High-resolution, high-throughput imaging with a multibeam scanning electron microscope

Abstract

Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.

Keywords: High-throughput imaging; multibeam; parallel data acquisition; scanning electron microscopy.

Figure 1

The multibeam SEM uses multiple beams in parallel to image a hexagonal sample area 100-μm wide. Primary electrons (solid lines, left) are focused onto the specimen and separated by a beam splitter from the secondary electrons (dotted lines, right) which are detected simultaneously. All electron beams form many individual images which are then merged into a single, large area micrograph.

Figure 2

Cerebral cortex of mouse brain (block-face), sample by Winfried Denk and Shawn Mikula, Max Planck Society, showing unmyelinated neuronal and glial processes and a neuronal nucleus (left of centre), acquired by the multibeam SEM at 0.45 GPixel/s and 3.8 nm pixel size, 26 nA total current, 270 electrons per pixel, scale bar: 10 μm. A 1–2 nm coat of palladium has been evaporated onto the block-face to dissipate charging (Titze & Denk, 2013). Within the cellular processes, mitochondria, microtubules, synapses and endoplasmic reticulum are visible. Inset lower right: 12 μm × 10 μm single-beam subimage, detail of the full multibeam image, scale bar: 5 μm.

Figure 3

Cortex of mouse brain (serial ultrathin section), sample by Jeff Lichtman and Richard Schalek, Harvard University, showing myelinated axons, plasma membranes, cell somata and dendrites, acquired by the multibeam SEM at 0.45 GPixel/s and 3.8 nm pixel size, 26 nA total current, 270 electrons per pixel, scale bar: 10 μm. Sample charging has been mitigated by placing the thin sections on a conductive surface such that no additional conductive coating is required. Within the cells, dendrites and axons, organelles such as mitochondria and endoplasmic reticulum are visible. Inset lower right: 12 μm × 10 μm single-beam subimage, detail of the full multibeam image, scale bar: 5 μm. Inset upper right: 3 μm × 2.6 μm detail of the single-beam subimage, scale bar: 1μm.

Figure 4

Femoral neck (PMMA-embedded and polished block-face), sample by Melissa Knothe Tate, University of New South Wales, and Ulf Knothe, Cleveland Clinic, showing an osteon comprising a bone capillary surrounded concentrically by osteocytes, acquired by the multibeam SEM at 0.18 GPixel/s and 11.3 nm pixel size, 40 nA total current, 420 electrons per pixel, scale bar: 10 μm. Inset lower right: 12 μm × 10 μm single-beam subimage, detail of the full multibeam image, showing one osteocyte, scale bar: 5 μm.

Figure 5

Test chip showing a hexagonal arrangement of calibration structures for tool adjustments. The structures are printed in an e-beam direct write lithography process with a high placement precision, etched in SiO₂ on a Si-substrate, and finally coated with a completely conductive layer, scale bar: 10 μm. Pixel size was 3.8 nm, acquisition speed 0.72 GPixel/s, 40 nA total current, 210 electrons per pixel. Inset lower right: 12 μm × 10 μm single-beam subimage, detail of the full multibeam image, scale bar: 5 μm.