Super-resolution brain imaging

Conventional fluorescence microscopy is limited by diffraction to about 250 nm — too coarse to resolve many neuronal structures. STED microscopy (Hell & Wichmann, 1994) breaks this limit by adding, on top of the excitation spot, a doughnut-shaped depletion beam that switches off fluorescence everywhere except at its very centre: the effective focal spot shrinks well below the diffraction limit, down to a few tens of nanometres. Shaping this depletion beam (2D, z or 3D STED) tailors the resolution gain in the lateral and axial directions.

Principle of STED (a); the STED microscope (b); the improvement in resolution achieved with STED (c); and the resulting depletion and effective PSFs (d–e) (Bancelin et al., 2023).

Applied in vivo, STED makes it possible to image dendritic spines — the tiny postsynaptic compartments that underlie synaptic plasticity — directly in the brain of a living mouse, through a cranial window and at nanoscale resolution (Bancelin et al., 2023). Chronic, time-lapse imaging then reveals how individual spines change shape and turn over in their native environment.

In vivo STED imaging of dendritic spines along a dendrite in the living mouse brain (top, 14×7 µm²), and a 3D comparison of a spine imaged with two-photon vs STED microscopy (bottom).

A complementary approach, super-resolution shadow imaging (SUSHI) (Tønnesen et al., 2018), flips the contrast: instead of labelling sparse structures, the extracellular space is filled with a fluorescent dye, so that every cell and process appears as a dark “shadow” against the bright background. A single STED acquisition then reveals the complete, densely-packed anatomy of the tissue — neurons, glia, blood vessels and the extracellular space itself — non-invasively and in vivo.

SUSHI (super-resolution shadow imaging): the extracellular space is labelled so that cells and processes appear as dark shadows, revealing the full tissue anatomy in vivo.