Bioimaging of metallothioneins in ocular tissue sections by laser ablation-ICP-MS using bioconjugated gold nanoclusters as specific tags

An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma -mass spectrometry (ICP-MS). Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 mu m thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MSwas performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 mu m. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method.