凍結割断法

freeze fracturing

細胞や組織等の試料内部を露出させるための手法。試料を液体窒素等で凍結後、真空下でナイフを用いて衝撃を与えて試料を割断する。その後、割断面のレプリカを作製して電顕観察する。その際には、そのまま割断面のレプリカを作製する凍結レプリカ法と、割断面の氷を昇華させ、露出した内部構造のレプリカを作製する凍結エッチング法がある。通常、レプリカは真空下で白金や白金パラジウムを試料に蒸着し、その上から炭素を蒸着して作製する。蒸着後、試料をアルカリ系試薬に浸漬して溶解、除去し、レプリカのみをメッシュに載せ観察を行う。

Freeze fracturing is a technique to cut and expose the inner face of a biological specimen such as cells and tissues. This technique freezes the specimen with liquid nitrogen, etc., and then fractures the specimen under vacuum by giving an impact on the frozen specimen using a knife. After the specimen is fractured, a replica of a fractured surface is made for TEM observation. To observe the fractured surface with a TEM, either of two replication techniques is applied. One is freeze replication to make a replica of the fractured surface without any processing. Another is freeze etching by which ice on the fractured surface is sublimated, followed by making a replica of the exposed inner structure without ice.
A replica is usually made under vacuum by depositing platinum or platinum palladium onto the specimen and then by depositing carbon onto the already deposited surface. The specimen is immersed and dissolved in an alkali reagent. Only the replica is mounted on a mesh (grid) for TEM observation.