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The overall goal of this technique is to fabricate an array of protein nano-clusters in a supported lipid bilayer, which is compatible with sensitive surface microscopy techniques for cell biology applications. This method can help answer key questions in the field of cell biophysics, in particular, how the nano-clustering of ligands influences T cell activation and adhesion. The main advantage of this technique is that it is easily reproducible in standard biophysical lab and it is compatible with advanced surface sensitive microscopy technique, such as TIRF and RICM.

Though this technique is designed to provide insight into immunology, it can be applied to other cell types with potential applications in cell biology, oncology and tissue engineering. To begin this procedure, clean the glass cover slides and observation chambers as outlined in the text protocol. Then, deposit 70 microliters of 2%silica bead suspension drop by drop onto a cover slide held at a 15 degree incline.

Flip the glass 90 degrees every 15 seconds for one minute while the suspensions dries. It is critical to create a closely-packed monolayer of beads and to avoid the formation of multilayers and oil clusters. That's why the concentration and the volume of the bead solution as well as the hydrophilicity of the slide need to be optimized.

After the liquid has evaporated, place the slide inside a radio frequency magnatron sputtering device onto a rotating table situated 105 millimeters away from an aluminum silicon target. After this, use a turbo-molecular pump to decrease the pressure in the deposition chamber to 2.6 times 10 to the negative fourth pascals. Introduce a pure argon atmosphere with a flux of 10 standard cubic centimeters per minute and a pressure of 0.8 pascals.

Next, switch on the radio frequency power generator. After the plasma is stabilized, sputter for two minutes while keeping the shutter closed, to remove possible impurities from the target surface. Open the shutter, and allow the sputtering to continue for 60 minutes to deposit a 200-nanometer thick layer of aluminum on the slide.

Then cut the flow of argon. Close the gate valve to isolate the turbo-molecular pump from the deposition chamber. After this, vent the chamber with clean nitrogen to reach ambient pressure.

Recover the aluminum-coated slide from the chamber. Immerse the recovered slide in ultra-pure water at room temperature and ultra-sonicate at 50 watts and 50 hertz for 30 seconds. Next, deposit 0.5 milliters of APTES at the bottom of a desiccator.

Place the glass slide on a ceramic grid. Place the grid into the desiccator. Connect the desiccator to a membrane pump.

Then run the pump at maximum power for 30 minutes to generate a low vacuum. Close the valve of the desiccator, and switch off the pump. Heat to 50 degrees celsius for one hour.

After this open the desiccator and collect the slide. Place the collected slide on a PTFE support. Deposit 2 milliliters of 25 micrograms per milliliter biotin dissolved in PBS.

Allow the slide to rest for 30 minutes at room temperature. Then rinse 10 times with PBS. Incubate the slide in a solution of sodium hydroxide and PBS at room temperature overnight.

After this, rinse the slide 10 times with ultra-pure water. After cleaning the Langmuir trough, place PTFE trays in the trough's PTFE enclosure. Then fill it with ultra-pure water.

Set the measured pressure to zero millinewtons per meter. Using a gas-tight glass metal syringe, deposit 30 microliters of 1 milligram per millimeter DOPC and chloroform solution on the water's surface. Close the PTFE barrier until the desired pressure of 27 millinewtons per meter is reached to compress the lipid monolayer.

Next, using a motorized clamp, dip the prepared glass slide into the PTFE enclosure. Hold the slide in the clamp perpendicular to the air-water interface. Raise the slide through the interface at a rate of 15 millimeters per minute while maintaining a constant pressure of 27 millinewtons per meter.

Then, place the glass slide horizontally on the surface of the water above a PTFE tray. Next, using metal tweezers, push the slide down into the PTFE tray, immersing it in the ultra-pure water. Use the tweezers to transfer the PTFE tray containing the slide into a crystallizer filled with ultra-pure water.

Transfer the coated slide underwater into an observation chamber. After this, close the chamber, ensuring that approximately 1 milliliter of water is trapped inside. Another delicate step is the assembly of the sample chamber underwater.

And one should absolutely avoid contact with the air of the deposited bilayers, so to ensure this use a large volume of water and ensure that the whole assembly process can occur underwater. Remove the assembled chamber from the water, check to ensure that the chamber is watertight and free from leaks. Add 500 microliters of PBS, then remove 500 microliters of liquid from the chamber.

Repeat this process 10 times to fully replace the one milliliter of ultra-pure water in the chamber with PBS. Next, introduce 100 microgram per milliliter of bovine serum albumen to the observation chamber. Incubate at room temperature for 30 minutes.

After this, rinse the bilayer by removing and adding 500 microliters from the chamber, 10 times. Then, functionalize with ligands, deposit cells, and observe the proteolipidic nanopattern and SLB fluidity as outlined in the text protocol. In this protocol, a bead mask is used to create a secondary metal mask by controlled deposition of aluminum.

Then, the bead mask is removed, leaving a layer of aluminum with holes. Next, an organo-amino siline, called APTES, is deposited in the vapor phase, followed by deposition of BSA-Biotin in aqueous phase. Then, aluminum is removed, revealing nanometric protein patches.

Finally the inter-dot space is back-filled with a supported lipid bilayer. Epiflourescence images are then taken of the nano-dots and supported lipid bilayer. A composite image shows perfect complementarity with the lipid bilayer deposited uniquely around the protein dots, but not on them.

In an epiflourescence image of a freshly-deposited supported lipid bilayer, the protein dots appear as dark spots in a bright sea of lipids. However, after continuous photo bleaching for 50 seconds, the image shows a halo inside the region delimited by the field diaphragm, indicating that the lipids are mobile. Analysis of the average intensity profile along the edge of the field diaphragm, and the decay of this intensity over time during the bleaching process, reveals that the diffusion constant is five micrometers squared per second.

This technique can be done in two days if it is performed properly. Its's important to maintain very clean conditions during aluminum deposition, and to carefully calibrate the deposition curve. Following this procedure, the patterned slide can be further functionalized.

For example, by adding another biomolecule in the bilayer. We use this to mimic the antigen plant-eating cells, in order to study the T cell activation. So after its development, this technique should interest diverse audiences.

For example, tissue engineers may want to use it as scaffold to grow artificial tissues, and oncologists may use this as a platform for posing fundamental questions about addition of cancer cells. After watching this video you should have a good understanding of how to fabricate an area of protein nano-clusters in supported lipid bilayer, which is compatible with an advanced microscopy technique. While we use this to study T cells, we believe that this substrate have the potential to become the platform of choice to study the interaction of any kind of cells with controlled pattern proteolipidic membrane.