Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity

Abstract Quantitative analysis of fluorescence signals from cells reacted with fluorescently labeled probes is a widely-used method for assessing cell biology. This method has become especially powerful for screening novel nanostructured materials for their influence on cell behavior. However, the e...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Christopher A. R. Chapman, Xiangchao Zhu, Hao Chen, Ahmet A. Yanik, Pamela J. Lein, Erkin Seker
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/97d04f455ed3442cabc7ebe53a47535f
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:97d04f455ed3442cabc7ebe53a47535f
record_format	dspace
spelling	oai:doaj.org-article:97d04f455ed3442cabc7ebe53a47535f2021-12-02T11:40:58ZNanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity10.1038/s41598-017-00447-72045-2322https://doaj.org/article/97d04f455ed3442cabc7ebe53a47535f2017-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-00447-7https://doaj.org/toc/2045-2322Abstract Quantitative analysis of fluorescence signals from cells reacted with fluorescently labeled probes is a widely-used method for assessing cell biology. This method has become especially powerful for screening novel nanostructured materials for their influence on cell behavior. However, the effect of nanostructured surface on fluorescence intensity has largely been ignored, which likely leads to erroneous conclusions about cell behavior. This paper investigates this possibility by using fibroblasts cultured on nanoporous gold (np-Au) as a model nanostructured material system. We found that fibroblasts stained for f-actin using phalloidin conjugated with common fluorophores display different levels of fluorescence on np-Au, planar gold, and glass, suggesting different levels of f-actin composition. However, direct quantification via western blots indicates that the actin expression is the same across all conditions. We further investigated whether the fluorescence intensity depended on np-Au feature size, complementing the findings with reflection dark field measurements from different np-Au surfaces. Overall, our experimental measurements in agreement with our electrodynamic simulations suggest that nanostructured surfaces alter the fluorescence intensity of fluorophores by modulating both the excitation and light emission processes. We conclude that comparison of fluorescence on materials with different nanostructures should be done with a quantification method decoupled from the nanostructure's influence.Christopher A. R. ChapmanXiangchao ZhuHao ChenAhmet A. YanikPamela J. LeinErkin SekerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christopher A. R. Chapman Xiangchao Zhu Hao Chen Ahmet A. Yanik Pamela J. Lein Erkin Seker Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
description	Abstract Quantitative analysis of fluorescence signals from cells reacted with fluorescently labeled probes is a widely-used method for assessing cell biology. This method has become especially powerful for screening novel nanostructured materials for their influence on cell behavior. However, the effect of nanostructured surface on fluorescence intensity has largely been ignored, which likely leads to erroneous conclusions about cell behavior. This paper investigates this possibility by using fibroblasts cultured on nanoporous gold (np-Au) as a model nanostructured material system. We found that fibroblasts stained for f-actin using phalloidin conjugated with common fluorophores display different levels of fluorescence on np-Au, planar gold, and glass, suggesting different levels of f-actin composition. However, direct quantification via western blots indicates that the actin expression is the same across all conditions. We further investigated whether the fluorescence intensity depended on np-Au feature size, complementing the findings with reflection dark field measurements from different np-Au surfaces. Overall, our experimental measurements in agreement with our electrodynamic simulations suggest that nanostructured surfaces alter the fluorescence intensity of fluorophores by modulating both the excitation and light emission processes. We conclude that comparison of fluorescence on materials with different nanostructures should be done with a quantification method decoupled from the nanostructure's influence.
format	article
author	Christopher A. R. Chapman Xiangchao Zhu Hao Chen Ahmet A. Yanik Pamela J. Lein Erkin Seker
author_facet	Christopher A. R. Chapman Xiangchao Zhu Hao Chen Ahmet A. Yanik Pamela J. Lein Erkin Seker
author_sort	Christopher A. R. Chapman
title	Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
title_short	Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
title_full	Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
title_fullStr	Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
title_full_unstemmed	Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity
title_sort	nanostructure introduces artifacts in quantitative immunofluorescence by influencing fluorophore intensity
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/97d04f455ed3442cabc7ebe53a47535f
work_keys_str_mv	AT christopherarchapman nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity AT xiangchaozhu nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity AT haochen nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity AT ahmetayanik nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity AT pamelajlein nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity AT erkinseker nanostructureintroducesartifactsinquantitativeimmunofluorescencebyinfluencingfluorophoreintensity
_version_	1718395474539446272

Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity

Ejemplares similares