data are also provided in spreadsheets for each step for further analysis.</li>
	<li>Once the measuring head is fully retracted, raise the protective cover, discard the sample and carefully remove the plates. Clean and disinfect the plates using warm water and soap. 
	NOTE: Dry the geometry set thoroughly before repeated use.</li>
</ol>

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The unique viscoelastic properties of mucus are essential in maintaining healthy airways. Internal and external factors can alter airway mucus biophysical properties, causing clinical complications characteristic of muco-obstructive diseases. Hence, monitoring changes in mucus viscoelasticity might be considered during assessments of disease status and exploration of therapies that reduce mucus viscoelasticity. Empirical studies from the 1980s demonstrated a strong correlation between mucus rheology and airway clearance ...

Muco-obstructive airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and other respiratory conditions, such as viral and bacterial pneumonia, are prevalent health concerns worldwide. While the pathophysiology varies greatly between each condition, a common key feature is abnormal mucociliary clearance. In healthy lungs, mucus lines the airway epithelium to trap inhaled particles and provide a physical barrier against pathogens. Once secreted, airway mucus, composed of ~97.5% water, 0.9% salt, ~1.1% globular proteins, and ~0.5% mucins, is gradually transported toward the glottis by the coordinated beating of cilia1,2. Mucins are large O-linked glycoproteins that interact via non-covalent and covalent bonds to provide the distinct viscoelastic properties of mucus, which is required for efficient transport3. Changes in the ultrastructure of the mucin network caused by altered ion transport, mucin unfolding, electrostatic interactions, cross-linking, or changes in composition can significantly affect mucus viscoelasticity and impair mucociliary clearance4,5. Hence, identifying changes in the biophysical properties of airway mucus is essential to understanding disease pathogenesis and testing novel mucoactive compounds6.
Various factors can lead to the production of aberrant mucus in the lungs. In COPD, chronic inhalation of cigarette smoke triggers mucus hypersecretion as a result of goblet cell metaplasia, as well as airway dehydration via the downregulation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, causing mucus hyperconcentration and small airway obstruction7,8. Similarly, CF, a genetic disorder associated with mutations in the CFTR gene, is characterized by the production of viscous, adherent mucus that is inadequate for transport8,9. In brief, CFTR dysfunction induces airway surface liquid depletion, polymeric mucin entanglement, and increased biochemical interactions, which result in chronic inflammation and bacterial infections. In addition, inflammatory cells trapped in static mucus further exacerbate the viscoelasticity of mucus by adding another large molecule, DNA, into the gel matrix, worsening airway obstruction5. One of the best examples of the importance of mucus rheology on the overall health of the lungs is provided by the example of recombinant human DNFase (rhDNase) in the treatment of cystic fibrosis patients. The effects of rhDNase were first demonstrated ex vivo on expectorated sputum, which showed a transition from viscous mucus to a flowing liquid within minutes10,11. Clinical trials in CF patients demonstrated that reducing airway mucus viscoelasticity with rhDNase inhalation decreased the rate of pulmonary exacerbations, and improved lung function and overall patient well-being12,13,14. As a result, rhDNase inhalation aimed to facilitate clearance became the standard of care for CF patients for more than two decades. Similar clinical benefits were observed with the use of inhaled hypertonic saline for mucus hydration in CF, which correlated with changes in rheological properties and resulted in mucociliary clearance acceleration and improved lung function15,16. Hence, a rapid and reliable protocol to measure mucus viscoelastic properties in clinical settings is important to optimize therapeutic approaches.
