All the confocal microscopy images were obtained using the Nikon A1RHD Multiphoton upright confocal microscope. We acknowledge the guidance and assistance of the support staff, especially Guillermo Marques, at the University Imaging Center at the University of Minnesota. This work was supported by NIH Grant HL51177. SI was supported by a Ruth L. Kirschstein NRSA Institutional Research Training Grant F32 HL151128.

1. Preparation of capillary tubes
<ol>
	<li>Place the capillary into a capillary puller and run the desired pulling program to make two tapered capillaries with an outside diameter (OD) of ~1 &#181;m at the tip. 
	NOTE: The OD of the capillary before pulling must be the OD specified to fit in the capillary holder in the microtensiometer cell. The inner diameter (ID) of the capillary can vary, but will affect the critical radius of the capillary following pulling. A pulling program is chosen so tha...

The combined CPM/CFM is a powerful tool for examining interfacial dynamics, equilibria, and morphology. This protocol describes the steps necessary for obtaining data with CPM/CFM.
Figure 2 shows the cell design with channels for the capillary, solvent, and heat exchange indicated. The inlet for solvent exchange should be at the bottom of the cell while the outlet should be at the top, allowing for the cell to not overflow during the exchange. In practice, the inl...

Air-water interfaces covered by surfactant films are ubiquitous in daily life. Surfactant-water injections are used to enhance oil recovery from depleted fields and are used as hydraulic fracturing solutions for shale gas and oil. Gas-liquid foams and liquid-liquid emulsions are common to many industrial and scientific processes as lubricants and cleaning agents and are common in food. Surfactants and proteins at interfaces stabilize antibody conformations during packaging, storage, and administration1,2,3,4,5, tear film stability in the eye6,7,8, and pulmonary mechanics9,10,11,12,13,14,15.
The study of surface-active agents or surfactants adsorbing to interfaces and their properties has a long history with many different experimental techniques16,17,18,19,20,21,22,23,24,25,26,27. A recent development is the capillary pressure microtensiometer (CPM), which allows the examination of interfacial properties on highly curved interfaces, at much smaller length scales, while using significantly fewer materials than other common methods9,23,24,25. Confocal fluorescence microscopy (CFM) can be used to study the morphology of lipids and proteins at the air-water interfaces in the CPM22 or on Langmuir troughs20,26,27,28,29. Here a CPM and CFM have been combined to connect morphological phenomena to dynamic and equilibrium interfacial properties to develop structure-function relationships for biological and technological interfaces.
There are numerous parameters of importance in interfacial surfactant systems accessible to the CPM-CFM. In the CPM, a 30-200 &#181;m diameter air bubble is pinned to the tip of a glass capillary tube. In earlier versions of the CPM, the capillary pressure difference between the inside and outside of the bubble was controlled via a water column and oscillatory syringe pump9,30 ; the new version described here replaces these with a higher precision, computer-controlled microfluidic pump. The surface tension (&#947;) is determined via the Laplace equation, &#916;P = 2&#947;/R, from the pressure drop across the interface set by the pump, &#916;P, and optical analysis of the radius of curvature of the bubble, R. The dynamic surface tension of the interface can be determined with 10 ms time resolution following the generation of a new bubble