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PLoS One. 2016 Jul 8;11(7):e0158884. doi: 10.1371/journal.pone.0158884. eCollection 2016.

Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.

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Cell Biophysics group, Department of Cell biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Molecular Biophysics group, Utrecht University, Utrecht, the Netherlands.
Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands.
Van Leeuwenhoek Centre for Advanced Microscopy, Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.


Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off ("blink"), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording.

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