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

Abstract

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 μm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was 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 μm. 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. Graphical abstract Gold nanoclusters (AuNCs) conjugated to a primary specific antibody serve as a label for amplified bioimaging of metallothioneins (MTs) by laser ablation coupled to inductively coupled plasma - mass spectrometry (ICP-MS) in human ocular tissue sections.

Laser ablation ICP-MS for simultaneous quantitative imaging of iron and ferroportin in hippocampus of human brain tissues with Alzheimer's disease

“…Although several studies can be found nowadays in the literature related to the imaging of biomolecules by LA-ICP-MS, most of them only collect qualitative distributions of sought proteins. In this context, we have recently proposed the use of small metal nanoclusters (NCs) with fluorescent properties as elemental labels for specific and absolute quantitative imaging of proteins in biological tissues by LA-ICP-MS [21,22]; considering that each NC has several hundreds of metal atoms, a high signal amplification in LA-ICP-MS measurements can be achieved. Therefore, these metal NCs offer possibilities for multimodal imaging (through fluorescence and MS analysis) in the same tissue section.…”

Super-Resolution Reconstruction for Two- and Three-Dimensional LA-ICP-MS Bioimaging

“…10 A major difficulty for both of these imaging approaches is the square relationship between signal intensity and spot size, which limits the practical spot size for quadrupolebased instruments to approximately 4 to 10 μm. 5,11 Below this, there is not enough material in each sampling event for detection.…”

Multi-Functionalized Nanomaterials and Nanoparticles for Diagnosis and Treatment of Retinoblastoma

“…AuNPs can act as perfect contrast agents for imaging, in addition to QDs, and several authors have used AuNPs for imaging eye cancers over the past few years [ 82 ]. For example, Cruz-Alonso et al described an immunohistochemical approach for visualizing the distribution of metallothionein 3 (MT3) and metallothionein 1/2 (MT 1/2) in human ocular tissue [ 74 ]. In this methodology, Au nanocluster (AuNC)-connected antibodies are used as markers and can be coupled with ICP-MS. Water-soluble fluorescent AuNCs with an average size of 2.7 nm were prepared by carbodiimide coupling and then covalently linked to antibodies.…”