Biology Reference
In-Depth Information
is used to impart electron density to the specimen, the electron-dense line is
thought to represent a row of phosphorus atoms in the phospholipid head group.
The result directly demonstrates that the LD surface is made of a phospholipid
monolayer.
The diameter of isolated LDs observed by cryomicroscopy is significantly smal-
ler than that of LDs in intact cells observed by conventional EM of ultrathin sections
(
Tauchi-Sato et al., 2002
). The difference suggests that LDs are disrupted into small
ones during the isolation procedure and that the structure could have been modified
from the native one. To circumvent this potential problem, cryoelectron microscopy
of intact cells using vitreous cryosections (CEMOVIS) is needed (
Al-Amoudi et al.,
2004
). This is a promising technique for observing biological structure and macro-
molecular organization in a cellular context and should also provide important infor-
mation on LDs.
13.3
FREEZE-SUBSTITUTION
13.3.1
Rationale
In freeze-substitution, frozen cells are immersed in fixatives in an organic solvent at a
very low temperature so that ice in the sample is dissolved without the formation of
secondary hexagonal ice crystals. Osmium tetroxide and/or uranyl acetate, which re-
act with lipids, is usually used as the fixative. Freeze-substitution is advantageous in
comparison with conventional methods in several respects: (1) because fixation and
dehydration are done at low temperature, ultrastructural changes induced by fixation
and dehydration at room temperature are likely to be avoided; (2) because fixatives
penetrate at a low temperature and begin to react only after the temperature rises,
fixation occurs homogenously throughout the specimen.
To harness the benefit of freeze-substitution maximally, cells are quick-frozen
without chemical fixation and directly transferred to precooled fixative. Quick-
freezing can be done by several different methods. High-pressure freezing is pre-
ferred for observation of intracellular structures because the method can freeze
samples up to 200
m in thickness without ice crystal formation (“vitrification”)
(
Dahl & Staehelin, 1989; Vanhecke, Graber, & Studer, 2008
). After freeze-
substitution, the specimen is warmed, rinsed, and embedded in plastic resin in the
same manner as conventional EM samples.
m
13.3.2
Methods
Cells are quick-frozen by using a high-pressure freezing apparatus (e.g., EM PACT2
HPF [Leica], HPM100 [Leica]) and kept in 2% osmium tetroxide in acetone contain-
ing 1-5% distilled water for 2 days at
85 to
90
C. The samples are then slowly
warmed to 0
C (e.g., over 16-22 h), rinsed with acetone, and embedded in epoxy
resin. There are many different protocols for the freeze-substitution procedure and
