Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

This work explores the potential of multi-color Photoactivated Localization Microscopy (PALM) imaging to probe sub-diffraction limit interactions between proteins with spectrally separated labels. Using a PALM setup built around a commercial microscope axially stabilized to nm-level, we determined the ultimate registration accuracy that could be achieved (10 nm) and compared the performance of three different pairs of fluorescent proteins that can be used in dual color PALM. Fusion constructs were cloned and imaged either in vitro or at the cell plasma membrane, allowing to identify a current limit to co-localization precision of approximately 30-40 nm. We identified the better performing pair and present a concluding perspective application to a co-clustering study. © 2012 Annibale et al.