Biomedical Engineering Reference
In-Depth Information
7. Once considerations for specifi c localization of the cell and the
organelle have been optimized, the precise 3D structure of
the organelle and its connections with other organelles at dif-
ferent moments of its life circle can be identifi ed. EM analysis
of serial sections can be supported by digital 3D serial recon-
struction or high-voltage EM tomography. This method is
applicable for CLEM even if cells were looked at as video
CLEM. EM tomography represents an alternative to the serial
section TEM approach for imaging tissue. The method there-
fore relies on the mechanical microtome approach, with a dia-
mond knife cutting thin sections from the block face. Unlike
TEM, in which the contrast in the images is generated by the
elastic scattering of electrons in regions of higher density, the
block-face scanning approach captures electrons backscattered
from below the surface of the block itself. More backscatter-
ing occurs in regions of higher electron-dense stain, making it
possible to image conventionally prepared biological tissue.
This technique can be highly automated, removing many of
the problems associated with manual serial section manipula-
tion and imaging in the TEM, automatically generating
aligned serial images. In EM from FEI, available 3D imaging
software (Xplore3D) automatically collects tomography tilt
series and reconstructs detailed 3D models of intricate bio-
logical structures.
Finally, the EM analysis of serial sections can be supported
by high-voltage EM tomography and/or digital 3D recon-
struction. In electron tomography, a biological specimen of
thickness from 0.1 to 0.5
m is imaged by EM over a range of
tilt angles to provide a series of projections onto planes perpen-
dicular to the beam direction. By back-projecting the images
and summing over all the orientations, it is possible to obtain a
3D reconstruction of the specimen. In addition to correlative
imaging at multiple scales by EM, it is imperative that methods
for correlating 3D light microscopy data with high-resolution
tomography data of the same region at the ultrastructural level
are properly developed, together with techniques for mapping
antigens of interest in 3D reconstructions.
Recent success in electron tomography has depended on
advances in automated data acquisition, which have greatly
reduced the collection times for tilt series and lowered the
required electron doses [
1
]. This powerful technique allows
for 3D reconstructions of intracellular objects with extremely
high spatial resolution, and its use in CLEM is currently being
investigated, with satisfactory results obtained so far. At the
end of the procedure just described, the result is equivalent to
a “snapshot” of the 3D ultrastructure at high resolution of a
single moving intermediate taken at any time chosen by the
experimenter while monitoring the carrier in vivo under light
microscope.
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