Figure 5: In cuvette validation of the protein motion model.

(a) (Upper panel) Pictured GFP dynamics in the presence of colliding crowders. (a) (Lower panel) Experimental iMSD of GFP diffusing in BSA solution with different excluded volume (ϕ). For both tested excluded volume, the iMSD is not distinguishable from free diffusion and increasing excluded volume decrease molecular diffusivity (for ϕ=0.1, D=46±2 μm² s⁻¹ and N=7 measurements; for ϕ=0.2, D=24±2 μm² s⁻¹ and N=7 measurements). (b) (Upper panel) Pictured GFP dynamics in the presence of spatially organized obstacles. Fast scanning allows measuring GFP dynamics in the free space between obstacles. Decreasing scanning speed allows measuring the effect of boundaries on the GFP dynamics. (b) (Lower panel) The complete iMSD of GFP in Sephacryl beads is reported, as average of N=5 beads (black dots), and compared with the iMSD in solution outside the beads (dashed green line). The local dynamics (below 2 × 10⁻⁵ s) can be described well by GFP diffusion outside the Sephacryl beads (χ²=0.8, P<0.025). However, above 2 × 10⁻⁵ s, the latter is not representative for the whole iMSD (χ²>25, P>0.995) because of the reduction of long-range GFP mobility by the solid structure of the polymeric beads. The inset shows a representative scanning electron microscopy image of a Sephacryl beads, in which many submicrometer cavities are visible. Scale bar, 200 nm. Data are mean values±s.d.