Array tomography reveals synaptic distinctions in imaging brain connections

Brain circuitry is incredibly complex. Even the best of traditional light microscopes have been unable to reliably resolve the tiny, tightly packed synapses that link a single neuron by as many as tens of thousands of connections.

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Brain circuitry is incredibly complex. Even the best of traditional light microscopes have been unable to reliably resolve the tiny, tightly packed synapses that link a single neuron by as many as tens of thousands of connections. But researchers at the Stanford University School of Medicine, applying an imaging system called array tomography, say they have been able to locate and count myriad such connections in unprecedented detail—quickly and accurately—using brain-tissue samples from mice.1 The work has allowed them to capture and catalog a "surprising variety" of neuronal connections.

Array tomography, invented by professor Stephen Smith and senior scientist Kristina Micheva, combines high-resolution photography with specialized fluorescent molecules that bind to different proteins and relies on massive computing power to convert captured data into imagery. In this study, the researchers carefully sliced tissue from a mouse's cerebral cortex into 70 nm thick sections, which they stained with antibodies designed to match 17 different synapse-associated proteins. The sections were further modified by conjugation to molecules that respond to light by glowing in different colors.

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Brain samples from a mouse, bioengineered so that particularly large neurons express fluorescent protein, allowed researchers to visualize synapses against the background of the neurons they linked.

The antibodies were applied in groups of three to the brain sections. After each application, huge numbers of extremely high-resolution photographs were automatically generated to record the locations of different fluorescing colors associated with antibodies to different synaptic proteins. The antibodies were then chemically rinsed away and the procedure was repeated with the next set of three antibodies, and so forth. Each individual synapse thus acquired its own protein-composition "signature," enabling the compilation of a very fine-grained catalog of the brain's varied synaptic types.

The information was recorded and processed by software—designed for the most part by graduate student Brad Busse—which virtually stitched together 2-D images of the slices into a 3-D rendering that the researchers can rotate, penetrate and navigate.

The study was designed to showcase the technique's application to neuroscience; the team discovered distinctions within a class of synapses previously assumed to be identical. They are now using array tomography to tease out more such distinctions, and with support from the National Institutes of Health, they are examining tissue samples from Alzheimer's brains.

Smith and Micheva are founding a company that is gathering investor funding; Stanford's Office of Technology Licensing has obtained one U.S. patent on array tomography and filed for a second.

1. K. Micheva et al., Neuron 68 (4): 639-653 (2010)

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