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

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 mathematicall...

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Veröffentlicht in:PloS one Jg. 11; H. 7; S. e0158884
Hauptverfasser: Nahidiazar, Leila, Agronskaia, Alexandra V., Broertjes, Jorrit, van den Broek, Bram, Jalink, Kees
Format: Journal Article
Sprache:Englisch
Veröffentlicht: United States Public Library of Science 08.07.2016
Public Library of Science (PLoS)
Schlagworte:
Biology
Biophysics
Blinking
Buffers
Cancer
Color
Color imagery
Diagnostic imaging
Dyes
Engineering and Technology
Fluorescent Dyes - chemistry
Image acquisition
Image processing
Image quality
Imaging, Three-Dimensional - methods
Labels
Lasers
Localization
Longevity
Methods
Microscopy
Microscopy, Fluorescence, Multiphoton - methods
Optimization
Physical Sciences
Power
Research and Analysis Methods
Science
Stitching
Storms
Netherlands
ISSN:1932-6203, 1932-6203
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Zusammenfassung: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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Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: LN KJ. Performed the experiments: LN AVA JB. Analyzed the data: LN KJ AVA BvdB. Contributed reagents/materials/analysis tools: AVA BvdB KJ. Wrote the paper: LN KJ.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0158884