Figure 5. Simultaneous iSCAT and fluorescence tracking of myosin 5a.
(A) Tracking of the scattering signal from the gold nanoparticle attached to the N-terminus (black) and fluorescence signal from the GFP moiety located at the C-terminus of the same myosin 5a molecule (green). Movement of the tail correlates with the labeled head taking its step (74 nm displacement) and with the AB transition (power stroke of the labeled head). Green arrows represent the tail movement, which corresponds to the step of either the labelled or unlabeled head. When the unlabeled head takes its step it coincides with the AB transition within the labeled head (red arrows). Static localization precisions determined as in Figure 2: 1.6 nm (iSCAT), 8 nm (GFP). (B) Labelling scheme and schematic of the stepping mechanism and the corresponding observables. ATP concentration: 1 μM. Imaging speed for both channels: 20 frames/s.