FIB-SEM · Lobeworks/17

FIB-SEM (focused ion beam scanning electron microscopy) is a volume imaging method that alternately mills a few nanometres off the face of a resin-embedded sample with a beam of gallium ions and photographs the fresh face with a scanning electron microscope, building a stack of images whose resolution is the same in ev

FIB-SEM. FIB-SEM (focused ion beam scanning electron microscopy) is a volume imaging method that alternately mills a few nanometres off the face of a resin-embedded sample with a beam of gallium ions and photographs the fresh face with a scanning electron microscope, building a stack of images whose resolution is the same in every direction.

The technique came from the semiconductor industry, where ion beams are used to open chips and inspect their layers. On a block of stained brain the ion beam works like a sandblaster one atom deep, and since the milling step can be made as fine as the pixel, the voxels are cubes, 8 nm on a side in the fly connectomes, where a cut section gives bricks as tall as the section is thick. Cubes matter for fine processes that run at a shallow angle to the cut, which a sectioned volume smears.

It is slow and it eats the sample. Every face is photographed once and then milled away, so a mistake cannot be re-imaged, and a whole fly nervous system took seven microscopes thirteen months.

It has a depth limit. Beyond about a hundred micrometres along the beam the milling streaks and the images degrade, so the brain is first cut into 20 µm slabs with a heated, oil-lubricated diamond knife (the hot knife), each slab is imaged on its own machine, and the slabs are stitched back together, losing about 30 nm at each seam.

It built two fly connectomes. The hemibrain of 2020 (13 slabs of a series of 37) and the male central nervous system published in 2026, brain and nerve cord together, were imaged this way; the female brain of FlyWire was imaged by sectioning and transmission electron microscopy instead.

FIB-SEM trades speed for voxels that are the same size in every direction.

Cutting with an ultramicrotome is faster and finer in the plane, and milling is finer in depth.

Questions: When is milling a brain with ions better than cutting it with a diamond? When the fine processes run in every direction and the volume is small enough to wait for. A diamond knife cuts sections about 40 nm thick, so a neurite running nearly parallel to the cut is smeared across one thick slice; an ion beam mills a few nanometres at a time, giving voxels of 8 nm on every side. The price is speed, the loss of the sample as it is imaged, and a depth limit of about 100 µm, which is why whole fly brains are first split into 20 µm slabs with a heated knife and milled on several machines at once.