title: Double-layer coating for high-resolution low-temperature scanning electron microscopy.
abstract: 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 e-nm-2). The consequences of this beam damage are significantly 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 of platinum) that is in close contact with the biological surface (double layer coating). This heavy metal layer can be visualized in field-emission scanning electron microscopy 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, structural information comparable to that obtained with the transmission electron microscopy freeze-fracture replica technique can be achieved.
mesh_terms: D002244:Carbon; D015925:Cryopreservation; D005613:Freeze Etching; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D010984:Platinum; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*; D012867:Skin / Q000648:ultrastructure*
chemical_list: D010984:Platinum; D002244:Carbon