in contact with a bulk liquid containing a soluble surfactant. The surfactant adsorption dynamics can be described by the classic Ward-Tordai equation10,31 to determine essential properties of the surfactant, including the diffusivity, surface coverage, and the relationship between bulk concentration and equilibrium surface tension. Once an equilibrium surface tension is achieved, the interfacial area can be oscillated to measure the dilatational modulus, <img alt="Equation 1" src="/files/ftp_upload/64110/64110eq01.jpg" style="margin: auto;" />, by recording the changes in surface tension, induced by small changes in the bubble surface area, A32. For more complex interfaces that develop their own internal structures such as entangled polymers or proteins, the surface tension, , is replaced by a more general surface stress4,33, <img alt="Equation 2" src="/files/ftp_upload/64110/64110eq02.jpg" style="margin: auto;" />.
Lung stability during breathing may be directly tied to maintaining both a low surface tension and a high dilatational modulus at the alveolar air-liquid interface9,10. All internal lung surfaces are lined with a continuous, microns-thick film of epithelial lining fluid to maintain tissue hydration34. This epithelial lining fluid is primarily water, with salts and various other proteins, enzymes, sugars, and lung surfactant. As is the case for any curved liquid-vapor interface, a capillary pressure is induced with the pressure higher on the inside of the alveolus (or bubble). However, if the surface tension was constant everywhere within the lungs, the Laplace equation, &#916;P = 2&#947;/R, shows that smaller alveoli would have a higher internal pressure relative to larger alveoli, forcing the gas contents of the smaller alveoli to flow to larger, lower pressure alveoli. This is known as &#34;Laplace Instability&#34;9,35. The net result is that the smallest alveoli would collapse and be filled with liquid and become difficult to re-inflate causing part of the lung to collapse, and other parts would over-inflate, both of which are typical symptoms of acute respiratory distress syndrome (ARDS). However, in a properly functioning lung, the surface tension changes dynamically as the air-epithelial fluid interface in the alveolus interfacial area expands and contracts during breathing. If <img alt="Equation 3" src="/files/ftp_upload/64110/64110eq03.jpg" style="margin: auto;" />, or <img alt="Equation 4" src="/files/ftp_upload/64110/64110eq04.jpg" style="margin: auto;" />, the Laplace pressure decreases with decreasing radius and increases with increasing radius so as to eliminate the Laplace instability, thereby stabilizing the lung9. Hence, <img alt="Equation 5" src="/files/ftp_upload/64110/64110eq05.jpg" style="margin: auto;" />, and how it depends on frequency, monolayer morphology and composition, and alveolar fluid composition may be essential for lung stability. The CPM-CFM has also provided the first demonstrations of the effects of interfacial curvature on surfactant adsorption25, monolayer morphology22 and dilatational modulus9. The small volume (~1 mL) of the reservoir in the CPM allows for the quick introduction, removal, or exchange of the liquid phase and minimizes the required quantity of expensive proteins or surfactants10.
