The authors are grateful to the Pioneer Oil Company (Vincennes, IN) for financial support.

1. Minimum instrument specifications
<ol>
	<li>Prepare a tensiometer for the EBM with the following specifications: (i) a dispensing system for controlling the dispensing gas volume; (ii) a camera for capturing the bubble image; (iii) an image analysis software for solving the Laplace-Young equation (LY equation) with the axisymmetric bubble shape analysis algorithm11,12; and (iv) a temperature-controlled sample chamber. 
	NOTE: Usually, the instrument for the EBM can also be used for the pendant drop method, in which a small drop is formed and hangs vertically from the end of a syringe needle.</li>
	<li>Prepare a tensiometer for the SBM with the following specifications: (i) a sample tube holder that is capable of spinning a sample tube holder horizontally at high rotation frequencies of at least 6,000 rpm; (ii) a camera for capturing the image of the spinning bubble in the tube; and (iii) an image analysis software to solve the general LY equation and Vonnegut equation13. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Materials and sample preparation
<ol>
	<li>Obtain pure water from a water purification apparatus. The resistivity of the water at 25 &#176;C at the device output should be 18.2 M&#8486;&#183;cm or close.</li>
	<li>Clean all borosilicate vials, quartz cells, glassware, and magnetic stirring bars by soaking them in pure water for at least 8 h and repeat the soaking process at least one more time. 
	NOTE: The soaking process is aimed at removing residual ions from the glass containers, which can affect the surface tension values significantly.</li>
	<li>Prepare a surfactant solution of interest in the cleaned glassware. 
	NOTE: The surfactant concentration should be lower than its solubility limit in the water.</li>
	<li>Wash each container that will be used for the tension measurements with the sample solution that will be used for the actual measurements prior to sample loading.</li>
	<li>Measure the densities of the liquid samples prior to the tension measurement to three or four significant figures. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Surface tensiometry with the emerging bubble method (EBM)
<ol>
	<li>Calibrate the image-acquiring device of the tensiometer according to the vendor&#39;s user manual. 
	NOTE: The protocol can be paused here.</li>
	<li>Select an inverted stainless-steel needle based on the estimated maximum bubble diameter from the estimated surface tension values. 
	NOTE: the maximum bubble diameter can be estimated from the capillary length, dc ( <img alt="Equation 1" src="/files/ftp_upload/59818/59818eq1.jpg" />, where &#947; is the surface tension (N&#183;m-1), &#916;&#961; is the density difference of the liquid phase and the air (kg&#183;m-3), and g is the gravitational acceleration (m2&#183;s-1)). The maximum bubble volume (Vmax) can be estimated as &#960;dc3/6. 
	NOTE: The protocol can be paused here.</li>
	<li>Place the inverted stainless-steel needle, obtained from the same vendor of the tensiometer, at the tip of the dispensing device. 
	NOTE: An automated dispenser is recommended compared to a manual syringe, because it is easier and more accurate for the users to produce the desired volume, and then, volume and area perturbations to the surface. The smallest volume step of the dispenser is recommended to be less than 1 &#181;L, from 0.2-0.5 &#181;L, in order to produce precise area perturbations. The protocol can be paused here.</li>
	<li>Determine the volume of the liquid sample for the tension measurements such that the depth of the liquid sample is long enough to have the entire inverted part of the dispensing needle submerged, and to have an additional ~20 mm depth of liquid sample between the inverted needle tip and the liquid sample surface.</li>
