*5.2.2 Differential quenching*

Differential quenching is the method that is utilized to gain insights into the localization of peptides in the lipid bilayer membrane [61]. The method takes advantage of simple diffusional quenching notions to the bilayer membrane's constrained dimensions. The membrane bilayer serves as a slab in which fluorophores and quenchers are distributed uniformly. The quenchers' distribution has been described using simulations of single-molecule Brownian dynamics, whereas the fluorophores' distribution has been determined using quenchers' pairs in phospholipids that are generally in different orientations of the acyl chain in the phospholipids [62]. Because the relative degree of quenching between quenchers and fluorophores is dependent on their propinquity, and the information on the peptides' current location in the membrane will be provided by fluorescence intensity [61]. A water-soluble quencher, like acrylamide, can also be employed to detect whether the fluorophore is not affected by the aqueous conditions, which is a useful addition to this assay [63].
