NEAR-FIELD SCANNING OPTICAL MICROSCOPY OF BIOLOGICAL MEMBRANES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 199

9.5 CONCLUSIONS

The past few years have witnessed tremendous technical advances in super-
resolution optical microscopy using both far- and near-ield methods. This
has in turn further increased our understanding on the compartmentalization
of the cell membrane and its implications in cellular function and diseases.
However, a signiicant number of questions are still open and awaiting for
techniques that combine high spatial and temporal resolution in one and
the same instrument. Far-ield super-resolution methods have already
demonstrated the possibility of following the dynamics of slowly moving
receptors on the cell membrane on small ields of views or in combination
with a FCS approach. Further developments of probes and instrumentation
will certainly lead to improvement of these techniques.
Within the context of near-ield super-resolution, irst demonstrations
of NSOM measurements on living cells have been reported although high-
resolution dynamics on the membrane of living cells is yet to be demonstrated.
Obviously, if the scanning speed is not signiicantly faster than protein
diffusion, the optical signal will be blurred. Nevertheless, the promising
demonstration of subwavelength-FCS 57 opens the way for probing dynamics
at relevant spatial scales potentially revealing the driving mechanisms for
nanodomain formation and evolution during cell activation. Additionally,
multicolour cross-correlation should indicate if certain proteins are diffusing
in identical or separate domains. The combination of capabilities that is
offered by NSOM makes the technique a worthy and essential asset in the
spectra of biophysical techniques available nowadays.

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