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.
