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Direct evidence of lack of colocalisation of fluorescently labelled gold labels used in correlative light electron microscopy

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@article{55ab3dff96894c49b8145ea3435c09f2,
title = "Direct evidence of lack of colocalisation of fluorescently labelled gold labels used in correlative light electron microscopy",
abstract = "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.",
keywords = "Correlative Light Electron Microscopy, Cross-Polarization Imaging, Gold nanoparticles, Fluorescent Labels, single particle detection, single molecule detection, Fluorescent Quenching, Colocalisation",
author = "Miles, {Benjamin T.} and Greenwood, {Alexander B.} and David Benito-Alifonso and Hugh Tanner and Galan, {M. Carmen} and Paul Verkade and Henkjan Gersen",
year = "2017",
month = "3",
day = "20",
doi = "10.1038/srep44666",
language = "English",
volume = "7",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Springer Nature",

}

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TY - JOUR

T1 - Direct evidence of lack of colocalisation of fluorescently labelled gold labels used in correlative light electron microscopy

AU - Miles, Benjamin T.

AU - Greenwood, Alexander B.

AU - Benito-Alifonso, David

AU - Tanner, Hugh

AU - Galan, M. Carmen

AU - Verkade, Paul

AU - Gersen, Henkjan

PY - 2017/3/20

Y1 - 2017/3/20

N2 - 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.

AB - 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.

KW - Correlative Light Electron Microscopy

KW - Cross-Polarization Imaging

KW - Gold nanoparticles

KW - Fluorescent Labels

KW - single particle detection

KW - single molecule detection

KW - Fluorescent Quenching

KW - Colocalisation

UR - http://www.scopus.com/inward/record.url?scp=85015809396&partnerID=8YFLogxK

U2 - 10.1038/srep44666

DO - 10.1038/srep44666

M3 - Article

VL - 7

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

M1 - 44666

ER -