Improved handling of structural fragile cell-biological specimens during electron microscopic preparation by the...

An exact method of preparation of soft biological specimens for electron microscopic analysis of surface fine structures is described. It allows routine preparations of fragile specimens for SEM and TEM imaging modes. With this procedure physical preparation parameters such as mechanical loads on the specimen surface or changes of temperature are controlled. The wet specimens are premounted in cheap disposable BEEM-containers or glass boats and are constantly kept under liquid in a closed system. The exchange of preparation media is performed continuously and, if necessary, over gradients. For comparative investigations with different EM-modes, at each step of the procedure parts of the specimens may be removed for individual processing. Conventionally prepared critical-point dried specimens are compared to those processed by the exchange technique and preservation of surface fine structures is demonstrated. Shadow-casted clathrin cages and stereo-replicas of virus infected cell cultures are shown in TEM preparations. For SEM, coverslip cell cultures and isolated glomerulus basement membranes are prepared and an additional flat embedding for TEM ultrathin sections is demonstrated.