Low-temperature field-emission scanning electron microscopy of high-pressure frozen samples reveals structural information below 10 nm

Abstract

High-pressure-frozen samples can be imaged in the frozen hydrated state with a low temperature scanning electron microscope (LTSEM) thereby preventing many of the fixation and drying artifacts related to chemical fixation and dehydration. One limiting factor of high resolution LTSEM, however, is mass loss of the hydrated specimen due to electron beam irradiation. This effect is significantly reduced by using double layer coating and backscattered electron imaging (Walther et al., 1995). Thereby the sample is first coated with a contrast forming heavy metal layer (1 - 3 nm thick) and afterwards with a stabilising carbon coat (5 - 10 nm; Walther and Hentschel, 1989). The heavy metal layer is imaged with backscattered electrons that penetrate the carbon layer. Using this method structural information comparable to the TEM-replica (“freeze-etch”) technique can be achieved, without the need of replica cleaning. The method has been especially useful for samples where replica cleaning is difficult; e. g. investigation of new materials for filling cavities in dentistry, or the effects of drug application to human skin, or investigation of new materials in polymer science.