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. 2008 Mar 19;28(12):2959-64.
doi: 10.1523/JNEUROSCI.3189-07.2008.

Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling

Graham Knott  1 , Herschel Marchman, David Wall, Ben Lich
Affiliations

Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling

Graham Knott et al. J Neurosci. .
No abstract available

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Figures

Figure 1.
Figure 1.
a, Three-dimensional drawing of the resin block showing the arrangement of the gallium ion beam (white) that scans parallel to the block (indicated by double-headed arrow) and mills away a layer of resin, creating an imaging face. The electron imaging beam (gray) at an angle of 52° from this vertical face is then used to image the embedded tissue at this surface. b, Scanning electron micrograph of a tissue block in the same orientation as a with a dotted line indicating the milled region, which is shown in c in reverse contrast. Scale bar, 20 μm.
Figure 2.
Figure 2.
a, Reverse-contrast backscattered electron micrograph of an imaging face milled to expose a field of view ∼120 × 115 μm. For this initial preparation phase, a small amount of “curtaining” can be seen in the bottom right corner. This imaging artifact can be removed once serial milling and imaging begins. The image was taken with a beam voltage of 5 keV, beam current of 0.40 nA, and pixel dwell time of 10 μs. The resolution of the image is 30 nm/pixel. Within this image, cell bodies (cb) and blood vessels (bv) are easily visible. b, Reverse-contrast backscattered scanning electron micrograph taken from the imaging face shown in a, of the neuronal cell body indicated as cb, using the same beam parameters, but a resolution of 10 nm/pixel. With these settings, the microscope can generate images that clearly show the stained membranes and many features within the neuropil, e.g., dendrites (de). c, The same image of the region enclosed in the white square in b, which has been enlarged to illustrate the detail that can be seen with these imaging parameters. The image shows an axonal bouton (ab) and dendritic spine head (sp). Scale bars: a, 20 μm; b, 5 μm.
Figure 3.
Figure 3.
a, Reverse-contrast backscattered electron micrograph of a region of neuropil with a resolution of 4 nm/pixel. The electron beam energy was 5 keV. The image shows limited contrast between the stained membranes and intracellular space. This effect appears to blur the image. b, Reverse-contrast micrograph of the same region of the imaging face that was imaged in a. All the imaging parameters are the same, except that the electron beam energy has been lowered to 2 keV. In this image, the membrane contrast is improved and the depth of imaging is decreased. Arrows in both images indicate a myelinated axon. The inset images in both panels show a dendritic spine head (sp), but only in b can the cluster of vesicles be seen along with the presynaptic and postsynaptic densities (arrowheads).
Figure 4.
Figure 4.
a–c, Serial scanning electron micrographs taken from the imaging face each time that 40 nm of resin had been milled away. The beam voltage was 2 keV, the beam current was 0.4 nA, and the dwell time was 100 μs. The magnification was set at 4 nm/pixel, giving a field width of 8.4 μm. In each image, the inset shows a digitally magnified region that is indicated with the white square showing a bouton making an asymmetric synapase (white arrowhead) and a bouton making a symmetric synapse (black arrowhead). The dotted line in c indicates a portion of the field of view that was selected from the stack of 120 images that is shown in supplemental movie 1 (available at www.jneurosci.org as supplemental material).
See this image and copyright information in PMC

References

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