Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Miles, Benjamin T., Greenwood, Alexander B., Benito-Alifonso, David, Tanner, Hugh, Galan, M. Carmen, Verkade, Paul, Gersen, Henkjan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
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
_version_ 1782516105436200960
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
work_keys_str_mv AT milesbenjamint directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT greenwoodalexanderb directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT benitoalifonsodavid directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT tannerhugh directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT galanmcarmen directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT verkadepaul directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy
AT gersenhenkjan directevidenceoflackofcolocalisationoffluorescentlylabelledgoldlabelsusedincorrelativelightelectronmicroscopy