The benchtop rheometer tested herein offers a fast and convenient alternative for performing comprehensive viscoelastic measurements of mucus/sputum samples. Using dynamic oscillations with controlled angular displacement, the instrument provides deformation via a pair of adjustable parallel plates (e.g., rough or smooth geometries) to measure the torque and displacement with resolutions of 15 nN.m and 150 nm, respectively17. A default standardized calibration combined with user guidelines adapted for non-rheology specialists allows for straightforward measurements and reduces the risk of operator errors. The device produces a strain sweep curve that is processed and analyzed in real-time (within ~5 min) and automatically provides both linear viscoelastic (G&#39;, G&#34;, G*, and tan &#948;) and gel point (&#947;c, and &#963;c) characteristics (see Table 1).The elastic or storage modulus (G&#39;) describes how a sample responds to stress (i.e., the ability to return to its original shape), while the viscous or loss modulus (G&#34;) describes the energy dissipated per cycle of sinusoidal deformation (i.e., the energy lost due to the friction of molecules). The complex or dynamic modulus (G*) is the ratio of stress to strain, which describes the amount of internal force buildup in response to a shearing displacement (i.e., the overall viscoelastic properties). The damping factor (tan &#948;) is the ratio of the viscous modulus to the elastic modulus, which indicates the ability of a sample to dissipate energy (i.e., a low tan &#948; indicates an elastic-dominant/solid-like behavior, while a high tan &#948; indicates a viscous-dominant/liquid-like behavior). For gel point characteristics, the crossover strain (&#947;c) is the measure of the shear strain, calculated by the ratio of the deflection path to the shear gap height, at which the sample transitions from a solid-like to a liquid-like behavior and occurs, by definition, at oscillation strain where G&#39; = G&#34; or tan &#948; = 1. The crossover yield stress (&#963;c) is a measure of the amount of stress applied by the device at which the elastic and viscous moduli cross. In healthy sputa, elasticity dominates the mechanical response to strain (G&#39; &#62; G&#34;). In muco-obstructive diseases, both G&#39; and G&#34; increase as a result of pathological mucus changes17,18,19. The operational simplicity of the device facilitates onsite measurements and circumvents the need for sample storage/transportation/shipment to an offsite facility for analysis thus avoiding the time and freeze-thaw effects on the properties of these biological samples.
In this study, 8 MDa polyethylene oxide (PEO) solutions of different concentrations (1%-3%) were used to validate the measuring range of a commercial benchtop rheometer (Table of Materials) and the obtained concentration-dependent curve was directly compared to measurements acquired with a traditional bulk rheometer (Table of Materials). The repeatability of rheological measurements was then assessed using bronchoscopically harvested mucus from an intubated patient suffering from status asthmaticus (SA), an extreme form of asthma exacerbation characterized by bronchospasm, eosinophilic inflammation, and mucus hyperproduction in response to an environmental or infectious agent8,20. In this case, the SA patient had been intubated for severe respiratory failure and required ECMO (extracorporeal membrane oxygenation) due to the inability to support the patient effectively and safely with mechanical ventilation alone, despite aggressive standard asthma therapies. During a clinically-indicated bronchoscopy for lobar collapse, thick, clear, tenacious secretions were noted to be obstructing lobar bronchi and were aspirated using saline washings. Immediately following collection, excess saline was removed from the aspirate and the viscoelastic properties of the remaining SA sample were analyzed using the benchtop device. Additional sample aliquots were treated with a reducing agent, tris (2-carboxylethyl) phosphine hydrochloride (TCEP), to determine whether this protocol might be used to characterize therapeutic compound efficacy ex vivo.
The results showed that this protocol and the benchtop device can be used effectively in a clinical setting. The rheological properties determined from PEO concentration-dependent curves (Figure 1A)&#160;were indistinguishable between the tested benchtop device and a traditional parallel plate rheometer (Figure 1B). Triplicate measurements of the SA mucus were repeatable, with a 10% coefficient of variation for G*, G&#39;, and G&#34; endpoints and reflected the substantial abnormalities in mucus viscoelasticity that were clinically apparent in this patient&#39;s case (Figure 1D). Finally, ex vivo treatment with TCEP resulted in a significant reduction in G&#39; and G&#34;, and an increase in tan &#948;, demonstrating responsiveness to the treatment by alterations in the mucin network (Figure 2). In conclusion, this protocol using a benchtop rheometer provides a simple and effective approach to assess viscoelastic properties of mucus samples obtained from the clinic. This capability may be used to facilitate precision medicine approaches to care, as clinicians can test the efficacy of approved mucoactive drugs onsite, which can help identify alternative treatment options. In addition, this approach can be used in clinical trials to examine the effects of investigational drugs.

