Figure 3. Evaluating the fraction of... | Rockefeller University Press

Figure 3.

$Figure 3. Evaluating the fraction of incidentally overlapped spots as a function of the NDDS: experimental validation of Eq. 1. (A) Representative single-molecule TIRF images of monomer reference molecules—ACP(DY547)-TM (left) and Alexa-DOPE (middle)—and a dimer reference molecule, CD28-mGFP (green arrow, tetramer-like spot). Yellow arrowheads and red arrows indicate the spots with monomeric and dimeric intensities, respectively. (B) Distributions of the signal intensities of individual spots, fitted with the sum of two Gaussian functions (apparent monomers and apparent dimers), giving PDspotT+A. (C and D) PDspotT+A (C) and PDspotT (equal to PDspotT+A – PDspotA; D) plotted as a function of NDDS, for ACP(DY547)-TM (red) and Alexa-DOPE (blue), showing that these plots agree with the simulation result (black keys and the best fitting curve, PDspotA = 0.075 × NDSS + 0.00057 × NDDS2. For each data point, 1,550 < nspot < 1,800.$

Evaluating the fraction of incidentally overlapped spots as a function of the NDDS: experimental validation of Eq. 1. (A) Representative single-molecule TIRF images of monomer reference molecules—ACP(DY547)-TM (left) and Alexa-DOPE (middle)—and a dimer reference molecule, CD28-mGFP (green arrow, tetramer-like spot). Yellow arrowheads and red arrows indicate the spots with monomeric and dimeric intensities, respectively. (B) Distributions of the signal intensities of individual spots, fitted with the sum of two Gaussian functions (apparent monomers and apparent dimers), giving P_Dspot^T+A. (C and D) P_Dspot^T+A (C) and P_Dspot^T (equal to P_Dspot^T+A – P_Dspot^A; D) plotted as a function of NDDS, for ACP(DY547)-TM (red) and Alexa-DOPE (blue), showing that these plots agree with the simulation result (black keys and the best fitting curve, P_Dspot^A = 0.075 × NDSS + 0.00057 × NDDS². For each data point, 1,550 < n_spot < 1,800.