Biology Reference
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in many cases to contradictory results.
11
There is therefore a need for new
high-resolution methodologies capable of directly imaging domains within
the plasma membrane of intact cells.
Fluorescence microscopy has become one of the most prominent and
versatile research tools used in modern cell biology and in principle ideal to
investigate cell membrane organization in living cells.
The reasons for it are
essentially twofold. First, light-based microscopy allows the study of living
specimens in their native environment in a non-invasive manner. Additionally,
luorescence microscopy offers chemical speciicity by exploiting polarization,
lifetime and spectral contrast.
12
Furthermore, progress in detector technology
has recently pushed luorescence microscopy to its ultimate level of sensitivity:
the detection of individual molecules.
13
Second, enormous progress on the
development of speciic and highly eficient luorescent probes for exogenous
labelling has been achieved. In parallel to external antibody labelling, the
advent of green luorescent protein (GFP) technology has revolutionized live
cell imaging because an autoluorescent molecule can be genetically encoded
as a fusion with the c-DNA of interest.
17
Indeed, the spectral variants of GFP
and the unrelated red luorescent protein (DsRed) make it possible to perform
nowadays multicolour imaging in living cells.
17,18
14-16
Figure 9.1.
Comparison of spatial resolution techniques for biological imaging.
WF: wide-ield microscopy; TIRF: total internal relection luorescence microscopy;
STED: stimulated emission depletion; PALM: photoactivated localization microscopy;
STORM: stochastic optical reconstruction microscopy; EM: electron microscopy; AFM:
atomic force microscopy. STED, PALM and STORM belong to far-ield super-resolution
techniques while NSOM is a near-ield super-resolution technique.
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