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Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part o...

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Autores principales: Boudreau, Colton, Wee, Tse-Luen (Erika), Duh, Yan-Rung (Silvia), Couto, Melissa P., Ardakani, Kimya H., Brown, Claire M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971503/
https://www.ncbi.nlm.nih.gov/pubmed/27485088
http://dx.doi.org/10.1038/srep30892
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author Boudreau, Colton
Wee, Tse-Luen (Erika)
Duh, Yan-Rung (Silvia)
Couto, Melissa P.
Ardakani, Kimya H.
Brown, Claire M.
author_facet Boudreau, Colton
Wee, Tse-Luen (Erika)
Duh, Yan-Rung (Silvia)
Couto, Melissa P.
Ardakani, Kimya H.
Brown, Claire M.
author_sort Boudreau, Colton
collection PubMed
description It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity.
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spelling pubmed-49715032016-08-11 Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity Boudreau, Colton Wee, Tse-Luen (Erika) Duh, Yan-Rung (Silvia) Couto, Melissa P. Ardakani, Kimya H. Brown, Claire M. Sci Rep Article It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity. Nature Publishing Group 2016-08-03 /pmc/articles/PMC4971503/ /pubmed/27485088 http://dx.doi.org/10.1038/srep30892 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Boudreau, Colton
Wee, Tse-Luen (Erika)
Duh, Yan-Rung (Silvia)
Couto, Melissa P.
Ardakani, Kimya H.
Brown, Claire M.
Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title_full Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title_fullStr Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title_full_unstemmed Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title_short Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity
title_sort excitation light dose engineering to reduce photo-bleaching and photo-toxicity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971503/
https://www.ncbi.nlm.nih.gov/pubmed/27485088
http://dx.doi.org/10.1038/srep30892
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