	<li>Load a liquid sample in the quartz cell and place the cell on top of the sample platform. In our example, the liquid sample volume was 40 mL.</li>
	<li>Adjust the height of the inverted needle such that the tip of the needle is at least 20 mm below the surface of the liquid sample.</li>
	<li>Adjust the position of the inverted needle such that the boundary of the needle tip is parallel to the liquid-air surface.</li>
	<li>Inject ~1 mL of air through the submerged inverted needle to remove impurities that could possibly be present on the tip of the syringe. This procedure is used to improve the surface chemical purity of the air/liquid interface.</li>
	<li>Estimate the maximum bubble volume (Vmax) with a procedure described as follows. First, dispense ~2 &#181;L of air to form a bubble at the tip of the syringe and observe the bubble shape. Then, increase the bubble volume by ~0.5 &#181;L and observe the bubble shape. Repeat the two previous steps until the bubble detaches from the needle tip. This step specifies the Vmax.</li>
	<li>Determine the appropriate range of the bubble volume, based on the previous set of observations. 
	NOTE: The bubble shape should be non-spherical, substantially deformed by gravity, to allow accurate use of the axisymmetric drop shape analysis algorithm, and the bubble volume should be quite smaller than the Vmax to avoid bubble detachment from the needle tip. For the syringe tip with the inner diameter of 0.84 mm, the preferred initial bubble volume is about 4 &#181;L.</li>
	<li>Determine the initial bubble volume based on the bubble volume range determined from the previous step. The initial bubble volume should be close to the middle of the bubble volume range so that the volume, and area, perturbations produce bubbles inside the range.</li>
	<li>Dispense the predetermined initial bubble volume from the previous step to form a bubble at the tip of the inverted syringe tip. Make sure that the bubble is in hydrostatic equilibrium, which means that the surface tension forces balance the gravity (buoyancy) forces. 
	NOTE: It is important to have the bubble pinned outside of the needle tip perimeter to prevent the presence of surfactant solution inside the syringe needle. If the bubble is pinned inside of the needle tip, repeat the step 3.8 to purify the needle tip.</li>
	<li>Measure the dynamic surface tension based on the shape of the produced air bubble at the tip of the needle tip every 1 s, or another predetermined time interval. The recommended numerical algorithm for calculating surface tension is one based on the axisymmetric drop shape analysis method of the LY equation11,12.</li>
	<li>Compare the actual shape of the bubble with the calculated shape. If the two shapes overlap completely, or nearly, one infers that the equilibrium LY equation is valid for each dynamic and slowly-varying shape. This inference is completely valid when the bubble stops moving, and the ST stops changing, to have hydrostatic equilibrium. 
	NOTE: The criterion that the surface tension value is uniform throughout the interface and that hydrodynamic effects are not important is that the calculated bubble interface shape based on the optimal inferred surface tension values overlaps visually with the actual bubble interface shape. More quantitative tests are possible but will not be considered in this article.</li>
	<li>Measure the surface tension as a function of time until the first steady-state surface tension (SST1) is achieved. The SST is defined as a plateau value beyond which the surface tension changes by less than 1 mN&#183;m-1 (or by less than 5%) in several (10 to 100) consecutive dynamic surface tension measurements.</li>
	<li>Record the bubble volume (V1) and the surface area (A1)</li>
	<li>Decrease the bubble volume by removing ~1 &#181;L of air, and record the new bubble volume, V2 and area, A2 (see Figure 1).</li>
	<li>Continue measuring the DST and the areas until the DST reaches the second SST (SST2) at the bubble volume of V2.</li>
