Fluorescence Quenching by Colloidal Heavy Metals Nanoparticles: Implications for Correlative Fluorescence and Electron Microscopy Studies

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-cAu10, were also tested and proved to be a mixture of IgG-AF (unbound to cAu) and cAu-IgG-AF.

Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy

“…However, we observe no colocalisation between scattering and fluorescent signatures hinting that metallic nanoparticle-single fluorophore systems should be treated cautiously when selected as probes for CLEM1. This result confirms earlier results10 in bulk solution that binding single fluorophores to metallic nanoparticles results in strong quenching of the fluorescence. Further work applying the technique presented herein is in progress to aid in the development of CLEM labels and that would allow for fast characterization at the level of the individual probe.…”

“…Assessment of this quenching behaviour has to date largely been conducted by fluorometric analysis of the bulk probe solution10, however it is important to conduct this analysis at the single CLEM label level to remove any ambiguity in the analysis that may arise from fluorescent contributions from unbound fluorophores. To directly assess the fluorescent signal colocalised with individual CLEM probes, we use a recently introduced technique, Interferometric Cross-Polarization Microscopy (ICPM), which has shown detection of the weakly scattered signal from individual metallic nanoparticles down to 5 nm at low optical powers (<1 μW)11.…”

Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function

“…In order to distinguish several proteins in the same sample, immuno-gold particles of variable sizes directed against different fluorescence tags can be used. However, since sample fixation may interfere with genetically encoded fluorescence markers (Muller-Reichert and Verkade, 2014) and gold particles may quench the fluorophore in parallel fluorescence/electron imaging (Kandela and Albrecht, 2007), additional markers are desirable. One such alternative relies on quantum dots (QDs; Figure 2B ), which were shown to have a 10 times higher labeling efficiency than immuno-gold (Giepmans et al, 2005; Kuipers et al, 2015).…”

Probing the future of correlative microscopy

“…Developed at the beginning of the 1990s, it combines a fluorescent moiety with a very small nanogold particle. As the quenching of fluorescence is strongly influenced by the size (and distance) of the gold particle to the fluorophore [2], this probe does not seem to suffer serious quenching. A disadvantage is that the 1.4-nm gold is too small to be directly visible in the EM against the background contrast of cells or tissues.…”

“…a gold particle next to a fluorescent molecule decreases or completely masks the fluorescence emission (see, e.g. [2]). Hence, there have been major efforts to create the 'optimal' CLEM probe, one that is visible directly in the LM and EM and can be easily tagged to a protein of interest.…”