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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doaj-b81a9c0f8e8e497bbb75f60fb10c99972020-11-25T02:47:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01117e015888410.1371/journal.pone.0158884Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy.Leila NahidiazarAlexandra V AgronskaiaJorrit BroertjesBram van den BroekKees JalinkSingle 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.http://europepmc.org/articles/PMC4938622?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Leila Nahidiazar Alexandra V Agronskaia Jorrit Broertjes Bram van den Broek Kees Jalink |
spellingShingle |
Leila Nahidiazar Alexandra V Agronskaia Jorrit Broertjes Bram van den Broek Kees Jalink Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. PLoS ONE |
author_facet |
Leila Nahidiazar Alexandra V Agronskaia Jorrit Broertjes Bram van den Broek Kees Jalink |
author_sort |
Leila Nahidiazar |
title |
Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. |
title_short |
Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. |
title_full |
Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. |
title_fullStr |
Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. |
title_full_unstemmed |
Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy. |
title_sort |
optimizing imaging conditions for demanding multi-color super resolution localization microscopy. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2016-01-01 |
description |
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. |
url |
http://europepmc.org/articles/PMC4938622?pdf=render |
work_keys_str_mv |
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