<table><tbody><tr><td>Capillary Pistons Tips</td><td>Gilson</td><td>CP1000</td><td></td></tr><tr><td>Discovery Hybrid Rheometer-3</td><td>TA Instruments</td><td></td><td>DHR-3 Bulk Rheometer manufactured&lt;br/&gt; by TA Instruments in New Castle, DE: Used to preform rheological tests.</td></tr><tr><td>Graphing Software</td><td>GraphPad Prism</td><td></td><td>GraphPad Software (San Diego, CA) used for data analysis</td></tr><tr><td>Microcentrifuge Tube</td><td>Costar</td><td>3621</td><td></td></tr><tr><td>Peltier plate</td><td>TA Instruments</td><td></td><td>Temperature control system manufactured&lt;br/&gt; by TA Instruments in New Castle, DE</td></tr><tr><td>Polyethylene oxide</td><td>Sigma</td><td>372838</td><td>8 MDa polymer used as mucus simulant</td></tr><tr><td>Positive Displacement Pipette</td><td>Gilson</td><td>M1000</td><td>Pipette used for handling viscous solutions</td></tr><tr><td>Rheomuco</td><td>Rheonova</td><td></td><td>Benchtop Rheometer manufactured by Rheonova in France: Used to preform rheological tests.</td></tr><tr><td>Rough Lower Geometries</td><td>Rheonova</td><td>D-1811-007</td><td>25mm Diameter</td></tr><tr><td>Rough Upper Geometries</td><td>Rheonova</td><td>U-1811-007</td><td>25mm Diameter</td></tr><tr><td>Smooth Upper Parallel Plate</td><td>TA Instruments</td><td></td><td>20mm Diameter</td></tr><tr><td>tris(2-carboxyethyl)phosphine</td><td>Sigma</td><td>646547-10X1ML</td><td>TCEP: Potent reducing agent.</td></tr></tbody></table>

rapid viscoelastic characterization of airway mucus using a benchtop rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

Figure 1 shows the accuracy and repeatability of rheological measurements using concentration-dependent curves of viscoelastic control, i.e., polyethylene oxide (PEO) solution, and status asthmaticus (SA) mucus. Measurements of viscoelastic characteristics of 8 MDa PEO at five different concentrations (1%, 1.5%, 2%, 2.5%, and 3%) were directly compared between the evaluated benchtop rheometer and a traditional bulk rheometer (Table of Materials). In contrast with SA mucus, P...

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Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

rapid-viscoelastic-characterization-airway-mucus-using-benchtop

Research

JoVE Journal

Medicine

3.2K Views.  The University of North Carolina at Chapel Hill. Muco-obstructieve ziekten worden gekenmerkt door abnormale viscositeit van het luchtwegslijm. Het protocol laat zien hoe reologie kan worden gebruikt om de viscositeit van slijm te kwantificeren en gegevens te analyseren om slijmgerichte behandelingen te testen. Slijmreologie biedt specifieke kwantitatieve biomarkers.Het gebruik van een benchtop-apparaat vereenvoudigt de logistiek van het meten van deze biomarkers in klinische monsters zoals menselijke sputummonsters aanzienlijk. Het g...

Video: Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

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 ...

Engineering

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Immunology and Infection

Challenges in Rheological Characterization of Highly Concentrated Suspensions &#8212; A Case Study for Screen-printing Silver Pastes

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the manifold requirements during processing and application of such complex fluids. In this investigation, measuring protocols for a conclusive assessment of different process relevant rheological parameters have been evaluated. This includes the determination of yield stress, viscosity, wall slip velocity, structural recovery after large deformation and elongation at break as well as tensile force during filament stretching.
The importance of concomitant video recordings during parallel-plate rotational rheometry for a significant determination of rheological quantities is demonstrated. The deformation profile and flow field at the sample edge can be determined using appropriate markers. Thus, measurement parameter settings and plate roughness values can be identified for which yield stress and viscosity measurements are possible. Slip velocity can be measured directly and measuring conditions at which plug flow, shear banding or sample spillover occur can be identified clearly. 
Video recordings further confirm that the change in shear moduli observed during three stage oscillatory shear tests with small deformation amplitude in stage I and III but large oscillation amplitude in stage II can be directly attributed to structural break down and recovery. For the pastes investigated here, the degree of irreversible, shear-induced structural change increases with increasing deformation amplitude in stage II until a saturation is reached at deformations corresponding to the crossover of G' and G'', but the irreversible damage is independent of the duration of large amplitude shear. 
A capillary breakup elongational rheometer and a tensile tester have been used to characterize deformation and breakup behavior of highly filled pastes in uniaxial elongation. Significant differences were observed in all experiments described above for two commercial screen-printing silver pastes used for front side metallization of Si-solar cells.

A protocol for a robust and application relevant rheological characterization of highly concentrated suspensions is presented. Silver pastes used for screen-printing application in solar cell production are employed as model systems.

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the ...

