Imaging of proteins in biological tissues by fluorescence microscopy and laser ablation-ICP-MS using natural and isotopically enriched silver nanoclusters

Abstract

Small water soluble silver nanoclusters (AgNCs) were synthesized either using silver of natural isotopic composition or isotopically enriched silver (^natAgNCs and ¹⁰⁹AgNCs) and employed as labels for specific protein bioimaging. Both types of AgNCs emit a strong fluorescence at 660 nm. Distributions, independently and simultaneously, of metallothioneins 1 and 2 (MT1/2) and superoxide dismutase 1 (SOD1) in human retinal layers from 5 μm thick ocular tissue sections of post-mortem donors were investigated as a proof of concept. For such a purpose, anti-MT1/2 and anti-SOD1 were respectively labelled with ^natAgNCs and ¹⁰⁹AgNCs, and an immunohistochemical protocol was developed. Detection was carried out by fluorescence microscopy and laser ablation (LA) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). In the case of fluorescence microscopy, a high autofluorescence signal was observed at the outer segments of photoreceptors within the retina thus limiting the analytical information in such a region. Additionally, since both types of AgNCs emit at the same wavelength it was not possible to obtain the simultaneous distribution of both proteins by fluorescence microscopy. However, simultaneous specific bioimaging of the two proteins at the same tissue section was possible using both types of AgNCs as labels combined with ICP-MS detection. For such a purpose, the potential of an isotope pattern deconvolution mathematical tool in combination with LA-ICP-MS was successfully evaluated.