Contrast in a CPM-CFM image is due to the distribution of small fractions of fluorescently tagged lipids or proteins at the interface16,27. Two-dimensional surfactant monolayers often exhibit lateral phase separation as a function of surface tension or surface pressure, <img alt="Equation 6" src="/files/ftp_upload/64110/64110eq06.jpg" style="margin: auto;" /> &#960; is the difference between the surface tension of a clean fluid-fluid interface,&#160;&#947;0, and a surfactant-covered interface, &#947;. &#960; can be thought as the 2-D &#34;pressure&#34; caused by the interactions of surfactant molecules at the interface that acts to lower the pure fluid surface tension. At low surface pressures, lipid monolayers are in a liquid-like disorganized state; this is known as the liquid expanded (LE) phase. As the surface pressure increases and the area per lipid molecule decreases, the lipids orient with each other and can undergo a first order phase transition to the long-range ordered liquid condensed (LC) phase16,20,27. The LE and LC phases can coexist at various surface pressures and can be visualized as fluorescently tagged lipids are excluded from the LC phase and segregate to the LE phase. Thus, the LE phase is bright and the LC phase is dark when imaged with CFM16.
The goal of this manuscript is to describe the steps necessary to build and operate the combined confocal microscope microtensiometer. This will allow the reader to perform adsorption studies, measure surface tension, rheological behavior, and examine interfacial morphology simultaneously on a micron-scale air/water or oil/water interface. This includes a discussion of how to pull, cut and hydrophobize the required capillaries, instructions for using pressure, curvature, and surface area control modes, and interfacial transfer of insoluble surfactant to the microtensiometer curved interface.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 O.D. Tygon tubing</td>
			<td>Fischer Scientific</td>
			<td></td>
			<td>Tubing</td>
		</tr>
		<tr>
			<td>A1RHD Multiphoton upright confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal Microscope</td>
		</tr>
		<tr>
			<td>Acid Cleaning Solution</td>
			<td></td>
			<td></td>
			<td>Sulfuric acid and Alnochromix diluted with water 50% by volume, wait until clear befor diluting</td>
		</tr>
		<tr>
			<td>Alnochromix</td>
			<td>Alconox</td>
			<td>2510</td>
			<td>Mixed with sulfuric acid to package instructionand diluted to make acid cleaning solution</td>
		</tr>
		<tr>
			<td>Ceramic glass cutter</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>650471</td>
			<td>HPLC Plus</td>
		</tr>
		<tr>
			<td>Curosurf</td>
			<td>Chiesi</td>
			<td></td>
			<td>&#160;Lung Surfactant</td>
		</tr>
		<tr>
			<td>Di Water</td>
			<td></td>
			<td></td>
			<td>18.5 M&#937; - cm</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>any</td>
			<td></td>
			<td>200 proof used for hydrophobization, denatured used for cleaning</td>
		</tr>
		<tr>
			<td>Fiber-Lite Model 190 fiber optic illuminator</td>
			<td>Dolan-Jenner Industries Inc.</td>
			<td>281900100</td>
			<td>Light source; other light sources should work as well</td>
		</tr>
		<tr>
			<td>Flow EZ F69 mbar w/Link Module</td>
			<td>Fluigent</td>
			<td>LU-FEZ-0069</td>
			<td>Microfluidic Pump</td>
		</tr>
		<tr>
			<td>Fluigent SDK VIs</td>
			<td>Fluigent</td>
			<td></td>
			<td>Required for CPM virtual Interface</td>
		</tr>
		<tr>
			<td>Fluoroelastomer gaskets</td>
			<td></td>
			<td></td>
			<td>Machined from 1 mm thick Viton sheet, See figure 3</td>
		</tr>
		<tr>
			<td>Gas filter</td>
			<td>Norgren</td>
			<td>F07-100-A3TG</td>
			<td>Put between microfluidic pump and pressure regulator</td>
		</tr>
		<tr>
			<td>Gas regulator</td>
			<td>Norgren</td>
			<td>10R0400R</td>
			<td>Steps down pressure from sorce to range of pump, connected to gas filter range 2-120 psi</td>
		</tr>
		<tr>
			<td>Glass Capilary</td>
			<td>Sutter Instruments</td>
			<td>B150-86-10</td>
			<td>Borosilicate glass O.D. 1.5 mm I.D. 0.86 mm</td>
		</tr>
