Cargando…

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study....

Descripción completa

Detalles Bibliográficos
Autores principales: Schmidt, Gregor W., Cuny, Andreas P., Rudolf, Fabian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718758/
https://www.ncbi.nlm.nih.gov/pubmed/33023973
http://dx.doi.org/10.1534/g3.120.401465
_version_ 1783619553646870528
author Schmidt, Gregor W.
Cuny, Andreas P.
Rudolf, Fabian
author_facet Schmidt, Gregor W.
Cuny, Andreas P.
Rudolf, Fabian
author_sort Schmidt, Gregor W.
collection PubMed
description Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.
format Online
Article
Text
id pubmed-7718758
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Genetics Society of America
record_format MEDLINE/PubMed
spelling pubmed-77187582020-12-17 Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe Schmidt, Gregor W. Cuny, Andreas P. Rudolf, Fabian G3 (Bethesda) Investigations Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms. Genetics Society of America 2020-10-06 /pmc/articles/PMC7718758/ /pubmed/33023973 http://dx.doi.org/10.1534/g3.120.401465 Text en Copyright © 2020 Schmidt et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Schmidt, Gregor W.
Cuny, Andreas P.
Rudolf, Fabian
Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title_full Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title_fullStr Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title_full_unstemmed Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title_short Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe
title_sort preventing photomorbidity in long-term multi-color fluorescence imaging of saccharomyces cerevisiae and s. pombe
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718758/
https://www.ncbi.nlm.nih.gov/pubmed/33023973
http://dx.doi.org/10.1534/g3.120.401465
work_keys_str_mv AT schmidtgregorw preventingphotomorbidityinlongtermmulticolorfluorescenceimagingofsaccharomycescerevisiaeandspombe
AT cunyandreasp preventingphotomorbidityinlongtermmulticolorfluorescenceimagingofsaccharomycescerevisiaeandspombe
AT rudolffabian preventingphotomorbidityinlongtermmulticolorfluorescenceimagingofsaccharomycescerevisiaeandspombe