Biology Reference
In-Depth Information
In the last few years, a number of luorescent-based techniques have
been applied to study the organization of the cellular plasma membrane.
In particular, confocal, wide-ield and total internal relection microscopy
can resolve structures on the cell membrane and track proteins and other
biomolecules in living cells ( Fig. 9.1 ) . However, a major drawback of standard
light microscopy is the fundamental limit of the attainable spatial resolution,
which is dictated by the laws of diffraction. This diffraction limit originates
from the fact that it is impossible to focus light to a spot smaller than half
its wavelength. In practice, this means that the maximal resolution in optical
microscopy is ~250-300 nm. Since a large body of evidence indicates that
dynamic cell-signalling events start by oligomerization and interaction
of individual proteins (i.e., on the molecular scale), the need for imaging
techniques that have a higher resolution is growing.
Traditionally, high-resolution cell biology has been the arena of
electron microscopy ( Fig. 9.1 ), which offers superb resolution but lacks
the aforementioned advantages of luorescence microscopy. The advent of
scanning probe microscopy ( Fig. 9.1 ) , and especially atomic force microscopy
(AFM), in which an atomically sharp probe attached to a cantilever is scanned
over the surface of interest, has made nanometre resolution also attainable
on living cells.
However, although AFM produces a high-resolution
topographical image of the sample, it lacks biochemical speciicity. Hence,
although individual molecules can be seen, their identities cannot be deined.
This seriously limits the usefulness of AFM for high-resolution imaging on
cells. A promising way around the problem relies on speciic labelling of
the AFM probe with biomolecules (e.g., with antibodies or ligands). This
introduces a contrast mechanism based on speciic interactions between
the probe and a certain type of molecules in the specimen. 21 More recently,
molecular recognition imaging using AFM and biofunctionalized probes has
been successfully implemented by the Hinterdorfer group (see Chapter 7 ). 22
Although extremely sensitive, the experimental approach is, so far, restricted
to a single type of interaction being probed. The combination of scanning
probe microscopy with an optical contrast mechanism, affording spatial
super-resolution imaging and spectroscopy, biochemical speciicity and
versatility, and ultra-fast time response, is the domain of near-ield scanning
optical microscopy (NSOM) and the main topic of this chapter.
As a side note, it is worth mentioning that in recent years, several new far-
ield super-resolution imaging techniques have also broken the diffraction
limit of light, producing luorescence images in the nanometre range, not only
laterally but also in three dimensions ( Fig. 9.1 ) . In short, these techniques
19,20
 
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