DOUBLE-LAYER COATING FOR HIGH-RESOLUTION LOW-TEMPERATURE SCANNING ELECTRON-MICROSCOPY

Specimen damage caused by mass loss due to electron beam irradiation i s a major limitation in low-temperature scanning electron microscopy o f bulk specimens. At high primary magnifications (e.g. 100 000x) a hyd rated sample is usually severely damaged after one slow scan (about 30 00 e(-) nm(-2)). The consequences of this beam damage are significantl y reduced by coating the frozen-hydrated sample with a 5-10-nm-thick c arbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in fi eld-emission scanning electron microscopy with the material-dependent backscattered electron signal. The method allows for routine observati on of large frozen-hydrated samples. By use of an in-lens field-emissi on SEM and a sensitive backscattered electron detector, structural inf ormation comparable to that obtained with the transmission electron mi croscopy freeze-fracture replica technique can be achieved.