Paper BI+AS-WeA7
Protein Imaging from the Subcellular Level to the Single Protein Level

Wednesday, November 1, 2017, 4:20 pm, Room 12

Most of biological story tellings are mainly based on proteins and their interactions. Therefore protein imaging and their interaction studies have been the key interest in bio imaging. Most of protein bioimaging have been based on confocal fluorescence microscopy for 2 or 3 proteins. We have developed a new multiplex protein imaging method for TOF-SIMS with metal oxide nanoparticle (MONP) conjugated with proteins up to 9 proteins, in theory, several tens, and a single protein imaging technique based on He Ion Microscopy (HIM)

In SIMS analysis, MONPs provide high secondary ionization yield and amplification of ion yields. We systhesized 9 MONPs working right such as CoO, CdO, Fe3O4, TiO2, PbO, In2O3, SiO2, Al2O3, La2O3. In addition to protein imaging, SIMS intrinsically provides tens of bio-molecular imaging including lipids and metabolites, and metals with a TOF mass analyzer, which makes this new methodology to be an omni-molecular mass spectrometric imaging technique. Sliced and cultured mouse hippocampal tissues were imaged with typical spatial resolution of 2 µm, which can be improved down to 300 nm for 9 neuronal proteins. Proteins chosen to image mouse hippocampal tissues are NeuN for all nuclei, Cav1,3 for neuron cells, Iba1 for microglia cells, GFAP for astrocytes, AMPA receptor, phosphorylated Tau, amyloid beta (Aß) 1-42, amyloid precursor protein, and APOE, which were selected to visualize important proteins as landmarks of Alzheimer Disease (AD). With multiplex proteins imaging, we could estimate the proximity of associated proteins in mouse hippocampal tissues, which changes with aging and AD progression.

Since HIM has a spatial resolution of 0.5 nm, HIM can observe single proteins in theory but in practice, it may be very dfficult to observe a single protein molecule due to the similar secondary electron yields of proteins compared to other proteins or extracellular matrix molecules. We demonstrated that HIM can image each MONP conjugated with proteins from a mouse hippocampal tissue revealing the distribution of single proteins in synapses, neuronal soma, amyloid plaques, and neurofibrillary tangels with their changes along aging and AD.

With the co-development of multiplex protein SIMS imaging and single protein HIM imaging technology, I expect we can improve our understanding on the role of proteins and their interactions in biology, biomaterials, and medicine.