The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure

Abstract

The rapid accumulation of neuroproteomics data in recent years has prompted the emergence of novel antibody-based imaging methods that aim to understand the anatomical and functional context of the multitude of identified proteins. The pioneering field of ultrastructural multiplexed proteomic imaging now includes a number of high resolution methods, such as array tomography, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy and automated transmission electron microscopy, which allow a detailed molecular characterization of individual synapses and subsynaptic structures within brain tissues for the first time. While all of these methods still face considerable limitations, a combined complementary approach building on the respective strengths of each method is possible and will enable fascinating research into the proteomic diversity of the nervous system.

Figure 1. Different proteomic views of the synapse

Array tomography, STORM and ATEM provide detailed snapshots of multitudes of individual synapses, while single particle tracking reveals the dynamic nature of proteins at smaller subsets of synapses. Array tomography: synapse visualization as volume renderings (A) or as “synaptograms” (B). Synaptograms display high dimensional proteomic data with columns representing individual serial sections through a synapse and rows representing each marker. The two synaptograms show examples of a glutamatergic synapse with glutamatergic markers boxed in red and a GABAergic synapse with the respective markers boxed in blue. Reproduced with permission from [16]. STORM: A synaptic coordinate system is defined by the positions of Bassoon (presynaptic) and Homer 1 (postsynaptic). Triple immunolabeling allows the localization of other synaptic proteins along these coordinates. Reproduced with permission from [21]. ATEM: A fragment of the mammalian AII amacrine cell network is visualized (A) with all synaptic and gap junction contacts mapped. Examples of synaptic connections between the reconstructed cells are shown in B. ATEM datasets contain both ultrastructural and molecular markers information. E, glutamate, G, glycine, γ, GABA. Reproduced with permission from [27]. Single particle tracking: The trajectories of single quantum dot-labeled α7-nicotinic acetylcholine receptors (white) are shown in relation to glutamatergic synapses (red, mCherry-Homer1c) and GABAergic synapses (green, EGFP-gephyrin) in cultured hippocampal interneurons. Reproduced from [31].