For more than 50 years, thin section Transmission Electron Microscopy (TEM) has been a mainstay of cell biology. Early electron microscopists quickly realized the value of obtaining serial sections to gather three-dimensional (3D) information1
. The application of serial section technique to the study of 3D microcircuitry of the nervous system began more than 30 years ago 2
. New techniques have recently been developed to image larger volumes of tissue with the goal of understanding connectivity in the nervous system3
. With the advent of serial block face (SBF-SEM) 4
and Focused Ion Beam (FIB) 5
methods for fully automated electron microscopic volume imaging and techniques for collecting long series of ultrathin sections onto large glass coverslip 6
substrates, reflection mode Scanning Electron Microscopy (SEM) is moving to the forefront of electron microscopic 3D reconstruction. While traditional TEM methods have provided higher resolution and contrast imagery, they pose greater challenges for the collection and imaging of large specimens volumes due to the small grid sizes and the inherent fragility of the thin film supporting substrates required for transmission EM. Moreover, new Field-Emission SEM tools (FESEM) provide useful resolution that begins to approach that obtained by TEM, while the optimized en bloc
staining methods we describe here provide contrast that also begins to rival that achieved by TEM.
The advantages of FIB- and SBF-SEM, which allow the collection of hundreds or thousands of automatically cut, perfectly aligned thin sections, over serial section TEM, which requires highly skilled collection and imaging techniques 8
, is evident. However, these sections are lost permanently lost due to the destructive nature of the process of FIB and SBF-SEM. In contrast, the automatic collection of thin sections onto tape, and array tomography, where serial thin section ribbons are picked up onto carbon-coated coverslips (), allow structural and multi-marker immunolabeling studies of the same section. Additionally, both tape and coverslip mounted samples permit repeated multi-site imaging of full sections without the interference of grid bars or unstable formvar films for the FESEM (). Finally, the large chamber size of the FESEM easily accommodates serial sections mounted on these large specimen carriers i.e. 60 mm coverslips and 4-inch silicon wafers.
Figure 1 Preparation of serial thin sections. (a) Block face with parallel top and bottom edges. (b) Carbon coated coverslip submerged in Jumbo Histo knife. Use eyelash tool to detach ribbon (arrow) from knife-edge (1) and move it to attach to metal bar holding (more ...)
While major advances have been made in the field of imaging and cutting of sections, the same basic tissue preparation techniques, fixatives and heavy metal stains have not changed significantly in many years. We have combined and compared several classical EM preparation methods to achieve the best staining combination for segmentation of neuronal membranes in FESEM images. Here, we describe a method that can be applied to ultrastructural studies of many tissue types, both for the FESEM and TEM. For 3D serial reconstructions of different tissue types, one should consider which tissue elements are to be enhanced for imaging and choose the appropriate heavy metal stain to do so.
En bloc staining is applicable to both TEM and FESEM imaging, and it advantageous to be able to go directly to the electron microscope to view thin sections without the bother of further post staining. The main limitation of this method can be inadequate contrast due to uneven stain penetration and fragility of the tissue because of heavy metal infusion. Careful attention to solution preparation, incubation times and adequate rinsing steps will minimize background. Precise specimen handling at every step to lessen contamination and tissue loss is imperative when using the osmium impregnation technique.