		<tr>
			<td>Glass Slide</td>
			<td>any</td>
			<td></td>
			<td>75 mm x 25 mm</td>
		</tr>
		<tr>
			<td>Glass Syringe</td>
			<td>Hamilton</td>
			<td>84878</td>
			<td>25 &#956;L glass syringe</td>
		</tr>
		<tr>
			<td>Hydrophobizing Agent</td>
			<td>Sigma-Aldrich</td>
			<td>667420</td>
			<td>1H,1H,2H,2H-Perfluoro-octyltriethoxysilane 98%, other hydrophobic triethoxysilane can be substituted</td>
		</tr>
		<tr>
			<td>Insoluble surfactant</td>
			<td>Avanti</td>
			<td>850355C-200mg</td>
			<td>16:0 DPPC in chloroform</td>
		</tr>
		<tr>
			<td>LabVIEW Software</td>
			<td>National Instruments</td>
			<td></td>
			<td>2017</td>
		</tr>
		<tr>
			<td>Longpass Filter</td>
			<td>ThorLabs</td>
			<td>FEL0650</td>
			<td>650 nm Longpass filter, wavelength must remove excitation lazer frequence</td>
		</tr>
		<tr>
			<td>Lyso-PC</td>
			<td>Avanti</td>
			<td>855675P</td>
			<td>16:0 Lyso PC 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine</td>
		</tr>
		<tr>
			<td>Masterflex L/S variable speed analog consol pump system w/&#160; Easy-Load II pump head</td>
			<td>Masterflex</td>
			<td>HV-77916-20</td>
			<td>Peristaltic Pump</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>R2019</td>
		</tr>
		<tr>
			<td>Micropipette Puller P-1000</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Capillary Puller</td>
		</tr>
		<tr>
			<td>Microtensiometer Cell and Holder</td>
			<td></td>
			<td></td>
			<td>Cell machined from PEEK, holder machined from aluminum, See Figure 3 and 4</td>
		</tr>
		<tr>
			<td>Microtensiometer Objective</td>
			<td>Nikon</td>
			<td></td>
			<td>Fluor 20x/0.50W DIC M/N2 &#8734;/0 WD 2.0 mm</td>
		</tr>
		<tr>
			<td>NI Vision Development Module</td>
			<td>National Instruments</td>
			<td></td>
			<td>Required for CPM virtual Interface</td>
		</tr>
		<tr>
			<td>PEEK finger tight fittings</td>
			<td>IDEX</td>
			<td>F-120x</td>
			<td>10-32 Coned Ports</td>
		</tr>
		<tr>
			<td>PEEK plug</td>
			<td>IDEX</td>
			<td>P-551</td>
			<td>10-31 Coned Ports</td>
		</tr>
		<tr>
			<td>pippette tips</td>
			<td>Eppendorf</td>
			<td>22492225</td>
			<td>100 &#956;L - 1000 &#956;L, Autoclaved</td>
		</tr>
		<tr>
			<td>Plastic Forceps</td>
			<td>Thermo Scientific</td>
			<td>6320-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Syringe</td>
			<td>Fischer Scientific</td>
			<td>14-955-459</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Plumbing parts</td>
			<td>Fischer Scientific</td>
			<td></td>
			<td>3-way valves and other plumbing parts to connect tubing.</td>
		</tr>
		<tr>
			<td>Research Plus 1-channel 100 &#956;L&#8211;1000 &#956;L</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td>Micro pipetter</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>any</td>
			<td></td>
			<td>Used for acid cleaning solution</td>
		</tr>
		<tr>
			<td>T Plan SLWD 20x/0.30 OFN25 WD 30 mm</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal Microscope Objective</td>
		</tr>
		<tr>
			<td>Texas Red DHPE triethylammonim salt</td>
			<td>Thermo Fischer Scientific</td>
			<td>1395MP</td>
			<td>Fluorophore</td>
		</tr>
		<tr>
			<td>Vaccum Pump</td>
			<td>Gast</td>
			<td>DOA-P704-AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A major source of measurement error arises from the capillaries that have defects either from the cutting process (Figure 5A,B) or the coating process (Figure 5D). Both types of defects lead to errors in determining the bubble shape and size by the optical image analysis system, leading to inaccurate surface tension values. It is important to carefully examine each new capillary after it is pulled and coated under the optical microscope before i...

