Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 229

this point is to keep the sample under liquid to preserve the
molecular structure. This technique is reproducible and
enables observing large sheets of organized molecules.
(b) Vesicle fusion method
The Vesicle Fusion Method requires to get first a vesicle solu-
tion in buffer. The latter is deposited onto mica or another
suitable substrate. During the incubation period, the vesicles
will settle, spread, and fuse to the surface at room tempera-
ture in a relatively short time (e.g. about 15 min for phos-
phatidylcholine vesicles on mica). Usually the lipid films are
not as large as with the LB technique. They form patches on
the surface or include “defects”, where the substrate is not
covered but exposed. This protocol is useful to integrate pro-
teins inside the film to more closely simulate cell membranes
and to study lipid-protein interactions.

3.2. Transmission
Electron Microscopy

The electron microscope (EM) extracts structural information
carried by the scattered electrons. In the fixed-beam transmission,
EM electrons scattered by the irradiated sample are collected over
a narrow solid angle and focused by the objective lens onto the
image plane. The elastically scattered electrons, that is, those
which have changed their direction but not lost any energy on
interaction with the specimen, interfere with the unscattered elec-
trons to produce a phase contrast image. The inelastically scat-
tered electrons, that is, those which have changed both direction
and energy on interaction with the specimen, generate a diffuse
background image that is in some EMs eliminated by an energy
filter. With a resolution of about 2 nm, the electron microscopy
offers a useful technique for the ultrastructural characterization of
the oligomeric aggregates and the more complex prefibrillar
structures eventually growing into protofibrils and mature amy-
loid fibrils The negative staining technique for specimen prepara-
tion has been widely used for transmission electron microscopy
(TEM), although alternative techniques such as high resolution
metal shadowing and cryo-TEM of unstained or cryo-negatively
stained fibrils can be used as well. Negative fibril staining is per-
formed using uranyl acetate, lead citrate, platinum palladium, and
phosphotungstic acid. Negative staining produces quite flat
images of fibrils characterized by two dark edges suggesting a
possible fibril structure of a ribbon formed by protofilaments run-
ning side by side. By using this technique, Tashima et al. investi-
gated the interaction of amyloid beta peptide (Abeta)1-40 and
Abeta1-42 with model membranes and the influence of choles-
terol and monosialganglioside on their deposition and aggrega-
tion ( 12 ). By TEM immunogold labelling of calcitonin, Diociaiuti
et al. observed that the peptide interacts with model membranes
and the binding is favoured by the presence of rafts. In addition,