	<li>Expand the bubble volume by injecting ~1 &#181;L of air so that V3 &#8776; V1 and A3 &#8776; A1. 
	NOTE: Having V3 and A3 exactly equal to V1 and A1 is not essential.</li>
	<li>Continue measuring DST values until a third SST (SST3) is reached. If the three SST values differ from each other by less than 1.0 mN&#183;m-1, or by 5%, then their average is defined as the equilibrium surface tension (EST). 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Surface tensiometry with the spinning bubble method (SBM)
<ol>
	<li>Calibrate the image-acquiring device of the tensiometer according to the vendor&#8217;s user manual. 
	NOTE: The protocol can be paused here.</li>
	<li>Fill the glass tube of the sample holder, compatible with the spinning tensiometer for the measurement, with a liquid sample and close the lid. No air bubbles should be present inside of the glass tube. 
	NOTE: It is recommended that the sample holder and the glass tube, which are provided by the instrument vendor or are compatible with the tensiometer, be used.</li>
	<li>Place the filled sample holder inside of the spinning chamber of the spinning tensiometer. 
	NOTE: The protocol can be paused here.</li>
	<li>Spin the tube at a low rate of ~500 rpm to prevent the injected bubble from migrating upward and/or attaching to the tube wall. 
	NOTE: The protocol can be paused here.</li>
	<li>Load ~2.0 &#956;L of air in the syringe. 
	NOTE: The protocol can be paused here.</li>
	<li>Insert the syringe needle piercing through the polymeric septum sealing the top of the spinning tube.</li>
	<li>Inject an air bubble of ~2.0 &#956;L into the spinning tube. 
	NOTE: The bubble volume usually remains constant, unless the bubble breaks. If the bubble breaks, it is better to start the process again.</li>
	<li>Increase the rotation frequency of the sample holder to &#957;1 so the bubble inside the glass tube is deformed such that the ratio of the horizontal bubble length (L) and the radius of the middle of the bubble (R) to reach a value of 8 or greater. 
	NOTE: If, with the available instrument, the sample tube cannot be spun at a sufficiently high rotation frequency to allow substantial bubble deformation and have a L/R ratio of 8 or greater, the general LY equation can be used to calculate DST values.</li>
	<li>Adjust the tilt angle of the measuring chamber containing the tube, if necessary, to position the sample tube oriented horizontally, to prevent bubble movement, and to help achieve gyrostatic equilibrium (hydrostatic equilibrium in a rotating fluid) for an axisymmetric shape assumed in the LY equation and algorithm used. 
	NOTE: Gyrostatic equilibrium is defined for rotating bubbles, analogously to the hydrostatic equilibrium of non-rotating bubbles, when the bubble is not moving.</li>
	<li>Measure the DST values at a predetermined time interval. The typical value is 1 s.</li>
	<li>Continue to measure the DST at a fixed rotation frequency, &#957;1, until it reaches a steady-state value (SST1) and record SST1 and the rotation frequency &#957;1 (see Figure 2).</li>
	<li>Record the bubble volume, V1 and area, A1.</li>
	<li>Alter the rotation frequency to a second rotation frequency, &#957;2, to vary the surface area.</li>
	<li>Continue to measure the DST at a fixed rotation frequency, &#957;2, until it reaches a second steady-state value (SST2) and the rotation frequency &#957;2.</li>
	<li>Record the bubble volume, V2 and area, A2. 
	NOTE: V2 should be very close to V1.</li>
	<li>Change the rotation frequency to &#957;3. 
	NOTE: Having &#957;3 exactly equal to &#957;1 is not essential.</li>
	<li>Measure DST values at a fixed rotation frequency, &#957;3, until the third steady-state value, SST3, is reached.</li>
	<li>Record &#957;3 and A3.</li>
	<li>4.19. When the three SST values differ from each other by less than 1.0 mN&#183;m-1 (or by less than 5%), their average is taken to be the &#34;EST&#34;.</li>
</ol>

