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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Transmission electron microscopy | 9/13 | https://en.wikipedia.org/wiki/Transmission_electron_microscopy | reference | science, encyclopedia | 2026-05-05T10:06:36.644553+00:00 | kb-cron |
As TEM specimen holders typically allow for the rotation of a sample by a desired angle, multiple views of the same specimen can be obtained by rotating the angle of the sample along an axis perpendicular to the beam. By taking multiple images of a single TEM sample at differing angles, typically in 1° increments, a set of images known as a "tilt series" can be collected. This methodology was proposed in the 1970s by Walter Hoppe. Under purely absorption contrast conditions, this set of images can be used to construct a three-dimensional representation of the sample. The reconstruction is accomplished by a two-step process, first images are aligned to account for errors in the positioning of a sample; such errors can occur due to vibration or mechanical drift. Alignment methods use image registration algorithms, such as autocorrelation methods to correct these errors. Secondly, using a reconstruction algorithm, such as filtered back projection, the aligned image slices can be transformed from a set of two-dimensional images, Ij(x, y), to a single three-dimensional image, I′j(x, y, z). This three-dimensional image is of particular interest when morphological information is required, further study can be undertaken using computer algorithms, such as isosurfaces and data slicing to analyse the data. As TEM samples cannot typically be viewed at a full 180° rotation, the observed images typically suffer from a "missing wedge" of data, which when using Fourier-based back projection methods decreases the range of resolvable frequencies in the three-dimensional reconstruction. Mechanical refinements, such as multi-axis tilting (two tilt series of the same specimen made at orthogonal directions) and conical tomography (where the specimen is first tilted to a given fixed angle and then imaged at equal angular rotational increments through one complete rotation in the plane of the specimen grid) can be used to limit the impact of the missing data on the observed specimen morphology. Using focused ion beam milling, a new technique has been proposed which uses pillar-shaped specimen and a dedicated on-axis tomography holder to perform 180° rotation of the sample inside the pole piece of the objective lens in TEM. Using such arrangements, quantitative electron tomography without the missing wedge is possible. In addition, numerical techniques exist which can improve the collected data. All the above-mentioned methods involve recording tilt series of a given specimen field. This inevitably results in the summation of a high dose of reactive electrons through the sample and the accompanying destruction of fine detail during recording. The technique of low-dose (minimal-dose) imaging is therefore regularly applied to mitigate this effect. Low-dose imaging is performed by deflecting illumination and imaging regions simultaneously away from the optical axis to image an adjacent region to the area to be recorded (the high-dose region). This area is maintained centered during tilting and refocused before recording. During recording the deflections are removed so that the area of interest is exposed to the electron beam only for the duration required for imaging. An improvement of this technique (for objects resting on a sloping substrate film) is to have two symmetrical off-axis regions for focusing followed by setting focus to the average of the two high-dose focus values before recording the low-dose area of interest. Non-tomographic variants on this method, referred to as single particle analysis, use images of multiple (hopefully) identical objects at different orientations to produce the image data required for three-dimensional reconstruction. If the objects do not have significant preferred orientations, this method does not suffer from the missing data wedge (or cone) which accompany tomographic methods nor does it incur excessive radiation dosage, however it assumes that the different objects imaged can be treated as if the 3D data generated from them arose from a single stable object.
== Sample preparation ==
Sample preparation in TEM can be a complex procedure. TEM specimens should be less than 100 nanometres thick for a conventional TEM. Unlike neutron or X-ray radiation the electrons in the beam interact readily with the sample, an effect that increases roughly with atomic number squared (Z2). High quality samples will have a thickness that is comparable to the mean free path of the electrons that travel through the samples, which may be only a few tens of nanometres. Preparation of TEM specimens is specific to the material under analysis and the type of information to be obtained from the specimen. Materials that have dimensions small enough to be electron transparent, such as powdered substances, small organisms, viruses, or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the specimen onto films on support grids. Biological specimens may be embedded in resin to withstand the high vacuum in the sample chamber and to enable cutting tissue into electron transparent thin sections. The biological sample can be stained using either a negative staining material such as uranyl acetate for bacteria and viruses, or, in the case of embedded sections, the specimen may be stained with heavy metals, including osmium tetroxide. Alternately samples may be held at liquid nitrogen temperatures after embedding in vitreous ice. In material science and metallurgy the specimens can usually withstand the high vacuum, but still must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Constraints on the thickness of the material may be limited by the scattering cross-section of the atoms from which the material is comprised.
=== Tissue sectioning ===