Open AccessFluorescence Quenching by Colloidal Heavy Metals Nanoparticles: Implications for Correlative Fluorescence and Electron Microscopy Studies
Author(s) -
Kandela Irawati K.,
Albrecht Ralph M.
Publication year - 2007
Publication title -
scanning
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.359
H-Index - 47
eISSN - 1932-8745
pISSN - 0161-0457
DOI - 10.1002/sca.20055
Subject(s) - fluorescence , quenching (fluorescence) , colloidal gold , alexa fluor , chemistry , immunolabeling , palladium , fluorescence microscope , microscopy , photochemistry , nanoparticle , analytical chemistry (journal) , materials science , biophysics , nanotechnology , chromatography , optics , organic chemistry , biology , physics , immunohistochemistry , immunology , catalysis
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 (cAu 18 and cAu 5 ) or 12 nm colloidal palladium particles (cPd 12 ) 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‐cAu 5 and IgG‐AF‐cAu 10 , were also tested and proved to be a mixture of IgG‐AF (unbound to cAu) and cAu‐IgG‐AF. SCANNING 29: 000‐V000, 2007. © 2007 Wiley Periodicals, Inc.