<ol><li>Shah, D. O., Schechter, R. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aShah%2C+DO+%22Improved+oil+recovery+by+surfactant+and+polymer+flooding%22">Improved oil recovery by surfactant and polymer flooding</a>. , Academic Press Inc. New York. (1977).</li>
<li>Hiemenz, P. C., Rajagopalan, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHiemenz%2C+PC+%22Principles+of+Colloid+and+Surface+Chemistry%22">Principles of Colloid and Surface Chemistry</a>. , Marcel Dekker, Inc. New York. (1997).</li>
<li>Adamson, S. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAdamson%2C+SW+%22Physical+Chemistry+of+Surfaces%22">Physical Chemistry of Surfaces</a>. , Wiley. New York. (1990).</li>
<li>Doe, P. H., El-Emary, M., Wade, W. H., Schechter, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surfactants+for+producing+low+interfacial+tensions%3A+II.+Linear+alkylbenzenesulfonates+with+additional+alkyl+substituents%5BTitle%5D)+AND+%22Journal+of+the+American+Oil+Chemists%27+Society%22%5BJournal%5D)">Surfactants for producing low interfacial tensions: II. Linear alkylbenzenesulfonates with additional alkyl substituents.</a> Journal of the American Oil Chemists' Society. 55 (5), 505-512 (1978).</li>
<li>Chung, J., Boudouris, B. W., Franses, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+Tension+Behavior+of+Aqueous+Solutions+of+a+Propoxylated+Surfactant+and+Interfacial+Tension+Behavior+against+a+Crude+Oil%5BTitle%5D)+AND+%22Colloids+and+Surfaces+A%3A+Physicochemical+and+Engineering+Aspects%22%5BJournal%5D)">Surface Tension Behavior of Aqueous Solutions of a Propoxylated Surfactant and Interfacial Tension Behavior against a Crude Oil.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 537, 163-172 (2018).</li>
<li>Miller, R., Lunkenheimer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+determination+of+equilibrium+surface+tension+values+of+surfactant+solutions%5BTitle%5D)+AND+%22Colloid+%26+Polymer+Science%22%5BJournal%5D)">On the determination of equilibrium surface tension values of surfactant solutions.</a> Colloid & Polymer Science. 261 (7), 585-590 (1983).</li>
<li>Miller, R., Lunkenheimer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adsorption+kinetics+measurements+of+some+nonionic+surfactants%5BTitle%5D)+AND+%22Colloid+%26+Polymer+Science%22%5BJournal%5D)">Adsorption kinetics measurements of some nonionic surfactants.</a> Colloid & Polymer Science. 264 (4), 357-361 (1986).</li>
<li>Lunkenheimer, K., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+homologous+series+of+surface-chemically+pure+surfactants+at+the+water-air+interface+Part+I%3A+Equilibrium+properties%5BTitle%5D)+AND+%22Abhandlungen+der+Akademie+der+Wissenschaften+der+DDR%2C+abteilung+Mathematik%2C+Naturwissenschaften%2C+Technik%22%5BJournal%5D)">Properties of homologous series of surface-chemically pure surfactants at the water-air interface Part I: Equilibrium properties.</a> Abhandlungen der Akademie der Wissenschaften der DDR, abteilung Mathematik, Naturwissenschaften, Technik. (1), 113(1986).</li>
<li>Franses, E. I., Basaran, O. A., Chang, C. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Techniques+to+measure+dynamic+surface+tension%5BTitle%5D)+AND+%22Current+Opinion+in+Colloid+%26+Interface+Science%22%5BJournal%5D)">Techniques to measure dynamic surface tension.</a> Current Opinion in Colloid & Interface Science. 1 (2), 296-303 (1996).</li>
<li>Hua, X. Y., Rosen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+surface+tension+of+aqueous+surfactant+solutions+1.+basic+parameters%5BTitle%5D)+AND+%22Journal+of+Colloid+and+Interface+Science%22%5BJournal%5D)">Dynamic surface tension of aqueous surfactant solutions 1. basic parameters.</a> Journal of Colloid and Interface Science. 124 (2), 652-659 (1988).</li>
<li>Rotenberg, Y., Boruvka, L., Neumann, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+surface+tension+and+contact+angle+from+the+shapes+of+axisymmetric+fluid+interfaces%5BTitle%5D)+AND+%22Journal+of+Colloid+And+Interface+Science%22%5BJournal%5D)">Determination of surface tension and contact angle from the shapes of axisymmetric fluid interfaces.</a> Journal of Colloid And Interface Science. 93 (1), 169-183 (1983).</li>
<li>Boyce, J. F., Schurch, S., Rotenberg, Y., Neumann, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Measurement+of+Surface+and+Interfacial+Tension+by+the+Axisymmetric+Drop+Technique%5BTitle%5D)+AND+%22Colloids+and+Surfaces%22%5BJournal%5D)">The Measurement of Surface and Interfacial Tension by the Axisymmetric Drop Technique.</a> Colloids and Surfaces. 9, 307-317 (1984).</li>
<li>Vonnegut, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rotating+bubble+method+for+the+determination+of+surface+and+interfacial+tensions%5BTitle%5D)+AND+%22Review+of+Scientific+Instruments%22%5BJournal%5D)">Rotating bubble method for the determination of surface and interfacial tensions.</a> Review of Scientific Instruments. 13 (1), 6-9 (1942).</li>
<li>Lin, S. -Y., Mckeigue, K., Maldarelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusion-controlled+Surfactant+Adsorption+Studied+by+Pendant+Drop+Digitization%5BTitle%5D)+AND+%22AIChE+Journal%22%5BJournal%5D)">Diffusion-controlled Surfactant Adsorption Studied by Pendant Drop Digitization.</a> AIChE Journal. 36 (12), 1785-1795 (1990).</li>
<li>Sheng, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modern+chemical+enhanced+oil+recovery%3A+theory+and+practice%5BTitle%5D)+AND+%22Gulf+Professional+Publishing%22%5BJournal%5D)">Modern chemical enhanced oil recovery: theory and practice.</a> Gulf Professional Publishing. , (2010).</li>
<li>Moody, C. A., Field, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfluorinated+surfactants+and+the+environmental+implications+of+their+use+in+fire-fighting+foams%5BTitle%5D)+AND+%22Environmental+Science+and+Technology%22%5BJournal%5D)">Perfluorinated surfactants and the environmental implications of their use in fire-fighting foams.</a> Environmental Science and Technology. 34 (18), 3864-3870 (2000).</li>
</ol>

