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Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy
Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed und...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357795/ https://www.ncbi.nlm.nih.gov/pubmed/28317888 http://dx.doi.org/10.1038/srep44666 |
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author | Miles, Benjamin T. Greenwood, Alexander B. Benito-Alifonso, David Tanner, Hugh Galan, M. Carmen Verkade, Paul Gersen, Henkjan |
author_facet | Miles, Benjamin T. Greenwood, Alexander B. Benito-Alifonso, David Tanner, Hugh Galan, M. Carmen Verkade, Paul Gersen, Henkjan |
author_sort | Miles, Benjamin T. |
collection | PubMed |
description | Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field. |
format | Online Article Text |
id | pubmed-5357795 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-53577952017-03-22 Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy Miles, Benjamin T. Greenwood, Alexander B. Benito-Alifonso, David Tanner, Hugh Galan, M. Carmen Verkade, Paul Gersen, Henkjan Sci Rep Article Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field. Nature Publishing Group 2017-03-20 /pmc/articles/PMC5357795/ /pubmed/28317888 http://dx.doi.org/10.1038/srep44666 Text en Copyright © 2017, 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 Miles, Benjamin T. Greenwood, Alexander B. Benito-Alifonso, David Tanner, Hugh Galan, M. Carmen Verkade, Paul Gersen, Henkjan Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title | Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title_full | Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title_fullStr | Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title_full_unstemmed | Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title_short | Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy |
title_sort | direct evidence of lack of colocalisation of fluorescently labelled gold labels used in correlative light electron microscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5357795/ https://www.ncbi.nlm.nih.gov/pubmed/28317888 http://dx.doi.org/10.1038/srep44666 |
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