Fig. 6: Multispectral light-sheet imaging of intracellular trafficking using computationally designed receptor binders.

a, Schematic depicting the use of fluorescently labelled, computationally designed receptor binders for imaging receptor trafficking. b, Single frame of the RLSU unmixed results (Supplementary Video 13) of a TagBFP-NLS-expressing HeLa Kyoto cell loaded with four fluorescently labelled binders (TfRB, IGF2RB, BMPR2B and Iα5β1B) and one fluorescently labelled receptor ligand (EGF). Top–down and orthogonal-view maximum intensity projections are shown (cell boundaries in cyan). c, Colour merge of the channels in b. d, Individual channels and colour merge of the endosomes indicated by yellow, green, magenta and cyan boxes in c. e, Individual channels and colour merge of a single endosome during cargo sorting. Frames from Supplementary Video 13 displaying fissioning of this compartment are shown. Magenta dashed circles indicate the parent endosome (labelled ‘1’). Cyan arrowheads indicate the elongation of an intermediate tubule. Red and cyan circles indicate the two daughter endosomes post-fissioning (labelled ‘2.a’ and ‘2.b’, respectively). f, Graph depicting the average proportion of total endosomes (n = 1,872) containing each cargo across the first five frames of Supplementary Video 13. Error bars denote s.d. g, Individual channels and colour merge of a highly motile endosome at t = 0. h, Colour merge of the endosome in g undergoing directional transport. i, Kymographs of the highly motile endosome shown in g and h. j, Graph depicting the cargo proportion transferred from the the parent endosome (magenta dashed circle, endosome 1) to each daughter endosome post-fissioning (red and cyan dashed circles, endosomes 2.a and 2.b, respectively) in e. Scale bars, 5 μm (b,c); 1 μm (d,e,g,h); 0.5 μm and 2.5 s (i).