Open Access Original article

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

Paolo Annibale1*, Marco Scarselli12, Mattia Greco1 and Aleksandra Radenovic1*

Author Affiliations

1 Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, Lausanne 1015, Switzerland

2 Present address: Department of Neuroscience, University of Pisa, Pisa, Italy

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Optical Nanoscopy 2012, 1:9  doi:10.1186/2192-2853-1-9

Published: 24 November 2012

Abstract

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.

Keywords:
Super-resolution; Photoactivated localization microscopy; Axial stability; Registration precision; Endocytosis