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Quantomix Ltd., Rehovot, Israel
* Corresponding author: opher{at}quantomix.com
We introduce Wet SEM, a new imaging technology that allows electron microscopy of wet samples. The samples are placed in sealed specimen capsules and are separated from the vacuum in the microscope chamber by an impermeable, electron-transparent membrane. Imaging is performed in a standard scanning electron microscope (SEM) using a backscattered electron detector (BSE) (Fig. 1A). The technique is described in a pending patent application (1).
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The method presents some additional beneficial features, some of which were not wholly anticipated. We obtain resolutions between 10–100 nm, an order of magnitude smaller than could be predicted from the volume of interaction of the electron beam with an aqueous sample. Contrast between materials of low atomic number, such as carbon and oxygen, can be readily detected. Thick samples, such as tissue biopsies, can be imaged without thin sectioning: only the top layer of up to 3 µm is seen. Both the global and high-resolution distribution of colloidal gold labels on cells can be readily determined.
The Wet SEM is uniquely suitable for samples, including lipid-rich structures and hydrated gels, which are adversely affected by standard processes of dehydration that use organic solvents. The method has several practical advantages over standard EM techniques that derive from the simplicity of sample preparation; these advantages include the ability to process and image numerous samples, the ability to look at whole cells, the imaging of tissue specimens (especially of epithelial tissues), and the imaging of myelin sheaths in neural tissue.
During our 2-month stay at the Marine Biological Laboratory, we have explored several applications of the technology with scientists and visitors. One example is shown in Figure 1. Clam egg nuclei were fixed in formaldehyde, then placed in the imaging capsule. A brief centrifugation (500 x g, 5 min) caused the nuclei to adhere stably to the poly-
-lysine-coated partition membrane, and all subsequent treatments were performed in the capsule.
Figure 1B shows a nucleus stained with uranyl acetate; the condensed chromosomes are clearly visible, as are the outline of the nucleus and diffusely stained nuclear proteins. The chromosomes seem to be surrounded by a dark "halo," the significance of which is not yet clear.
Figure 1C shows a portion of a nucleus stained with antibodies to the nuclear pore complex, followed by secondary antibodies that are conjugated to 0.8-nm colloidal gold particles. The silver-enhanced gold particles are visible as bright dots. Note the edge (asterisk) between the region of the nuclear membrane that is attached to the partition membrane of the capsule (att) and the region that is exposed to the solution (exp); the immunolabel, which recognizes the outer aspect of the nuclear pore, has bound only the exposed region (as depicted in the schematic distribution of gold labels in Fig. 1A).
Figure 1D shows a portion of a nucleus stained with antibodies to lamin. Note that, in contrast to Figure 1C, the immunolabeling extends through the entire visible region of the nucleus. We attribute the difference to the location of lamin inside the nucleus, so access by labeling antibodies is not blocked by adhesion to the capsule’s partition membrane.
These results show that the Wet SEM technique can derive meaningful information at high resolution from samples that were subjected to treatments comparable to those used to prepare for light microscopy.
This work was performed in collaboration with Yosef Gruenbaum of the Hebrew University and Robert Goldman of Northwestern University. The XL-30 scanning electron microscope was a generous loan from FEI Company, Peabody, MA.
Literature Cited
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