Watch this Scientific Journal Video about Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces at JoVE.com

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces

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 ...

The method combines a confocal microscope with a capillary pressure micro tensiometer creating a powerful tool which can be used to study curve fluid fluid interfaces at high spatial and temporal resolution. This technique can examine structure and function relationships for surface active materials by taking simultaneous measurements of surface properties and confocal images of surface morphologies on highly curved interfaces. We hypothesize products of inflammation inhibit lung surfactant causing breathing problems associated with accurate respiratory distress syndrome.This door can investigate lung surfactant properties and morphology and lung stability subjected to such materials. To begin, assemble the CPM cell by placing the large side of the capillary into the top of the cell until it pushes through to the underside of the cell. Gently tighten the peak connector to secure the capillary and then attach the tube from the microfluidic pump to the large side of the capillary.As necessary attach the solvent exchange reservoir and or temperature control bath to the respective inlets and outlets on the CPM cell. Otherwise, plug the unused inlets and outlets. Attach the CPM cell to the confocal microscope stage roughly aligning it with the CFM objective, CPM camera and CPM light source.Open the gas flow to the microfluidic pump at the recommended operating pressure of the pump and ensure the flow to the capillary is open. Start running the CPM virtual interface setting the baseline pressure to 25 millibars and switching to pressure control mode. Then fill the CPM cell with water using a pipette.Focus on the capillary tip using the micro tensiometer camera and arrange the annus to overlap the bubble. Bring the immersion objective of the confocal microscope in contact with the fluid in the cell and focus on the bubble using the confocal microscope. Click on reset bubble and make sure that a new bubble is formed.If the bubble does not pop increase the reset pressure or increase the reset delay time in the bubble reset tab below the viewing window. Take out the water via the direct to cell syringe empty it and reattach it. Fill the cell with the desired sample.Using an autoclaved pipette keeping the CPM software in pressure control mode, ensuring that the initial surface tension is around 73 milli Newton per meter when a new bubble interface is created. After determining the radius of the newly formed bubble input that value into the centerline area control and change the control type to area control by clicking on the area control tab. Start recording the confocal video, then click on reset bubble, and immediately click on collect data.Adjust the data recording rate according to the total absorption time of the sample by sliding the bar. After the end of the experiment. Save the file by choosing the correct file path and clicking on the save button.Stop and save the recording on the CFM as well. Enter the desired baseline value oscillation percent and oscillation frequency and select the appropriate tab by deciding whether oscillation will be a pressure oscillation, area oscillation, or curvature oscillation. Start recording the confocal video and click on collect data on the CPM software.Choose a data acquisition rate to give an adequate number of data points to each oscillation cycle. If other oscillation amplitudes or frequencies are desired, change the values during the experiment and save the results. First, insert the inlet tube of the peristaltic pump into the bottle of desired exchange solution and insert the outlet tube into the waste container.Start recording the video in confocal software then click on collect data on the CPM software. Next, set the peristaltic pump speed. If multiple fluids need to be exchanged, stop the peristaltic pump and connect the inlet to another exchange solution.After the exchange has finished save the results as demonstrated previously. Microtensiometer results for a constant pressure absorption demonstrated that the bubble surface area increases significantly throughout the study and leads to much slower absorption than the constant surface area case. During the absorption process, the fluorescent signal from the interface started out low and increased as surfactant absorbs to the interface.If the surfactant forms surface domains these domains can be observed forming and growing. When performing an oscillation study the oscillation is only truly sinusoid for the parameter that is being controlled. As shown here for a surface area controlled study this is important when calculating the surface dilatational modulous as the oscillation in the area must be sinusoidal.The surface tension and area data collected from an oscillation study was used to directly calculate the interfacial dilatational modulus of the surfactant layer. When oscillating a phospho lipid mono layer, the motion of the black liquid condensed domains can be observed throughout the continuous colored liquid expanded phase. The distinct domains on the interface reorganized into a branching network that grew to cover the interface as the oscillations took place on the curved bubble.This is corroborated by a simultaneous change in surface tension and surface dilatational modulus. During a solvent exchange study for a lung surfactant monolayer with buffer solution and then lasso PC solution the morphology of the domains changed drastically as the exchange took place. It is important to visually follow the oscillation and pinning of the bubble to make sure that the capillary is square and the bubble maintains its spherical shape.In addition to air-water interfaces, oil-water interfaces can also be studied to determine stability and properties of emulsions. This technique offers a single changes approach to constructing structure property relationships of curve interfaces. It allows for new exploration of factors governing interracial morphology previously only studied on flat interfaces.

microtensiometer-for-confocal-microscopy-visualization-dynamic

Research

JoVE Journal

Engineering

2.2K vistas.  University of Minnesota. El método combina un microscopio confocal con un microtensiómetro de presión capilar creando una poderosa herramienta que se puede utilizar para estudiar las interfaces de fluidos de curva a alta resolución espacial y temporal. Esta técnica puede examinar las relaciones de estructura y función para materiales tensioactivos tomando mediciones simultáneas de propiedades superficiales e imágenes confocales de morfologías superficiales en interfaces altamente curvadas. Nuestra hipótesis...