Fluorescence quenching by colloidal heavy metals nanoparticles: Implications for correlative fluorescence and electron microscopy studies

Irawati K. Kandela, Ralph M. Albrecht*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Labels for correlative immunolabeling in light (LM) and electron microscopy (EM) employing colloidal metal nanoparticles (gold or palladium) and fluorescent dyes (Alexa Fluor, AF) were investigated. The fluorescence signals from direct conjugates (cAu-IgG-AF) and from an indirect label system (cAu-IgG-anti IgG-AF) were studied using scanning spectrofluorometry and fluorescence light microscopy. Direct conjugation of protein - AF, IgG-AF or FGN-AF to 18 and 5 nm colloidal gold (cAu18 and cAu5) or 12 nm colloidal palladium particles (cPd12) resulted in nearly completely quenched fluorescence signals (>99 %) at excitation wavelengths of 488, 546 and 594 nm. In contrast, indirect conjugation, when colloidal metal particles and AF were conjugated to primary or secondary antibody, respectively (cAu-IgG-antiIgG-AF), sufficient fluorescence signal was detected. Commercially available conjugates, consisting of IgG-AF-cAu5 and IgG-AF-cAu 10, were also tested and proved to be a mixture of IgG-AF (unbound to cAu) and cAu-IgG-AF.

Original languageEnglish (US)
Pages (from-to)152-161
Number of pages10
JournalScanning
Volume29
Issue number4
DOIs
StatePublished - Jul 2007

Keywords

  • Alexa Fluor
  • Correlative labeling
  • Electron microscopy
  • Fluorescent dyes
  • Light microscopy
  • Multiple labeling

ASJC Scopus subject areas

  • Instrumentation
  • Atomic and Molecular Physics, and Optics

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