Fig. 2: Brightness-calibrated ratiometric imaging (BCRI).
From: Insights into receptor structure and dynamics at the surface of living cells
a Schematic side view of the confocal detection volume (cyan) during raster scanning (arrow) of a cell and static positioning in free solution. Stochastic movements (dashed line) of the fluorescent particles (stars) allow to determine their molecular brightness. b Conversion of pixel intensities associated with the plasma membrane (PM) into particle numbers based on the molecular brightness of the labels for ligands (L) and receptors (R). c Titrations of trisNTA-Alexa647 and d IL-4-ATTO647N on cells expressing IL-4Rα*. Fitting a model function for 1:1 binding reproduces published affinities of trisNTA (34 ± 8 nM) and IL-4 (0.14 ± 0.04 nM) towards IL-4Rα*28,39. Concentrations of free ligand (x-axis) stem from FCS measurements in the supernatant. Markers and error bars represent mean ± SD of 13–155 cells (y-axis, precise number of cells for each concentration provided in the Source Data file) or 18 FCS runs (x-axis). Experiments were performed once (c) or twice (d). e Varying average occupancy of BCNK bearing receptors at the plasma membrane at fixed concentrations of IL-4-ATTO647N (color code according to Fig. 1a). Bars and error bars represent mean ± SD of single-cell measurements pooled from one (Lyn-eGFP, T18B), three (IL-4Rα*) or two (else) independent experiments. Descriptive statistics containing the number of cells measured for each receptor mutant are listed in Supplementary Table 3. f Gaussian-shaped probability distributions of single-cell averages of receptor occupancy (averages denoted in the panels). IL-4Rα* and Lyn-eGFP (gray) show stable means at both concentration levels (Kd of natural ligand 0.15 nM28), whereas for non-saturated receptor mutants (lower affinity) the occupancy increases with higher IL-4 concentrations. Bimodal Gaussian distributions in or close to the activation loop (blue) indicate two co-existing receptor states with different affinity. Descriptive statistics of all mutants at both concentrations are listed in Supplementary Table 3 and Supplementary Table 4. Source data are provided as a Source Data file.
