NANOPHYSIOLOGY OF CELLS, CHANNELS AND NUCLEAR PORES - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 123

reduced luorescent events. This result indicates that the ∆F508-CFTR is
misprocessed also in RBC, where only a small amount of CFTR reaches the
cell surface. Within the diffraction limitation of luorescence microscopy, two
closely positioned Qdots (e.g. ~200 nm distance) would be detected as one
bright spot. Therefore, to achieve single-molecule detection, our next step
was to apply the high-resolution AFM to the Qdot-labelled membranes.

(b)

(a)

(d)

(c)

Figure 6.5. Immunostaining of CFTR in isolated RBC membrane patches with Qdot-
labelled antibodies. The upper panel represents luorescence images of non-CF (a)
and CF (b) RBC membrane patches. Each inset shows a single membrane with clearly
distinguishable bright luorescence events. The lower panel shows AFM images
of non-CF (c) and CF (d) RBC membrane patches. High-resolution scans, shown in
(c) and (d), identify the Qdot as high structures (~15 nm, colour code: white) with
speciic shapes. It is evident that the number of Qdot-labelled CFTR molecules is much
higher in non-CF RBC than in CF RBC. 10

The crystalline nature of the Qdots, however, allowed single-molecule
detection with AFM. In the AFM images, Qdots appear as structures with
uniform height and shape ( Fig. 6.5c,d ). Since the AFM provided the required
single-molecule resolution for further detailed quantiication of Qdot-labelled
CFTR proteins at the RBC membranes, we used the images taken with AFM.