Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

We have developed a cryo-STEM holder which is compatible with commercial cryo-transfer stages for scanning electron microscopes (SEM). The new cryo-STEM setup allows the image and analysis of frozen specimens in both transmission and conventional secondary electron modes down to liquid nitrogen temperatures.In these days most electron microscope manufacturers offer scanning transmission electron microscope (STEM) detectors for their SEMs. The solid state STEM detectors are based on doped semiconductors with low work-functions, where the incident electrons generate multiple electronhole (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. The STEM design includes a Peltier stage, where the specimen temperature can be lowered to around 0°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. The STEM detector consists of 14 diode segments as illustrated in Figure 1. Similar to a dedicated STEM instrument the arrangement of the diodes allows obtaining true bright and dark field images. 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 TM 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 humidity 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 the cryo-transfer chamber.Photographs of the cryo-holder and sledge are shown in Figure 2. The TEM grid sits on a rod which slides into a protective cavity during transport and transfer (Figure 2a). After the transfer into the SEM cryo-transfer chamber, the fracture knife is used to move the sample back into the open STEM position (Figure 2b). Another feature of the sledge is that the clamp, which holds the grid in position, is manufactured from a low background material to minimize the signal from the holder during the EDS analysis.In this presentation we will introduce the cryo-STEM/SEM design and show first results of frozen tissue, cell monolayers and polymers.

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