Fig. 4: Co-diffusion analysis of fPEG-Chol and CF®680R-BTX-labelled nAChR using detector channel ratio (DCR) discrimination in combination with intensity changes.

a Representative data of DCR and intensity values (as effective counts at offset (ECO)) over time steps (upper two panels) and corresponding tracks in the X-Y plane with colours indicating classification as fPEG-Chol (orange), CF®680R-BTX (blue) and co-diffusion (black) for cells with fPEG-Chol alone, CF®680R-BTX nAChR alone, and the 2-colour co-labelling experiments with both fPEG-Chol and CF®680R-BTX. Co-diffusion becomes apparent in the increased ECO values and simultaneous change in DCR values converging in a corridor between 0.40 and 0.55 (marked by the two horizontal dashed lines). Scale bar = 100 nm. b Histograms of DCR values for the tracking data of fPEG-Chol (orange) and CF®680R-BTX (blue) only, and of fPEG-Chol and CF®680R-BTX co-labelled cells (purple) with two-species Gaussian fits for experiments with only one probe without (upper left, control) and with (upper middle) actin modulation by CK-666, and with doubly labelled experiments without any treatment (lower left, control), with actin modulation by CK-666 (lower middle) and with cholesterol supplementation (50 nM: upper right, 100 nM: lower right). The histograms built from the data acquired with both probes simultaneously show only the mobile fraction. Source data are provided as a Source Data file.