Biology Reference
In-Depth Information
13.2.2.2
Cryoelectron microscopy
A small amount (
l) of the LD preparation is placed on a microgrid using a mi-
cropipette tip. After wicking off any excess fluid with filter paper, the preparation is
frozen quickly by rapidly plunging it into liquid ethane cooled by liquid nitrogen
(
Adrian, Dubochet, Lepault, & McDowall, 1984
). By this procedure, isolated LDs
are embedded in thin vitreous ice formed in microgrid holes. The frozen specimen
is transferred to a cold stage in the cryoelectron microscope using a cryotransfer de-
vice. The specimen is first scanned at a low magnification with a minimum electron
dose. The focus is adjusted at a locus distant from the object and micrographs are
taken by using the minimum dose system (
Fujiyoshi et al., 1980
). A defocus value
is set at 1-2
1
<
m
m
m to enhance the image contrast.
13.2.3
Results and considerations
Isolated LDs are observed as round structures. Their main body is observed in a low
electron density, whereas their periphery was seen as a single electron-dense line of
2-2.5 nm in width (
Fig. 13.3
A and B) (
Tauchi-Sato et al., 2002
). This peripheral
structure is different from a phospholipid bilayer, which is observed as two parallel
electron-dense lines (
Fig. 13.3
C) (
Tahara & Fujiyoshi, 1994
). Because no chemical
FIGURE 13.3
CryoEM of isolated LDs. (A) An isolated LD is observed as a round structure of low electron
density. (B) High magnification of the white box area in (A). A single electron-dense line
of 2-2.5 nm in width is seen in the LD surface (between arrows). Because either a fixative
or stain is used, the electron density is derived from a row of phosphorus in the
phospholipid head group. The result shows that a phospholipid monolayer covers the LD
surface. Reproduced in a modified form from
Tauchi-Sato et al. (2002)
with permission.
(C) The phospholipid bilayer of liposome is observed as two parallel electron-dense lines.
Reproduced from
Tahara and Fujiyoshi (1994)
with permission.