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

Benjamin T. Miles, Alexander B. Greenwood, David Benito-Alifonso, Hugh Tanner, M. Carmen Galan, Paul Verkade, Henkjan Gersen*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

9 Citations (Scopus)
313 Downloads (Pure)

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.

Original languageEnglish
Article number44666
Number of pages7
JournalScientific Reports
Volume7
DOIs
Publication statusPublished - 20 Mar 2017

Keywords

  • Correlative Light Electron Microscopy
  • Cross-Polarization Imaging
  • Gold nanoparticles
  • Fluorescent Labels
  • single particle detection
  • single molecule detection
  • Fluorescent Quenching
  • Colocalisation

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