Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

The overall goal of this technique is to observe two dimensional mixing of two different surfactants and extract the diffusion coefficient from that. This technique can help answer key questions in the biophysics and surface science field, such as fluidity and interactions of different species in cell membranes, as well as interaction between surface active engines. The main advantage of this technique is that we can directly observe mixing of any two surface count monolayers.

Both of them are independently controlled using a very simple setup To form a phospholipid monolayer. First, prepare a phospholipid solution by cleaning a four liter vial with a polytetrafluoroethylene coated cap app. Rinse with acetone, ethanol, and deionized water at least three times.

Then blow nitrogen gas thoroughly into the vial to get rid of the water, dissolve one milligram of phospholipid into one milliliter of chloroform in the vial to obtain a concentration of one milligram per milliliter. Next, add dag phospholipids with less than one more percent of the phospholipid solution. In order to visualize the phospholipid monolayer with a fluorescence microscope, wrap the vial with a polytetrafluoroethylene tape to prevent solvent evaporation and store the sample in a freezer at minus 20 degrees Celsius to deposit the phospholipids onto the air water interface.

First, clean a Petri dish with ethanol and deionized water at least three times. Then fill the Petri dish with 10 milliliters of deionized water to create an air water interface. Spread a few microliters of the phospholipid solution with a micro syringe onto a clean interface to achieve the desired surface pressure and wait for at least 30 minutes to evaporate the solvent completely prior to doing the experiment.

Measure the surface pressure of the phospholipid monolayer with a Wilhelm plate.Tensiometer. Use a cone-shaped apparatus that contains a three millimeter reservoir with two thin channels connected to a large section of the Petri dish to suppress the convective flow of the monolayer, which can disturb morphology, imaging, and surface pressure measurement. To coat the phospholipids at a tip end of a tapered glass capillary.

First, clean a glass slide using acetone, ethanol, and deionized water at least three times. Place the tapered capillary on a clean glass slide and tilt the capillary to facilitate touching the glass slide with a tip end. Drop a few droplets of the phospholipid solution onto the tip end of the capillary that is adhered to the glass slide using a glass syringe and wait at least 30 minutes to evaporate the solvent completely to form a droplet that contains a phospholipid monolayer at the curved surface.

Fill in the tapered capillary with 10 microliters of deionized water. Connect the capillary to an automated micro injector to provide the pressure to form the droplet. Mount the capillary that is connected to a micro injector to a micro manipulator to control the position of the capillary precisely.

Prepare a bright field microscope for imaging the lateral view of the capillary with a CCD camera. This serves as microscope one and enables observation of the precise position of the droplet along the Z axis, as well as estimation of the droplet size. Using a micro manipulator, move the tip end to a position with a lateral view of the tip.

End is well visualized by microscope one and apply a variable pressure with the tip end of the capillary until an appropriate size of droplet is formed. To monitor and control the location of the droplet, prepare an inverted microscope set to serve as microscope two as instructed in the text protocol using the micro manipulator. Move the droplet coated with a phospholipid to a flat air water interface along the Z axis, but do not merge the droplet yet.

Use microscope one. To visualize the lateral view of the droplet. Locate the droplet at the center of the top view of the flat monolayer.

Using the micro manipulator. Use the brightfield microscope mode of microscope two to visualize the top view of the flat monolayer. To calculate a diffusion coefficient more accurately, the Tacho region formed by merging a ness should be a circular shape, so it is important to restrict flow in the Petri dish and make sure that the tip end does not touch the interface.

To merge the droplet onto the flat air water interface, use the micro manipulator to move the droplet further toward the flat interface until the droplet merges onto the interface. Record the series of fluorescent images according to the time after merging the droplet using the fluorescence mode of microscope.Two. Refer to the text protocol for image analysis.

A series of fluorescent images was obtained with time during the recovery process. After merging a droplet coated with A-D-O-P-C monolayer onto a flat DOPC monolayer, a recovery process was observed at 23 millitant per meter of fixed surface pressure. A fit of the change of fractional intensity according to time is shown here.

The R squared value of this fit is 0.999. The diffusion coefficient of the DOPC monolayer obtained from this fit was 27.54 square microns per second at 23 millinewtons per meter of surface for further validation of the fluorescence recovery after merging or fram diffusion coefficients of the DOPC and DPPC monolayers were measured according to surface pressure as shown here. Fram captures the rapid decrease of diffusion coefficient of the DPPC monolayer at approximately nine millinewtons per meter of surface pressure, where a liquid condensed liquid expanded phase transition occurs.

Values of diffusion coefficients agreed well with previous measurements. In addition, an exponential decay of the diffusion coefficient with the surface pressure was observed in the DOPC monolayer consistent with previously published research. Once mastered, this technique can be done in two hours if it is performed properly.

Following this procedure. Mixing between two different lipid species also can be observed by forming two different monolayers, one at the German surface and the other at the flat arrow to interface in order to answer additional questions like interaction between different lipid species after each development. This technique paved the way for researchers in the field of biophysics and surface science to explore fluidity and interactions of different species in cell membranes, as well as interaction between surface active agents.

Don't forget that working with chloro can be extremely hazardous, and precautions such as use of film hood should always be taken while performing this procedure.