Visually Based Characterization of the Incipient Particle Motion in Regular Substrates: From Laminar to Turbulent Conditions

Two different experimental methods for determining the threshold of particle motion as a function of geometrical properties of the bed from laminar to turbulent flow conditions are presented. For that purpose, the incipient motion of a single bead is studied on regular substrates that consist of a monolayer of fixed spheres of uniform size that are regularly arranged in triangular and quadratic symmetries. The threshold is characterized by the critical Shields number. The criterion for the onset of motion is defined as the displacement from the original equilibrium position to the neighboring one. The displacement and the mode of motion are identified with an imaging system. The laminar flow is induced using a rotational rheometer with a parallel disk configuration. The shear Reynolds number remains below 1. The turbulent flow is induced in a low-speed wind tunnel with open jet test section. The air velocity is regulated with a frequency converter on the blower fan. The velocity profile is measured with a hot wire probe connected to a hot film anemometer. The shear Reynolds number ranges between 40 and 150. The logarithmic velocity law and the modified wall law presented by Rotta are used to infer the shear velocity from the experimental data. The latter is of special interest when the mobile bead is partially exposed to the turbulent flow in the so-called hydraulically transitional flow regime. The shear stress is estimated at onset of motion. Some illustrative results showing the strong impact of the angle of repose, and the exposure of the bead to shear flow are represented in both regimes.

Two different methods for characterizing the incipient particle motion of a single bead as a function of the sediment bed geometry from laminar to turbulent flow are presented.

Two different experimental methods for determining the threshold of particle motion as a function of geometrical properties of the bed from laminar to ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Studying Large Amplitude Oscillatory Shear Response of Soft Materials

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl sulfoxide (DMSO) and xanthan gum in water &#8212; two concentrated polymer solutions used as viscosifiers in foods, enhanced oil recovery, and soil remediation. Understanding the nonlinear rheological behavior of soft materials is important in the design and controlled manufacturing of many consumer products. It is shown how the response to LAOS of these polymer solutions can be interpreted in terms of a clear transition from linear viscoelasticity to viscoplastic deformation and back again during a period. The LAOS results are analyzed via the fully quantitative Sequence of Physical Processes (SPP) technique, using free MATLAB-based software. A detailed protocol of performing a LAOS measurement with a commercial rheometer, analyzing nonlinear stress responses with the freeware, and interpreting physical processes under LAOS is presented. It is further shown that, within the SPP framework, a LAOS response contains information regarding the linear viscoelasticity, the transient flow curves, and the critical strain responsible for the onset of nonlinearity.

We present a detailed protocol outlining how to perform nonlinear oscillatory shear rheology on soft materials, and how to run the SPP-LAOS analysis to understand the responses as a sequence of physical processes.

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biology

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size.
The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a &#8216;thick and sticky&#8217; state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are&#160;facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process.
The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer. &#160;
A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi ...

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant adsorption rates, evaluating equilibrium surface tensions as a function of bulk surfactant concentration, and relating how surface tension changes with changes in the interfacial area following equilibration. Simultaneous visualization of the interface using fluorescence imaging with a high-speed confocal microscope allows the direct evaluation of structure-function relationships. In the capillary pressure microtensiometer (CPM), a hemispherical air bubble is pinned at the end of the capillary in a 1 mL volume liquid reservoir. The capillary pressure across the bubble interface is controlled via a commercial microfluidic flow controller that allows for model-based pressure, bubble curvature, or bubble area control based on the Laplace equation. Compared to previous techniques such as the Langmuir trough and pendant drop, the measurement and control precision and response time are greatly enhanced; capillary pressure variations can be applied and controlled in milliseconds. The dynamic response of the bubble interface is visualized via a second optical lens as the bubble expands and contracts. The bubble contour is fit to a circular profile to determine the bubble curvature radius, R, as well as any deviations from circularity that would invalidate the results. The Laplace equation is used to determine the dynamic surface tension of the interface. Following equilibration, small pressure oscillations can be imposed by the computer-controlled microfluidic pump to oscillate the bubble radius (frequencies of 0.001-100 cycles/min) to determine the dilatational modulus The overall dimensions of the system are sufficiently small that the microtensiometer fits under the lens of a high-speed confocal microscope allowing fluorescently tagged chemical species to be quantitatively tracked with submicron lateral resolution.

This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

Adsorption of surface-active molecules to fluid-fluid interfaces is ubiquitous in nature. Characterizing these interfaces requires measuring surfactant ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

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