Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

The overall goal of the following experiment is to image and probe the physical properties of phase separating membranes using atomic force microscopy. This is achieved by first mixing dilio, phosphatidylcholine, fgo, myelin, and cholesterol at a two to two to one ratio to form a phase separating lipid mixture. Next, the supported lipid bilayers are prepared by depositing the liposome suspension into a MICA surface using atomic force microscopy.

The surface of the bilayer is then scanned, producing a topographic image where structural features like height can be measured. Next force spectroscopy is performed on specific areas of the bilayer to probe the physical properties of the bilayer, most notably the breakthrough force and membrane thickness. Ultimately, the results show differences in properties between different lipid phases based on a FM images and force curves.

The main advantage of this technique over existing methods like fluorescence microscopy, is the increase in resolution and additional properties a FM can probe on the sample. This method can help answer key questions in the field of biophysics, such as the structure of biological membranes and proteins embedded on such membranes. Furthermore, we can also look at the physical properties of the membrane and monitor how changes in the composition or additional of membrane active agents might change these properties.

Generally, individuals new to this method will struggle because of the fragile nature of the sample. This has implications on sample preparation, handling choice of cantilevers and a FM imaging and spectroscopy conditions Begin by dissolving DOPC, spino, myelin and cholesterol into chloroform at the desired concentrations. Then combine 18.38 microliters of DOPC 17.1 microliters of sing myelin and 11.3 microliters of cholesterol to make a solution with one milligram of total lipids and a molar ratio of two to two to one respectively.

Mix a solution by vortexing, dry the mixture using an inert gas such as nitrogen, and then dry it further under vacuum for at least one hour. Next, dissolve the dried lipids in 100 microliters of PBS to a concentration of 10 milligrams per milliliter. Vortex to mixture vigorously for about one minute.

To get a cloudy suspension containing multi laminar vesicles. Ensure that no lipid films are left attached to the sides of the vial. Take 10 microliters of the multi laminar lipid suspension and add 150 microliters of supported lipid bilayer buffer.

Then mix a solution and transfer it to a small two milliliter capped glass vial and seal it with para film sonicate. The mixture at room temperature in a bath sonicate for 10 minutes. During this time, the solution should become clear and will yield small uni or vesicles with a diameter of approximately 50 nanometers.

Wash glass cover slips in water followed by ethanol, and then allow them to air dry. Next, take Micah discs and peel off the outer layer using adhesive tape. Attach the MICA disc to a cover slip using a drop of transparent optically clear glue.

Then attach plastic cylinders to the MICA using vacuum grease and ensure that there are no leaks. Pipee the whole liposome solution directly onto the mica and add an additional 140 microliters of supported lipid bilayer buffer to reach a volume of 300 microliters. Next, add 0.9 microliters of one molar calcium chloride solution to reach a final concentration of three millimolar of calcium, incubate the samples for two minutes at 37 degrees Celsius, and then for at least 10 minutes at 65 degrees Celsius during this step, also prewarm the supported lipid bilayer buffer to 65 degrees Celsius.

Add buffer if necessary to ensure that the sample does not dry up. Covering the sample chamber with a cover slip will also keep the sample hydrated. Then rinse the sample gently with the prewarm supported lipid by layer buffer 10 to 15 times to remove the calcium and unfused vesicles.

Take extra care during the washing steps to keep the bilayer from ever drying out following the wash steps. Cool the supported bilayers to room temperature. Before measurements on the atomic force microscope, set up the atomic force microscope or a FM to perform imaging of the lipid bilayers in a solution.

Using the contact mode in accordance with the manufacturer's specifications, add buffer to the sample to keep it immersed in buffer during the whole experiment. Mount the sample on the A FM stage and make sure that all vibration isolation modules are turned on. Wait for at least 15 minutes for the system to equilibrate and reduce thermal drift.

Next, approach the sample in contact mode with a soft a FM cantilever tip until contact is made. As bilayers are very fragile, minimize the set point of the A FM tip during contact mode imaging to keep the force at minimum and to correct for thermal drift while maintaining contact with the sample. Once imaging is complete, process the images by fitting each scan line to polynomial leveling functions.

Perform line removal for scans that lose contact with the membrane. Using the proprietary software of the A FM manufacturing company, calibrate the cantilever by following the manufacturer's protocol in order to determine the correct conversion of the electrical signal into a force measurement. After imaging an area of the bilayer, select a five micron by five micron region in the bilayer to perform force spectroscopy.

Set up the AFMs that it takes force curves in a 16 by 16 grid of that region, resulting in 256 force curves per region. Then set up the Z piso displacement to 400 nanometers. This determines the maximum range of movement of the z piezo and ensures that the cantilever has fully retracted away from the sample in between measurements.

Next set approach speed to 800 nanometers per second and the retract speed to 200 nanometers per second. Also set the maximum target force for each force curve in this case, 12 nano Newton. After setting up all the parameters, acquire force curves by pressing the run button of the A FM software.

Acquire at least 500 force curves for each condition to ensure good statistics, plot a histogram of the values and then fit the histograms to a Gaussian distribution in order to get the peak and width of the distribution. Because of the lipid composition using this study, two main membrane regions are formed. There is a liquid ordered region that is composed of fing, myelin and cholesterol, and a liquid disordered region that is mainly composed of DOPC.

The height profile from the A FM imaging can provide important information on the membrane structure by looking at the height profile. The bilayer thickness can be measured by utilizing the presence of defects the membrane. The height map can also be used to measure the difference in height between the ordered and disordered phases.

The force curves derived from the force spectroscopy mode are used to measure the breakthrough force by vining the force value at the peak of the force curve as shown here. In addition, the membrane thickness can be derived by subtracting the distance value from the peak of the force curve to the distance value. When the force curve begins to rise again, While attempting this procedure, it's important to remember to keep the B layering solution and monitor the FM assemble constantly to correct for effects of thermal drift and D accumulation on the tip Following this procedure.

Other methods like confocal fluorescence microscopy can be performed in order to answer additional questions like membrane dynamics. After watching this video, you should have a good understanding of how to prepare supported by layers and use a FM to image and investigate the physical properties of these bilayers.