Cryosectioning and Cryo-EM

Cryo-sectioning is used primarily as an alternative to conventional sectioning in immuno-EM studies. Tissues or cells are fixed, cryo-protected, and frozen in a cryogen. They are then thin sectioned on a cryo-ultramicrotome while frozen. The sectiones are thawed on the EM grid, labeled with antibody (if desired), and then either negatively or positively stained. Because the specimen is not embedded in plastic, entry of the antibody into the tissue sections is more effective than labeling on conventional plastic sections.

Negatively stained longitudinal cryosection of muscle (myosin and actin filaments appear white)

Negatively stained longitudinal cryosection of muscle (myosin and actin filaments appear white). Photo: Craig Lab.

Cryo-EM is a state-of-the-art technique used to observe molecules, viruses and macromolecular assemblies in their native, hydrated state, without fixation or staining. Specimens on an EM grid are rapidly frozen (while still wet) by plunging into a cryogen (liquid ethane). They are then observed at low temperature in the CM120 cryo-electron microscope using a liquid nitrogen-cooled specimen holder.

Cryo EM image of frozen-hydrated myosin filaments (no stain, no fix)

Cryo-EM image of frozen-hydrated myosin filaments (no stain, no fix). Photo: Faqing Zhao, Craig Lab.