Measuring EGFR Separations on Cells with ∼10 nm Resolution via Fluorophore Localization Imaging with Photobleaching
Figure 1
Measuring the separation between two molecules.
(A) Example intensity v time course from a spot on cells showing a pair of Affibody-Atto 647N molecules photobleaching in two steps. Images were acquired every 0.28 s. The insets show the profile of the spot before and after the first bleaching event. (B) Original and decorrelated separations to illustrate the effect of anti-correlated positional error. The data of each spot is resampled and fitted 1200 times yielding 1200 sets of seven parameters that are samples of the 7-dimensional parameter distribution. In the decorrelated version the parameters have been independently randomly reordered, i.e. parameters from the same set end up in different sets. (C) Comparison of the error estimation of the Thompson, Larson and Webb [34] and sequential photobleaching NALMS analysis method [29] (red line) to our FLIP variant (black line) for synthetic data, depending on the SNR. SNR is the ratio of the measured mean intensity of a single-fluorophore trace to its standard deviation. The graph shows how often the real distance lies within the 68% confidence interval of the measurement. For [34], the confidence interval was taken as separation estimate ± standard deviation; for FLIP the confidence interval was taken as the shortest interval that contains 68% of the separation results from the bootstrap sampling (1,200 samples per measurement). (D) A probability density plot of the separations from the bootstrap samples of an individual measurement, showing the best fit value and the confidence interval.