SEM sample prep – freeze fracturing with liquid Nitrogen

The long awaited freeze fracturing with liquid Nitrogen is finally here! We were thrilled to get to do this procedure and to see the results (see post “Freeze fracture samples under the SEM“). A brief overview: we took infected leaves from a madrone, salal and Douglas-fir, fixed them in glutaraldehyde, dried them in ethanol, froze them in liquid nitrogen, smashed them to bits while frozen, dried them further in the critical point drier, stuck them on stubs and finally coated them with gold-palladium in the sputter coater. Wow – Quite a process! It took us the better part of a day (and some of the day before), and we learned through the process.

After the prep process, they are ready to be looked at with the SEM, or Scanning Electron Microscope.

A special thank you to SITs Ladd, Sina and Dan for all the help – and of course, thank you to Dave and Pranav in Dirks’ lab, who gave us the procedure and were patient with all our questions.

Here are our leaf samples in glutaraldehyde. Glutaraldehyde fixes the samples by crosslinking the proteins which kills the cells quickly. As opposed to drying, where cells shrivel up and lose their structure, fixing these leaves in 2% glutaraldehyde stabilized and preserved the cell structure. As you can see, the samples are floating. We wanted them to be fully immersed in the fixative, so we hooked up the bottle to a vacuum system to pull the air out of the same (see below!)

After the leaves sat in fixative overnight, we rinsed them with Millonig’s buffer and soaked them in increasingly more concentrated solutions of ethanol. This was easy, but time consuming.