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To the Editor: Fluorescent proteins are known to display in certain cases an on-off blinking and switching behavior1. We could not yet find a report investigating the impact of this phenomenon on superresolution techniques that are based on the sequential photoactivation and bleaching of individual emitters such as photoactivated localization microscopy (PALM) 2. In our hands, even monomeric (m) Eos2, one of the most promising photoconvertible fluorescent proteins reported in this journal3, had non-negligible light-induced fluorescence recovery4 (Supplementary Figs. 1 and 2). This is particularly important for imaging membrane receptors, for which phenomena such as oligomerization and clustering of proteins can be properly identified only if the number of their constituents is correctly estimated.

Here we compared a negative and a positive clustering control by using plasma membrane–bound proteins labeled …