Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

This procedure prepares glass cover slips with viruses for use in atomic force microscopy and super resolution microscopy, such as fluorescent photo activated localization microscopy. First, clean the glass substrate coat the glass with a thin film of partially biotinylated polyethylene glycol grafted to poly L lysine. Next, treat the surface with avadon to increase the binding affinity of the Biotinylated film.

Now treat the surface with biotinylated antibodies specific to the virus of interest. Then treat the antibody prepared surface with the virus of interest. Unlike spin coating, this technique has the advantage of distributing variants along the surface homogeneously.

In order to optimize microscopy, Place the cover slips vertically in an appropriate size Teflon cover slip rack, and submerge them in a beaker filled with filtered ethyl alcohol sonicate for 30 minutes. Then rinse the cover slips extensively with ultra pure water. Next place the cover slips in a separate clean Teflon rack and beaker filled with one molar sodium hydroxide sonicate for 30 minutes and rinse extensively with ultrapure water.

Now dry the rinsed cover slips under a nitrogen stream and transfer them to a clean dry Teflon rack. Verify that each cover slip is clean from any visible film or particles. Immediately after drying in the nitrogen stream horizontally, place one cover slip into a sterile Petri dish on the center of the cover slip pipette the appropriate amount of biotinylated Polylysine grafted polyethylene glycol in PBS.

Place another clean cover slip atop the fluid in a sandwich method. Make sure that the volume between the active faces of the two cover slips is completely filled with fluid. Cover the Petri dish and incubate at room temperature for 45 to 60 minutes.

Next, pick up the sandwich with a pair of tweezers without pinching out the excess fluid. Now using thumb and index finger, pinch the sandwich lightly and horizontally. Slide the cover slips in opposite directions and separate them without touching the active faces.

Rinse both active faces with 25 milliliters of ultra pure water. Now dry the cover slips with a nitrogen stream and note which cover slip faces are active into a sterile Petri dish. Place a treated cover slip with the active face up pipette an appropriate amount of thawed evidence solution onto the middle of the active face.

Place another cover slip with active face downward to sandwich the fluid, then incubate at room temperature for 25 to 30 minutes. Next, separate the two cover slips as shown earlier. Making sure not to touch the active faces.

Now rinse both active faces with 25 milliliters of ultra pure water and then dry the cover slips with a nitrogen stream. It is important to remember which cover slip faces are active. For future reference, immediately place an avid and treated cover slip active face up in a sterile Petri dish, pipette an appropriate amount of thaw biotinylated antibody onto the middle of the active face.

Place another cover slip with active face downward atop the fluid in a sandwich method. Then incubate at room temperature for 45 to 60 minutes After separating the two cover slips, rinse both active faces with 25 milliliters of PBS. Then place an antibody treated cover slip with active face up in a sterile Petri dish and pipette an appropriate amount of virus onto the middle of the active face.

Now sandwich with a second active cover, slip, cover, and incubate. Now separate the two cover slips. Rinse both active faces with 25 milliliters of PBS and transfer them to a Teflon rack and beaker filled with PBS.

Immediately use the prepared samples immediately or store at four degrees Celsius for up to three days.Here. Wild type varion of vesicular stomatitis virus were anchored to the glass surface and analyzed by atomic force microscopy. The width and length of the virion are 80 nanometers by 180 nanometers, showing minimal distortions from the original size of the virion.

In this experiment, the single virion was imaged by fluorescent photo activation localization microscopy. Using Alexis 6 47 labeled VSVG antibodies. The blue isosurface demonstrates the recovered surface of the virion using FOM microscopy.

After watching this video, you should have a good understanding of how to prepare glass cover slips with viruses for use with atomic force microscopy or super resolution microscopy. It is important to remember at all times which face of the cover slip has the active chemistry on it.