Microscopy, Staining and Tissue Clearing

From Nissl and Golgi stains to electron microscopy, confocal and two-photon imaging, rainbow-coloured Brainbow mice, see-through brains with CLARITY, and expansion microscopy that physically enlarges tissue.

Classic stains

Brain tissue is almost transparent and colourless, so neuroanatomy has always depended on stains. The Nissl stain (Franz Nissl, 1880s) colours the RNA-rich material in cell bodies, revealing the density and arrangement of neurons — the basis of Brodmann's cortical maps. The Golgi stain (1873) blackens a few whole neurons including all their branches. Myelin stains (Weigert) reveal fibre tracts. Later, tract-tracing methods injected substances that are carried along axons, mapping connections between regions.

Electron microscopy

Light microscopes can't resolve structures smaller than about 200 nanometres. Electron microscopy (EM), applied to the brain in the 1950s, revealed the synaptic cleft, synaptic vesicles and the fine structure of myelin — settling the neuron doctrine. Today, automated serial-section EM is the basis of connectomics.

Fluorescence and antibodies

Immunohistochemistry uses antibodies tagged with dyes to label specific proteins, identifying cell types. Green fluorescent protein (GFP) from jellyfish, recognised with the 2008 Nobel Prize in Chemistry, lets researchers make chosen cells glow. Confocal microscopy captures sharp optical sections, and two-photon microscopy (developed by Winfried Denk and colleagues in 1990) uses infrared light to image hundreds of micrometres deep into living brain tissue with less damage.

Brainbow

In 2007 Jeff Lichtman, Joshua Sanes and colleagues created Brainbow mice, in which genetic recombination gives neighbouring neurons random combinations of several fluorescent proteins, painting them in around 100 distinguishable hues — so individual axons could be traced through tangled tissue.

Clearing and expansion

Lipids scatter light, making brains opaque. In 2013 Kwanghun Chung and Karl Deisseroth introduced CLARITY, which replaces lipids with a transparent hydrogel, producing see-through whole mouse brains that can be labelled and imaged in 3D. Many other clearing methods followed. In 2015 Ed Boyden's lab introduced expansion microscopy: tissue is embedded in a swellable gel, then physically expanded several-fold (later, over twenty-fold), so nanoscale structures can be resolved with ordinary light microscopes.