Double layer coating for high resolution low temperature SEM

Specimen damage caused by mass loss due to electron beam irradiation is a major limitation in Low Temperature Scanning Electron Microscopy of bulk specimens. At high primary magnifications (e.g. 100 000 x) a hydrated sample is usually severely damaged after one slow scan (about 3000 enm-2). The consequences of this beam damage are remarkably reduced by coating the frozen-hydrated sample with a 5-10 nm thick carbon layer. Since this layer covers up surface details, the sample is first unidirectionally shadowed with a thin heavy metal layer (e.g. 2 nm platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in a field emission SEM with the material dependent backscattered electron signal. The method allows for routine observation of large frozen-hydrated samples. By use of an in-lens field emission SEM and a sensitive backscattered electron detector a structural information comparable to the TEM freeze fracture replica technique can be achieved.