Retinal Elements Revealed by SEM Cryomicrodissection

Abstract

The scanning electron microscope was used to study the surface of a frozen and then fractured mouse eye. The frozen fractured surface was microdissected in the scanning electron microscope and the freshly fractured surface thus exposed was observed in the microscope.In order to study the frozen surface it was necessary to develop a cold stage for the Cambridge Stereoscan microscope. This was achieved by fashioning a platform which rests on the standard stage and makes contact with it at three points, two on top and the third below on the back of the stage. It is connected by a braided copper cable to a 1/2-inch by 3-inch brass bar which hangs in front of the stage in the vacuum. The stage functions as a sort of cold block. It is demounted from the microscope and placed in liquid nitrogen and the vitriously frozen sample is attached by means of a set screw in a hole. The cold stage with specimen attached is moved quickly onto the waiting microscope stage. It is necessary to back the specimen chamber with dry N2 gas and to use a liquid N2 trap in the vacuum system in order to minimize ice formation on the stage and to facilitate pumpdown. The stage warms up to the etching temperature (approx -100°C) in 20 to 30 minutes and the ice contamination disappears gradually to reveal the underlying fractured surface. There is no problem in charging up of these samples.