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. 2016 Jul 4;5(7):1001–1009. doi: 10.1242/bio.019570

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© 2016. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 2. — Proper PFA fixation preserves small actin fibers as faithfully as GA fixation. (A) PFA-PEM fixation preserves small actin fibers as faithfully as GA fixation. Individual actin fibers (5 to 10 fibers per condition) were manually segmented and their brightness profiles were plotted and fitted to a Gaussian distribution as described in Materials and Methods. Representative fibers, distribution profiles with Gaussian fit and FWHM are shown. Scale bar: 300 nm. (B) Only PFA-PEM and GA fixation preserve very thin actin fibers. Distribution profiles with Gaussian fit and FWHM of the thinnest detected fibers are shown for each fixation method. Scale bar: 300 nm. (C) Localization precision is not affected by the fixation method. Localization precision of images of actin obtained after different fixations was calculated using the Thunderstorm plugin (Ovesny et al., 2014) of Fiji (Schindelin et al., 2012). Bar graph shows localization precision and intensity (mean±s.d.) as obtained from five independent images (PFA-PBS fixation), seven independent images (GA fixation) and nine independent images (PFA-PEM fixation) (one-way ANOVA was employed to compare the obtained results and no significant differences were found).