The authors have nothing to disclose.

The EBM and the SBM are simple and robust methods for determining tension values for air/water or oil/water interfaces at atmospheric pressure. Prerequisite information for these methods is the density of each phase, and no contact angle information is required for determining tension values9. A major limitation of the techniques is that the samples should have a low viscosity, and be single-phase or below the surfactant solubility. The two protocols, the EBM and the SBM, are used for measuring DS...

The determination of the equilibrium surface tension (EST), or the equilibrium interfacial tension (EIFT), of a given air/water or oil/water interface is a critical step for applications in a wide range of industrial areas such as detergency, enhanced oil recovery, consumer products, and pharmaceutics1,2,3,4. Such tension values should be determined indirectly from the dynamic surface tension (DST) or the dynamic interfacial tension (DIFT), because only dynamic tension values are directly measurable. Dynamic surface tension values (i.e., measuring tension values as a function of time) are determined at regular time intervals. Equilibrium tension values are deemed to be determined when the DST values are at steady state. True equilibrium surface tension values are better established when they are stable against perturbations5. Several observations of the tension relaxation after surface area compression have been previously reported by Miller and Lunkenheimer, who used &#160;two classical tensiometry methods, the Du No&#252;y ring and the &#160;Wilhelmy plate methods6,7,8. Those methods are less accurate than the ones used in this study, and those DSTs were measured every few minutes. Numerous techniques have been developed for measuring the surface tension (ST) or interfacial tension (IFT) values of interfaces, but there are only a handful of techniques that can be used to measure DST or DIFT values and allow one to apply perturbations to test the stability of the acquired steady-state tension values9. If the aqueous solution contains surfactant mixtures, and when one of the components adsorbs much faster than the others, then there may be a temporary plateau in the DST curves10. Then the presented methods may not work well in the short time-scales as for one component surfactants, but they still may work if the procedures are extended slightly to cover longer time-scales.
The protocols described here show representative data only for surface tension values of an air/aqueous solution. However, these protocols also apply for the IFT of an aqueous solution against a second liquid, such as an oil, which is immiscible with the aqueous solution and has a smaller density than that of the aqueous solution. Here, we present two robust methods that satisfy these criteria, the emerging bubble method (EBM) and the spinning bubble method (SBM). In both methods, one determines ST values that are based on bubble shapes and do not require contact angle information, which can introduce significant uncertainties and errors to the measurements. For the EBM, area perturbations are introduced by abruptly changing the volume of the bubble emerging from a syringe needle tip. For the SBM, changes in the rotation frequency of the samples are used for area perturbations. The detailed protocols are aimed to guide researchers in the field, such that they can avoid common mistakes or errors in dynamic and equilibrium tensiometry and help prevent inaccurate interpretations of the acquired data.

<table><tbody><tr><td>10 &amp;micro;L, Model 1701 SN SYR, Cemented NDL, Custom gauge, length, point style</td><td>Hamilton</td><td>80008</td><td>gauge: 26s, needle length: 2.5 inch, point style: 2</td></tr><tr><td>Anton Paar Density Meter</td><td>Anton Paar</td><td>DMA 5000</td><td></td></tr><tr><td>Barnstead MicroPure Water Purification System</td><td>Thermo Fisher Scientific</td><td>50132374</td><td></td></tr><tr><td>Emerging bubble tensiometer</td><td>Ram&amp;eacute;-Hart Instrument Company</td><td>Model 790</td><td></td></tr><tr><td>Spinning bubble tensiometer</td><td>DataPhysics Instruments</td><td>SVT 20</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr></tbody></table>

accurate determination of the equilibrium surface tension values with area perturbation tests

