Biomedical Engineering Reference
In-Depth Information
living cells, whereas light microscopy is not able to reach the same
high level of resolution as EM. In this case, one needs correlative
light-electron microscopy.
Correlative light-electron microscopy (CLEM) was proposed
in 1958 [
1
]; more than several improvements were developed [
2
,
3
].
The correlative video-light-electron microscopy was published in
2000 [
4
]. However, only after 2007, its development greatly accel-
erated (reviewed in ref.
5
). The typical CVLEM procedure includes
several stages: (1) observation of the structures labeled with fl uo-
rescent protein (FP, i.e., green FP) or other fl uorescent markers in
living cells, (2) immobilization (fi xation or freezing), (3) immune
labeling or other types of labeling with gold particles or other
markers suitable for EM, (4) embedding (or freezing), (5) identi-
fi cation of the just examined cell in the resin block or within the
frozen sample, (6) sectioning of thin or thick serial sections (or
analysis of the sample under FIBSEM) and identifi cation of the cell
on the resin block and cutting of thin or thick serial sections, and
(7) EM analysis and structure identifi cation. Each of these steps
could be performed by different ways, and all techniques have their
own advantages and disadvantages.
Recently, CLEM became the leading edge of EM where doz-
ens of protocols could be combined for different purposes. This
fi eld became extremely wide and includes now almost all existing
methods of light and electron microscopy [
6
]. Now, there exist
more than 10,000 different methods of CLEM because at each
step of CLEM, there are several methods which could be used (as
such, the full description of the CLEM within one chapter is equal
to the description of almost all the methods of EM). Therefore, it
is necessary to select the best combination of methods, which
could be used for the specifi c goal of one's study. It is also neces-
sary to use stereology for measurement [
7
].
The fi rst choice is to select what state of cells or tissue will be
examined under LM: in living or fi xed state. If it is necessary to
study living cells, one should select what kind of organelle will be
examined: quickly moving or static. This determines the type of
LM, which is necessary to select. The fi nal selection is what type
of 3DEM is necessary to use. If you need the highest level of reso-
lution along
Z
-axis, the selection is EM tomography. If you need
to have rather high level of resolution, but the most important is
to have high thickness of the volume examined, the best choice is
FIBSEM. The resolution of 3DEM based on serial images depends
on what kind of image is formed. If this image represent the pro-
jection of a structure localized within a defi ned layer of a sample,
the resolution will be a double thickness of serial layers (sections,
virtual layer, or layer, which is eliminated during milling [scraping
of a layer of sample with the help of focused ions]). If serial images
represent the image of the very superfi cial zone of the layer exam-
ined, the
Z
-resolution is equal to the thickness of the single layer
(section). In EM tomography, this resolution is equal to the
