Biology Reference
In-Depth Information
such as ethanol and acetone. At some steps in the above procedure, either before or
after dehydration, samples are often treated with uranyl acetate, which binds to the
phosphate head group of phospholipids and gives an additional electron density to
membranes (
Ginsburg & Wolosin, 1979; Hayat, 2000
). The samples are then infil-
trated with a nonpolymerized epoxy resin solution and are kept at a high temperature
(e.g. 60-70
C) for a few days to polymerize the resin. Finally, ultrathin sections are
sequentially treated with solutions of uranyl acetate and lead nitrate (or lead citrate).
This step is called counterstaining and is done to increase the contrast of cellular
structures.
How do LDs look in conventional TEM
In conventional TEM using ultrathin sections, LDs are observed as round structures
with a homogenous content (
Fig. 13.2
A). The electron density of the LD core varies
depending on cell type, culture condition, the method used to prepare the specimen,
etc. (see
Section 1
for details). In the LD surface, distinct structures cannot always be
observed, but an electron-dense line may be seen (
Suzuki et al., 2012
). Occasionally,
a whorl-like structure exists near the surface of cholesterol-rich LDs (
McGookey &
Anderson, 1983
), but this may be an artifact resulting from the sample preparation.
13.1
PROBING THE LD CORE
13.1.1
Rationale
Except for a few special cases, the LD core looks homogenous and reveals no internal
structure in ultrathin sections. The electron density of the LD core can vary, but gen-
erally it is “grayish,” which is not much different from that of the cytoplasmic matrix.
Along with the absence of the phospholipid bilayer membrane, this feature makes it
rather difficult to identify small LDs unambiguously.
The electron density of lipid esters can be increased by taking advantage of the
reaction of osmium tetroxide to unsaturated fatty acids (
Adams et al., 1967; Hayes
et al., 1963
). That is, by adding polyunsaturated fatty acids, such as docosahexaenoic
acid (DHA), to the cell culture medium, the electron density of LDs increases sig-
nificantly as triglycerides (TG) are synthesized from DHA (
Cheng et al., 2009
). By
using the same principle, it is possible to analyze whether newly synthesized TG
(e.g., electron-dense DHA-rich TG) is incorporated into preexisting LDs or makes
independent LDs on its own.
13.1.2
Methods
13.1.2.1
Observation of small LDs
Cells are first cultured in lipid-deficient medium to reduce existing LDs as much as
possible. By culturing for 1-2 days in a medium supplemented with 2% lipoprotein-
deficient serum (LPDS) (
Goldstein, Basu, & Brown, 1983
), instead of 10% fetal calf
