Electron Tomography Basics:
Transmission Electron Microscopy Tomography (TEMT) is a method to reconstruct a 3-dimensional model of a thin TEM sample using a tilt series.
Both our JEOL JEM-2200FS and FEI tecnaiT12 are quipped with a goniometer which allows tilting of a sample up to 70 degrees in both directions.
Before attempting to do TEMT a user should be comfortable with all the needed software for TEM operation, tilt series acquisition, reconstruction (tomography) and modelling. Expect for basic TEM operation we utilize the same software for tilt-series acquisition in both TEMs (Serial EM). Reconstruction and Modelling are TEM independent and only require a decent computer with sufficient speed and memory. All this software packages are not targeted at the average PC user and require self initiative to learn and understand the meaning of the individual steps. All the software packages described below are available for download for free. If you plan to install on your own PC make sure that it has enough power to be able to handle the large data files.
- After a careful alignment, where special care ids given to the eucentric height of the sample, of the TEM a tilt-series is acquired using Serial EM. Even so the actual tilt series acquisition is automatic there are several manual alignment steps before the software can take over.
- A tilt series typically consists of about 80 images recorded at 1.5 degree steps from -60 deg to +60 deg at a resolution of 2Kx2K (our camera and computer can also handle 4Kx4K which will result in a single image stack of 2.5GB).
- if everything goes well, the image stack will be copied to a tomography workstation for the actual tomograph calculation using ETomo which is part of the iMOD image processing package. You can find a very well written ETomo tutorial here which will guide you step-by-step through the procedure.
- Once the tomograph has been calculated various modelling software packages can be use to make create volume rendered images. Chimera is a very powerful 3-D rendering tool which can read stack files created by ETomo.
- Sample Thickness: For 200kV sample thicknesses of carbon based materials up to 70nm-80nm are practical. When tilting a sample the thickness relative to the incident beam increases with tilt angle. At 70 deg the actual thickness the e-beam has to penetrate is about 2.5x the actual film thickness. For 200kV acceleration voltage this still usable even so focusing will get difficult. For samples composed of heavier element the film thickness typically has to be smaller since the mean free path is decreasing. For the same reason the sample needs to be thinner when using 120kV acceleration voltage.
- Choice of Grid: The hole size of the TEM grid is critical for successful tomograph acquisition. Obviously if the grid holes are too small or the sample is located next to a grid bar the grid bar itself will cover the sample at even lower tilt angles which prohibits a tilt series acquisition. This can make the sample preparation the most time consuming and frustrating exercise for TEMT. Especially when preparing a polymer sample via cryo-microtomy on has very limited control over the final sample location on the grid - and to some extend a little bit of luck is needed. In order to stabilize a sample ultra-thin carbon or Lacey carbon can be used. Carbon will add its thickness to the overall sample thickness and Lacey carbon only is usefull when doing TEMT at rather high magnifications as the carbon skeleton will overlap with the sample.
- Sample stability: