Figure 2.

(A) The fluorescence (or Förster) resonance energy transfer (FRET) efficiency (E) is calculated by the relationship between donor (D, green circle) and acceptor (A, red circle) intensities (I_D and I_A). FRET efficiency has a strongly non-linear dependence (sixth power) on the distance between the donor and acceptor molecules (d) attached to an intrinsically disordered protein. R₀ is the Förster radius, which determines the length scale of the FRET coupling and is the value where the transfer efficiency is 50%. The donor and acceptor fluorescent dye properties determine R₀, which usually is around 4–7 nm. (B) A schematic of prism-type total internal reflection (TIR) illumination for smFRET. The green arrow shows an incident laser beam that is totally internally reflected at the quartz-water interface of a fluidic channel, producing an evanescent wave that excites fluorophores near the surface. The zoomed detail shows an IDP labeled with a D–A pair that is encapsulated in a liposome (not to scale). The liposome is attached to the quartz surface via a biotin-streptavidin linkage while additional liposomes are shown in solution in the flowcell diagram.