Abstract

Scanning transmission electron microscopy (STEM) has become a standard option in SEM . The STEM detectors are based on doped semiconductors with low work-functions, where the incident electrons generate multiple electron-hole (E-H) pairs and the free charge carriers can be collected and further processed. Currently, all commercially available STEM detectors are built for EM observations at room temperature. However, FEI Company recently introduced a so called ‘Wet-STEM’ detector for imaging of wet and hydrated specimens in environmental SEMs. The STEM design includes a Peltier stage, where the specimen temperature can be lowered to around 0°C. In collaboration with FEI we went a step further and developed a cryo-STEM holder which allows imaging and analyzing native specimens in both transmission and conventional secondary electron modes from room to liquid nitrogen temperatures (~ -190°C). The new cryo-STEM design involves two parts. First, the main cryo-holder in the SEM sample chamber has been modified to house a state-of-the-art second generation STEM detector. Second, the sledge has been redesigned to meet following four requirements: (1) The TEM grid can be loaded into the sledge in a protected and cooled environment, such as in a FEI Vitrobot™ or cryo-chamber of an ultramicrotome. Further, (2) the sample needs to be maintained at liquid nitrogen temperature during transport and transfer into the cryo-transfer chamber. Also, (3) the frozen sample has to be protected from moisture in air or liquid nitrogen, to avoid ice formation on the sample surface and finally, (4) the sledge needs to be compatible with the stage in a commercial cryo-transfer chamber. Another feature of the cryo-holder is that it can be used in Dualbeam FIB /SEM instruments. Electron transparent cross-sections of frozen biological specimens can be produced by focused ion beam (FIB) milling and subsequently viewed in the same microscope using the cryo-STEM detector. In this presentation I will introduce the cryo-STEM/SEM design and show first results of frozen tissue, cell monolayers and polymers.