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

Paolo Annibale*, Marco Scarselli, Mattia Greco, Aleksandra Radenovic

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Citations (Scopus)

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.

Original languageEnglish
Article number9
Pages (from-to)1-13
Number of pages13
JournalOptical Nanoscopy
Volume1
Issue number1
DOIs
Publication statusPublished - 2012

Keywords

  • Axial stability
  • Endocytosis
  • Photoactivated localization microscopy
  • Registration precision
  • Super-resolution

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