DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking
Dunne PD, Fernandes RA, McColl J, Yoon JW, James JR, Davis SJ, Klenerman D. (2009), Biophys J. 97, L5-7
We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.
(A) Experimental setup – full details of which can be found in the Supporting Material. T cells expressing proteins labeled with mCherry and Citrine are attached to a glass coverslip coated with nonactivating donkey anti-mouse antibody and the diffusion of the individual molecules on the basal surface of the cell is imaged after exciting simultaneously with overlapped 488 and 568 nm laser beams in total internal reflection geometry. Example raw data can be found as Movie S1. (B) Principle of the DySCo method. Associated molecules track within a short distance of one another for multiple frames (right) whereas unassociated molecules show no correlated motion (left). Unassociated molecules may track together by chance over a short distance (center), but the probability of this occurring for multiple frames is small.