(A) Tip taper sizes were estimated from the standard deviation of the survival function used to fit the fall off of intensity at the tip (σMT). To determine the minimum detectable taper, simulated images of blunt microtubules were generated by using model convolution based on our microscope setup and experimental measurement noise. The distribution of tip standard deviations from these simulated images is shown in orange. We defined the minimal experimentally detectable plus-tip taper as three times the standard deviation of this distribution (red dashed line at 140 nm). The corresponding distribution from experiments imaging blunt, taxol-stabilized GMPCPP seeds is shown in blue. Note that a small population of our blunt microtubules had σMT above the threshold; we attribute these apparent tapers to experimental noise, such as out of focus images that are not captured in the model convolution. (B) Comparison of experimentally measured tip standard deviations for taxol-stabilized GMPCPP seeds (blue), and microtubules growing in 1.3 μM free tubulin (orange) and 1.5 μM free tubulin (yellow) in the presence of GMPCPP. The detection limit of 140 nm is shown as a dotted red line. As the free tubulin concentration increased, the fraction of tip estimations above the detection limit increased. (C) Calibration curve relating tip standard deviation from the survival function fit, σMT, to the actual taper length. Model convolution was used to generate simulated images of microtubules with increasing uniform taper structures (Bohner et al., 2016; Demchouk et al., 2011; Maurer et al., 2014; Prahl et al., 2014; Ruhnow et al., 2011) where the taper length is defined as the distance between the longest and the shortest protofilament. The mean and SD of σMT for each set of images is plotted as a function of the taper length used to generate that set of images (Bohner et al., 2016; Maurer et al., 2014; Prahl et al., 2014). A theoretical relationship between σMT and taper length is calculated by adding in quadrature the standard deviation of the protofilament length, σPF, and the spread of the optical point spread function (PSF), σPSF. . The relationship between the standard deviation of the protofilament length, σPF, and taper should theoretically be , but due to increases in tracking error that occur at longer taper lengths the relationship was fit empirically using σPF = 0.35*Taper (Demchouk et al., 2011; Maurer et al., 2014). . This calibration curve was used to estimate taper length from the experimental σMT data. The threshold for a measurable taper (red dashed line at 140 nm) corresponds to a minimum detectable taper length of 265 nm. (D) Tip taper quantification on blunt microtubules and microtubules growing in GMPCPP. ‘% Over Detection’ is defined as the proportion of σMT measurements above the 140 nm detection limit defined in panel A. Note the increasing percent of measurable tips with increasing free tubulin; the 5% for the seeds represents measurement error. * ‘Mean Taper’ is calculated by converting the σMT values for each condition to taper lengths using the calibration curve in panel B, and taking the mean of this population. Note that these mean taper sizes are underestimates because any σMT below the detection limit was defined as having a taper of 0 nm. ** ‘Max Tapers’ is the average of the top 1% of tapers above the detection limit for each condition. (E) Example time courses of the spread of the survival function (σMT, left y-axis) and the corresponding estimated taper length in tubulin subunits (right y-axis) for stabilized seeds (top), and microtubules growing in 1.3 μM (middle) and 1.5 μM (bottom) free tubulin in GMPCPP. The red dotted line is the measurement detection limit. Note that for the taxol-stabilized GMPCPP seed (top panel-blue), the entire trace lies below the detection limit. The 1.3 μM free tubulin trace (middle panel – orange) shows fluctuations of the taper length occurring over 10 s of seconds. The 1.5 μM free tubulin trace (bottom panel – yellow) shows time-dependent fluctuations of taper size with a sustained period above the detection limit.