We demonstrate two robust protocols for determining the equilibrium surface tension (EST) values with area perturbation tests. The EST values should be indirectly determined from the dynamic surface tension (DST) values when surface tension (ST) values are at steady-state and stable against perturbations. The emerging bubble method (EBM) and the spinning bubble method (SBM) were chosen, because, with these methods, it is simple to introduce area perturbations while continuing dynamic tension measurements. Abrupt expansion or compression of an air bubble was used as a source of area perturbation for the EBM. For the SBM, changes in the rotation frequency of the sample solution were used to produce area perturbations. A Triton X-100 aqueous solution of a fixed concentration above its critical micelle concentration (CMC) was used as a model surfactant solution. The determined EST value of the model air/water interface from the EBM was 31.5 &#177; 0.1 mN&#183;m-1 and that from the SBM was 30.8 &#177; 0.2 mN&#183;m-1. The two protocols described in the article provide robust criteria for establishing the EST values.

Dynamic surface tension and equilibrium surface tension of an aqueous Triton X-100 solution with the EBM 
The SST values of the Triton X-100 solutions against air were measured, and their stability was tested for 5 mM aqueous solution; the CMC for this surfactant in water is 0.23 mM14. The SST1, 31.5 &#177; 0.1 mN&#183;m-1, was obtained approximately 20 s after the bubble was formed (Figure 3). After about 25 s, the surface ...

Watch this Scientific Journal Video about Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests at JoVE.com

Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests

Two protocols for determining the equilibrium surface tension (EST) values using the emerging bubble method (EBM) and the spinning bubble method (SBM) are presented for a surfactant-containing aqueous phase against air.

We demonstrate two robust protocols for determining the equilibrium surface tension (EST) values with area perturbation tests. The EST values should be ...

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7.4K Views.  Purdue University. Deze protocollen kunnen worden opgenomen in spanningsmeetprotocollen. Ze kunnen bijvoorbeeld worden gebruikt om evenwichtsinterfaciale spanning tussen water- en oliefasen te meten. Elk van de twee protocollen in deze video kan worden gebruikt om robuuste en betrouwbare evenwichtsspanningswaarden te verkrijgen.Deze waarden kunnen worden vastgesteld na het testen van hun stabiliteit tegen verstoringen van het gebied. Om te beginnen moet u de tensiometer en het monster voorbereiden zoals ...

Video: Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Bioengineering

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Chemistry

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Environment

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Studying Surfactant Effects on Hydrate Crystallization at Oil-Water Interfaces Using a Low-Cost Integrated Modular Peltier Device

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a temperature regulator, visualization techniques, and inner pressure measurements. The temperature control system contains a low-cost, programmable temperature regulator made with solid-state Peltier components. Along with the temperature control system, we incorporated visualization techniques and internal pressure measurements to study hydrate formation and inhibition in the presence of nonionic surfactants. We studied the hydrate-inhibiting ability of nonionic surfactants (sorbitane monolaurate, sorbitane monooleate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate) at low (i.e., 0.1 CMC), medium (i.e., CMC), and high (i.e., 10 CMC) concentrations. Two types of crystals were formed: planar and conical. Planar crystals were formed in plain water and low surfactant concentrations. Conical crystals were formed in high surfactant concentrations. The results of the study show that conical crystals are the most effective in terms of hydrate inhibition. Because conical crystals cannot grow past a certain size, the hydrate growth rate as a conical crystal is slower than the hydrate growth rate as planar crystal. Hence, surfactants that force hydrates to form conical crystals are the most efficient. The goal of the protocol is to provide a detailed description of an experimental system that is capable of investigating the cyclopentane hydrate crystallization process on the surface of a water droplet in the presence of surfactant molecules.

We present a protocol to study the formation of hydrates in the presence of nonionic surfactants on the interface of a water droplet submerged in cyclopentane. The protocol consists of building a low-cost, programmable, temperature regulator. The temperature control system is combined with visualization techniques and internal pressure measurements.

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

Developmental Biology

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 ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests

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