Chapman, CAR;
Zhu, X;
Chen, H;
Yanik, AA;
Lein, PJ;
Seker, E;
(2017)
Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity.
Scientific Reports
, 7
(1)
, Article 427. 10.1038/s41598-017-00447-7.
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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.
| Type: | Article |
|---|---|
| Title: | Nanostructure Introduces Artifacts in Quantitative Immunofluorescence by Influencing Fluorophore Intensity |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-017-00447-7 |
| Publisher version: | http://doi.org/10.1038/s41598-017-00447-7 |
| Language: | English |
| Additional information: | Copyright © The Author(s) 2017. 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/ |
| Keywords: | Biophotonics, Fluorescence imaging, Nanoscale materials |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1556762 |
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