Biology Reference
In-Depth Information
layer as 5-10 nm (this includes a phospholipid monolayer and bound proteins), the
lipid ester core should occupy more than 97% of the total volume.
The above architecture is vastly different from vesicular organelles (e.g., endo-
somes), in which the aqueous lumen is delimited by a phospholipid bilayer membrane.
The shape of those organelles is quite variable, ranging from tubular, vesicular, to cis-
ternal, whereas the shape of LDs is almost always spherical, which minimizes the
interface between the hydrophobic lipid esters and the aqueous cytosol. Because of
the unique architecture and the highly hydrophobic content, some caution is necessary
in preparing LD specimens for observation by electron microscopy (EM). On the other
hand, the uniqueness of LDs can be exploited to study its molecular constituents.
In this chapter, we will briefly introduce the basic principle of transmission EM
(TEM) for readers who are not necessarily familiar with the technique, and then dis-
cuss TEM methods that have been used and/or can be used to study LDs. It is im-
portant to remember that only a representative protocol will be described below
and that many variations in details (e.g., combination of fixatives) are possible for
each method.
How images are generated in TEM
In TEM, electron beams are transmitted through a thin specimen placed in a high
vacuum. Among incident electrons, some are scattered by the specimen, whereas
others passing through it are focused onto a detector, such as CCD camera or pho-
tographic film. The difference in the signal intensity between the specimen and the
surrounding area forms images by scattering contrast.
The amount of scattering is dependent on the mass density of the specimen, which
is correlated with the average atomic number. That is, materials of high atomic num-
bers scatter electrons more strongly and thus look darker in the TEM image than
those of low atomic numbers. The dark objects in the image are often depicted as
electron-dense.
Because biological molecules are largely made of atoms of low atomic numbers,
for example, H (hydrogen,
Z
¼
1), C (carbon,
Z
¼
6), N (nitrogen,
Z
¼
7), and O (ox-
ygen,
Z
8), only a low level of scattering occurs to incipient electrons. Moreover,
because resins used to prepare conventional ultrathin sections are polymers made of
carbon, hydrogen, and oxygen, the contrast between the specimens and surrounding
resin areas is too small to make clear images. Therefore, to observe cellular structures
by TEM, specimens are usually treated with various reagents to impart them a higher
electron density. Os (osmium,
Z
¼
¼
76), Pb (lead,
Z
¼
82), and U (uranium,
Z
¼
92) are
used most frequently for this purpose either as a fixative or a stain.
Sample preparation for conventional EM
For conventional TEM, cell samples are generally fixed with aldehydes (i.e., form-
aldehyde and glutaraldehyde). The aldehyde fixatives primarily react with the amino
residue of proteins and stabilize the cellular structure by cross-linking proteins.
