Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

The procedure presented here shows an easy method of preparing MICA supported lipid by layers suitable for high resolution imaging, such as tur single molecule tracking studies. The MICA surface is prepared by splitting off a sandwich of a single sheet of MICA glued between the glass microscope slide and the cover slip using optical adhesive that exposed a flat and extremely thin fresh mica surface. By mounting a small plastic chamber made from a one and a half milliliter vial cup, the volume of lipid vesicles required for bilayer formation can be as low as 30 microliters.

As a first step, take the multi-layer sheet of MICA as it comes from the supplier and cut it with scissors into smaller pieces. Al leaflet into two or three thinner leaflets using a fresh knife blade. Now clean the cover sleeps thoroughly.

This involves placing 0.17 millimeter cover. Sleeps into a glass training jar each cover. Sleep in a separate slot and sonicate in detergent for 30 minutes at 60 Celsius degrees.

Wash the cover. Sleeps 10 to 20 times with mini key water and blow dry with a stream of air or nitrogen. Take a microscope glass slide and clean it with place.

A small drop of about 15 microliters of low viscosity optical ahe on the microscope blast and the globe previously. Cut leaflet hasif on a UV light box for a few minutes. Use scotch tape to peel off few layers of mica.

First few layers will be fragmented, so do it until you see that layers attached to the scotch. Tape are uniform and fully. Cover the tape.

Place a drop of high viscosity optical adhesive on Micah Surface. Make sure there are no bubbles of air over there. Gently place clean cover sleep on the offensive droplet.

Use tweezers to tap down the glass gently to spread the ahe. Again, cure adhesive with UV light for a few minutes. Use knife to split the sandwich consisting of glass.

Slide MICA and cover sleep. In most cases, only very thin layer of MICA will remain glued to the cover sleep. Use dissecting microscope to inspect your MICA surface by making small scratch with the tip of a needle or tweezers.

You can easily say if the surface is actually mica, where the surface is uniformly scratched, like over here or on the optical adhesive, like here where the surface is glowy. Take a cup from one and a half milliliter glass vial, remove gas cant, and glue the cap to the MICA surface using nail polish. Let it dry for a few minutes.

Now you can prepare a lipid bilayer repair lipid by layer using a standard protocol here. A 30 microliters drop of lipid vesicles is placed into the vial cap chamber and will be incubated for 60 minutes before washing your buffer. After washing the sample is ready for imaging.

In this case, it is mounted into custom made sample holder. Single molecules can be easily observed on such prepared MICA surface. Here we compare A-D-O-P-C by layer supported on glass and mica.

Note that the type of movement differs substantially between these two surfaces. The path of single molecules traced in colors in the right panels show clearly that diffusion behavior on MICA differs from that on glass. There is, however, no significant difference in imaging quality.

The average weave of the straight function at half maximum intensity for 20 spots is almost the same for both substrates, indicating that the MICA surface is fully penetrable by light without introducing noticeable optical aberrations. Overall, MSDS shows obvious difference in diffusion behavior. However, for each case, two populations of particles diffusing at different rates can be observed.

Two different diffusion coefficients fast and slow were extracted from double exponential feed to cumulative probability of square displacement. For this DOPC bilayer, both diffusion components are faster on MICA surface, the slow moving particles on MICA are about 10 to 20 times slower than the fast one. Slow population on glass, however, is close to being immobile by here.

We showed that type of surface might make a difference in diffusion behavior of lipid bilayers. Thus, one has to be careful when comparing results between studies on bilayers supported on different substrate such as MICA and the glass imaging bilayers on MICA is usually quite difficult due to complicated preparation we showed however, simply yet effective way of preparing such high quality surfaces.