Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 225

4. Aliquots of Ab42 aggregates in F-12 Ham medium are
centrifuged at 14,000 × g for 10 min to remove insoluble
structures.
5. The supernatant, defined as the amyloid b-derived diffusible
ligand (ADDL) preparation, consists of a fibril-free solution
of globular structures, as assessed by tapping mode atomic
force microscopy (AFM) ( 7 ).

3. Methods

3.1. Atomic Force
Microscopy

The AFM is one of the most powerful tools for determining the
surface topography of biomolecules in their native state at sub-
nanometer resolution ( 8 ). Unlike X-ray crystallography and elec-
tron microscopy (EM), AFM allows biomolecules to be imaged
not only under physiological conditions, but also while biological
processes are at work. Because of the high signal-to-noise (S/N)
ratio, the detailed topological information is not restricted to crys-
talline specimens. Hence, single biomolecules lacking inherent
symmetry can be directly monitored in their native environment
( 9-11 ). The AFM works in the same way as our fingers in the dark
which touch and probe the environment we cannot see. The use of
a finger to “visualize” an object leads our brain to deduce its topog-
raphy while touching it. The resolution we can get by AFM is
determined by the radius of the fingertip. To achieve atomic scale
resolution, a sharp stylus (radius ~1-2 nm) attached to a cantilever
is used in the AFM to scan an object point by point and contouring
it while a constant small force is applied to the stylus. The forces
(e.g., Van der Waals, electrostatic, magnetic, etc.) generated
between the AFM tip and the sample are transmitted to an attached
flexible cantilever, causing it to bend. The bending of the cantilever
is monitored by the deflection of a laser beam reflected by the
cantilever. In the AFM, the role of the brain is taken over by a com-
puter, while scanning the stylus is accomplished by a piezoelectric
tube. The basic characteristics of AFM are as follows: (1) nearly
no limits to samples; (2) high-resolution sample morphology in
three dimensions; (3) working in various environments, such as
vacuum, air, fluid and low temperature; (4) enabling dynamic
observation in physiological environment. In the fluid, biological
samples can maintain their native state avoiding any damage.
According to the different ways of interaction between the AFM
tip and the sample, the operation modes are classified as contact-
mode, non-contact mode and intermittent contact mode.

The first and foremost mode of operation of AFM, contact mode,
is widely used. As the tip is raster-scanned across the surface, it is
deflected as it moves over the surface corrugation. In constant

3.1.1. Operation Modes

3.1.1.1. Contact Mode