NEAR-FIELD SCANNING OPTICAL MICROSCOPY OF BIOLOGICAL MEMBRANES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 192

9.3.2 Cell Membrane Compartmentalizaon Inspected by NSOM

Regarding cell membrane quantitative imaging, NSOM has been mainly
used to investigate the degree of clustering of different receptors on the cell
membrane. Since live cell imaging still remains a technological challenge for
NSOM, most of the work reported so far has been performed in dry conditions
and with cells being subject to several treatments before inspection: dry air,
several steps of methanol and then dried or paraformaldehyde. The latter
is advantageous when working in liquid conditions, since it chemically ixes
the cells, without altering cell membrane morphology (<2%) and therefore
prevents mobility of membrane components during imaging. In some cases,
the association of multiple components has also been investigated using
dual-colour NSOM. In the context of receptor clustering, our group has used
NSOM to image pathogen recognition receptors with high spatial resolution
on cells of the immune system, providing insight into the mechanisms
exploited by the cell to ensure high performance of these receptors 40,60 ( Fig .
9.5 ) . By labelling the pathogen recognition receptor DC-SIGN with a speciic
monoclonal antibody, we found that as much as 80% of DC-SIGN is clustered
on the cell membrane of immature dendritic cells, imaged either in dry or
buffer conditions. 40,60 These domains were randomly distributed over the
plasma membrane with a size distribution centered at ~185 nm. Interestingly,
we discovered a remarkable heterogeneity of the DC-SIGN packing density
within the clusters. This suggests that the large spread in DC-SIGN density
per cluster likely serves to maximize the chances of DC-SIGN binding to a
large variety of viruses and pathogens having different binding afinities. 60
Indeed, the organization of DC-SIGN in nanodomains appeared crucial for
eficient binding and internalization of pathogens. 7
Recently, Chen used NSOM in dry conditions in combination with

quantum dots to label the T cell receptor (TCR) of T cells in live animals
before and after cell stimulation. 61 In the resting state, the TCR complexes
were found monomerically organized on the T cell membrane. Upon T cell
stimulation, the TCR complexes reorganized and formed 270-390 nm sized
domains. Interestingly, these small-sized domains were not only formed
but also sustained for days. Additional experiments showed that although
unstimulated cells could produce an immune response, stimulated cells
produced signiicant higher levels of cytokines. 61 By means of these high-
resolution NSOM experiments, it was shown that the TCR reorganization
plays a signiicant role in antigen recognition and cytokine production.

et al.