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Understanding the Basics of 3D Electron Microscopy

Electron microscopy is an extremely powerful analytical tool used to characterize a near limitless range of sample types, regardless of their material class. This outstanding versatility is coupled with exceptionally high spatial resolutions that far outstrip the capabilities of conventional optical microscopy. In fact, it seemed for a while as though the technological advances of electron microscopy faced ever-increasing resolution with no obvious barriers to infinitely-improving performance.

A relatively novel frontier in improving the depth of metrological data that electron microscopy can provide is three-dimensional (3D) electron microscopy.

In 3D electron microscopy, multiple sets of stereoscopic images taken from a range of viewing angles are collated and digitally constructed into a three-dimensional dataset. It is similar to computed tomography (CT) where samples are imaged as a series of two-dimensional cross-sections. Consequently, 3D electron microscopy is sometimes referred to as electron tomography.

To understand the basics of 3D electron microscopy, it is worth recapping the fundamentals of the two primary types of electron microscopes: SEM and TEM.

The Basics of SEM & TEM

Optical microscopes that use visible light to illuminate sample materials are limited to resolving features that are no closer than 200 nanometres (nm). Electron microscopes, by comparison, can readily achieve spatial resolutions approaching the sub-nanoscale – or magnifications greater than 1,000,000x.

However, optimal spatial resolution varies depending on microscope configurations. Scanning electron microscopes (SEM) are typically limited to resolutions of around 0.5 nm while the resolution of high-power transmission electron microscopes (TEM) may extend into the picometre (pm) regime.

It is worth exploring the differences between SEM and TEM in more depth at a later date, but for the purposes of understanding how 3D electron microscopy works, it is enough to know that both techniques use a focused electron beam to probe samples and obtain images based on secondary, backscattered, or transmitted electrons.

SEMs can be used to acquire high-resolution 3D surface images for specimens of any thickness, and typically offer greater flexibility and lateral range than TEMs, which are limited to extremely thin samples. However, TEMs can provide a 2D projection image of the inner structures of various sample types with significantly better resolution than SEMs.

The Principles of 3D Electron Microscopy

Although the differences between SEMs and TEMs seem significant, the principles of 3D electron microscopy do not vary dramatically by microscope configuration. Datasets acquired from either type of microscope can be readily converted into a high-resolution 3D model using the right software. The simplest approach to 3D electron microscopy is to take a pair of stereo images of the same surface but with differing tilt angles (via physical tilt or more recently by utilizing segmented detectors). Intuitive software is used to identify homologous points between the two images, yielding highly accurate 3D coordinates that are used to create a true 3D digital representation of the sample.

The benefits of 3D electron microscopy are profound, enabling researchers to obtain extremely detailed height maps of surface topographies, or of the finest internal structures at the smallest possible range.

Would you like to learn more about the basic principles or applications of 3D electron microscopy? Contact a member of the JEOL team today with any questions.

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