Amyloid, The Amyloid B -Peptide and Alzheimer’s Disease: Structural Considerations - Molecules: Nucleation, Aggregation and Crystallization - page 296

Atomic force microscopy

Atomic force microscopy (AFM) can visualize fibrils and small aggre-
gates by using a thin tip moving over the sample surface enabling struc-
tures in the nanometer range to be visualized (Harper et al ., 1997). The
resolution is comparable to EM, but AFM has one clear advantage; fibril
growth can be visualized in real time in solution. Thus, the growth of
individual fibrils can be monitored. The technique is relatively slow, only
species deposited on the surface are detected, and the surface affects
polymerization.

Fluorescence correlation spectroscopy (FCS)

By using a confocal microscope setup, a sensitive detector, and an effi-
cient correlator/computer, it is possible to monitor the diffusion of sin-
gle fluorescent molecules through a tiny (femtoliter) volume element.
Since single molecules are detected, the method is highly sensitive. The
diffusion time is approximately proportional to the cubic root of the
mass, i.e. an octamer will have a diffusion time twice that of a monomer.
Therefore, it can be difficult to resolve heterogeneous samples with
species of similar masses. However, single large aggregates can easily
be detected in a mixture of low molecular weight species. This method
was used to follow A -polymerization, showing that large aggregates,

undetected by CD spectroscopy, preceded the formation of fibrils
(Tjernberg et al ., 1999). The formation of these aggregates was clearly
concentration-dependent, and occurred only at A

β

β concentrations above

40 M. Thus, these aggregates are probably not relevant to AD, since

the physiological levels of A

µ

β are around or below 1 nM. Their occur-

rence suggests that A concentrations should be kept below this critical

concentration to avoid non-relevant polymerization pathways. By using
peptides with different fluorescent labels and two lasers, more informa-
tion can be extracted from fluorescence correlation spectroscopy (FCS)
experiments. For instance, in a 1:1 mixture of “red”-labeled and
“green”-labeled A

β

β , 50% of the dimers will carry both labels resulting