Resolution:
## Figure 5.
Gaining insight into optimal experimental design. The approximate posterior probability distributions for K(A) have different shapes if the data analyzed was simulated from three cells containing
equal concentrations of donors and acceptors which are much lower than (left), higher
than (right) or about equal to _{d }K(centre). For the data analyzed in the left panel, for instance, the cells contained
the concentrations [_{d }D_{0}] = [A_{0}] = 0.2·10^{-3 }μM, [D_{0}] = [A_{0}] = 1·10^{-3 }μM, and [D_{0}] = [A_{0}] = 5·10^{-3 }μM. Insets show amount of complex formed as a function of Kfor the indicated concentrations, demonstrating that where complex formation is insensitive
to _{d }Kcorresponds to plateaus in the posterior probability distributions for _{d }K. In each plot (or inset), a vertical dashed line (or red circle) indicates 10_{d}^{-6}M, the true value of K. (A) had 36,000 steps/walk and 5% added noise. Exploring another aspect of fluorophore
concentrations, increasing the ratio [_{d}D_{0}] : [A_{0}] increases the uncertainty in fitting K_{d }(B). As the ratio was increased (by keeping [D_{0}] constant for the three cells at 0.2·10^{-6}M, 1.0·10^{-6}M and 5.0 10^{-6}M while decreasing [A_{0}] according to the ratio), posterior probability distributions for Kbroadened (true value indicated by dashed vertical line). Insets show data used for
fitting (bars marked '_{d }E= 0.4') from the donor channel (left) and FRET channel (right) contrasted with data
from the same cells simulated with _{fr }E= 0, demonstrating that the relative contribution of FRET decreases as [_{fr }D_{0}] : [A_{0}] increases. (B) had 50 measurements/cell/channel, 36,000 steps/walk and 3% added
noise. Bars show mean ± SD. For other parameters, see Methods.
Lichten and Swain |