FIGURE 3.

Simultaneous multi-color 3D single molecule tracking. (A) Simultaneous three colors 3D single particle tracking on living COS-7 cells. (i.) Experiment principle: NCAM membrane receptors in a COS cell are labelled with three populations of spectrally different QDots and tracked in 3D over time. (ii.) Reconstructed trajectories of the detected Qdots color-coded for their depth (upper right corner) or for their assigned wavelength (bottom left corner). (iii.) Computed median and range of diffusion coefficients for each QDots population (left) and histogram of the wavelengths detected during the acquisition revealing the three Qdot populations (right). (B) Simultaneous three colors 3D single particle tracking on living neurons. (i.) Experiment principle: Hippocampal dissociated neurons culture are transfected at DIV-10 with the plasmids coding for D1R-cfp, EphrinD2-Flag and soluble GFP. In between DIV12 and DIV15, expressed D1R-cfp proteins are labelled with two populations of spectrally different Qdots (Qdot 655 and Qdots705) and expressed EphrinB2-flag proteins are labelled with Qdots605 before being imaged and analyzed by spectrally informed localization and tracking. (ii.) Left: Reconstructed trajectories of the three Qdots populations. Right: Successive zooms of the white dotted regions revealing the main and well distinct zones explored by the two receptors (top) and the trajectory of two spectrally different Qdots (Qd655 and Qd705) within a spine (bottom). (iii.) Median and range of diffusion coefficients for each of the three different Qdots population, revealing that the two Qdots conjugated to the same receptors (D1R) behave similarly whereas EphrinB2 receptors diffuse slower.