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Several super-resolution techniques exist, yet most require multiple lasers, use either large or weakly emitting fluorophores, or involve chemical manipulation. Here we show a simple technique that exceeds the standard diffraction limit by 5-15× on fixed samples, yet allows the user to localize individual fluorophores from among groups of crowded fluorophores. It relies only on bright, organic fluorophores and a sensitive camera, both of which are commercially available. Super-resolution is achieved by subtracting sequential images to find the fluorophores that photobleach (temporarily or permanently), photoactivate, or bind to the structure of interest in transitioning from one frame to the next. These fluorophores can then be localized via Gaussian fitting with selective frame averaging to achieve accuracies much better than the diffraction limit. The signal-to-noise ratio decreases with the square root of the number of nearby fluorophores, producing average single-molecule localization errors that are typically &lt;30 nm. Surprisingly, one can often extract signal when there are approximately 20 fluorophores surrounding the fluorophore of interest. Examples shown include microtubules (in vitro and in fixed cells) and chromosomal DNA.

Single-Molecule-Based Super-Resolution Images in the Presence of Multiple Fluorophores