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. 2011 Nov 16;101(10):2360–2369. doi: 10.1016/j.bpj.2011.10.014

Figure 3.

Figure 3

Single-color FCS on the cell surface. (A) Dimensions of the detection volume as positioned in free solution or at the apical membrane. Above the nucleus (N), fluorescence from filopodia (FN), the extracellular compartment (EC), and the cytoplasm (CP) may also be detected. (B) Confocal cross sections of the stable cell line H4G in the absence or presence of ligand IL4-A647. A typical position for single-point FCS is indicated at the top (cross hair). (C) Normalized correlation curves (data gray) measured in the supernatant (blue) and at the top of the apical membrane (red). Binding isotherm (inset) obtained by evaluating the molar fractions with a numerical example assuming 1:1 binding (Kd = 10 nM, Rmax = 100 nM). (D) Receptor diffusion. Correlation curve (data gray) and fitting with two correlation times (red, arrows). The molecular brightness of the eGFP-tagged receptors for saturating IL-4 binding as measured by the photon counting histogram (inset).