Electron Tomography (ET)

Overview

The fundamental concept behind tomographic reconstruction of all sorts is that it is possible to construct a three-dimensional (3d) representation of a 3d object by combining two-dimensional (2d) projection Johann Radonimages of the 3d object. It will be useful for the rest of this discussion to present some of the history of this field, both to clear up certain misconceptions about tomography and also because the field uses several different terminologies and formalisms when dealing with the relationship between a 3d object and its 2d projections. The "N-dimensional" mathematical proof behind the relationship between objects and projections and the "Radon transform" itself were introduced by the Austrian mathematician Johann Radon in a hard-to-locate article published in 1917. For various reasons, Radon's treatment of the problem had very little practical impact at that time but was of enormous interest to people working in areas of pure mathematics such as integral geometry and partial differential equations. Even today, although many people make reference to Radon and Radon transforms when talking about tomography, Radon's approach to the problem is seldom actually used.

In the 1950's, the physicist Allan Cormack developed the theoretical underpinnings that led to the field of X-ray computed tomography (CT) used in medical imaging. Alan Cormack's device for tomography of a human "phantom"CT was a practical demonstration of the relationship between a 3d object and 2d projections (X-ray images, in this case). Cormack's early work was done without any knowledge of Radon's mathematical treatment: he notes in his 1979 Nobel lecture that he only learned about Radon's work (and the work of various other people in the area we now call tomography) in the early 1970's when returning to CT-scanning problems. Cormack not only developed the mathematics for dealing with the problem of generating a volume based on a set of X-ray images collected at various angles relative to the original 3d object (various back-projection algorithms), but he also produced a practical demonstration of the theory using an X-ray source and a physical "phantom object" that mimicked the properties of the human body. In addition, Cormack described how noise in the X-ray images propagates into reconstructed volumes and developed practical ways of dealing with these effects. Cormack and the electrical engineer Godfrey Hounsfield (who built the first practical CT scanner) received the 1979 Nobel Prize in Physiology or Medicine "for the development of computer assisted tomography."

Another formalism that describes the relationship between 3d objects and 2d projections was developed by David DeRosier and Aaron Klug in the mid-1960's at the Medical Research Center's Laboratory for Molecular Biology in Cambridge, England. According to DeRosier, this work was done independently of both Radon's mathematics and Cormack's more practical work. The DeRosier and Klug description of the relationship between a 3d object and its 2d projections should be familiar to everyone in the field of cryoTEM and is known either as the projection theorem or the central section theorem (which more closely ties the name to the concepts behind it). In essence, the central section theorem states that the Fourier transform of a 2d projection of a 3d object is a central section (i.e., a section passing through the origin of the transform) of the Fourier transform of the 3d object. When the 3d reconstruction problem is considered using this framework, it is relatively easy to see how it should be possible to fill 3d Fourier transform space with the 2d Fourier transforms from a series of projection images at different tilt angles and to generate a 3d reconstruction simply by inverse Fourier transforming that (partially filled) volume.

Details of Electron Tomography

aligned tilt series of a porous silica sample

Electron tomography (ET) can be performed when a side-entry electron microscope is in either TEM or STEM mode. In both cases, the fundamental idea behind ET is that the user records an image of a particular area of the specimen, tilts the microscope specimen holder by a known amount and records a new image of the same area. This process is repeated until the tilt angle reaches the limit of a particular holder or until the specimen can no longer be imaged because the electron beam is blocked by a grid bar, by the holder itself, etc.

This tilting process can start at large positive or negative tilt and proceed to the opposite extreme. It can also start at zero tilt, record images as the holder tilts toward large positive tilt then jump back to zero and record images as the holder tilts towards large negative tilt or it can start at zero tilt, record images as the holder tilts toward large negative tilt then jump back to zero and record images as the holder tilts towards large positive tilt. There are advantages and disadvantages to all of these data collection schemes (and different automated data collection software packages may be restricted to one of them), but the major concern is that each image must have an associated tilt angle.

Although the sequence described above (record image, tilt specimen, record new image, tilt specimen further, etc.) is fundamentally extremely simple, things are not nearly so simple when a real specimen and a real electron microscope are used. The fundamental problems encountered during data collection for tomography are the imperfection of the microscope's goniometer (the mechanism in a side-entry (S)TEM instrument that allows the specimen holder to tilt) and the lack of absolute flatness for most (S)TEM grids. Lack of specimen flatness only exacerbates other problems caused by the imperfections of the goniometer, and most of the rest of this section will focus on the problems of the goniometer. Before dealing with goniometer issues, a brief description of certain microscope properties and features is useful.

aligned tilt series of plastic embedded Drosophila muscle

The optics of many (S)TEM's are designed to work best at a particular position along the beam path of the instrument. This position is coupled to empirically-determined objective lens conditions that JEOL microscopes call standard focus and that FEI microscopes call eucentric focus. When a specimen sits at this special position along the beam path and when the objective lens is put into the standard focus condition, all images will be essentially at focus. The images can be defocused either by moving the specimen closer to or further from the electron source, or by changing objective lens settings. A user can check the self-consistency of a particular electron microscope by moving the specimen a certain amount along the beam path (the z position, see below for a description of this geometry) and then returning the image to focus by adjusting the objective lens. When the microscope calibrations are self-consistent, movement in z (often measured with an accuracy of around 0.1 micrometers, μm) will closely match the change in focus (usually measured with an accuracy of a few nanometers, nm).

Since it is often possible to observe HRTEM image differences with changes in defocus smaller than 0.1 μm (i.e., the accuracy of the specimen position), this coupling between standard focus and position along the beam path is obviously limited by things like the precision to which the specimen can be moved along the beam path and how well the standard focus objective lens conditions have been determined. For all well-behaved (S)TEM's, standard focus should be well within a few μm of the optimal specimen position along the beam path.

diagram of the geometry of goniometer and optics around a TEM specimenReturning this discussion to goniometer issues, there is a line in 3d space that runs along and through the rod of a side-entry specimen holder that is called the specimen holder's rotation axis, the (holder's) mechanical axis or even more simply, the tilt axis. The specimen rotates around the tilt axis when the specimen holder's tilt is adjusted using the goniometer (as illustrated in the figure to the right, adapted from a presentation by Dr. Jacob Brink with JEOL USA). If the goniometer's geometry is described so that the x-axis runs parallel to and through the tilt axis and the y-axis is normal to both the x-axis and the electron beam (which becomes by default the direction of the z-axis), an adjustment of the entire goniometer in the y-direction (usually done by a service engineer) aligns the tilt axis of the holder to the optical axis, shown in magenta in the figure to the right (i.e., the tilt axis of the holder and the optical axis of the electron beam intersect). The user should then also adjust the goniometer in the z-direction (i.e., parallel to the beam path) so that the specimen is at the position described previously as the point along the beam path where the optics of the microscope perform best (which is also where standard focus should be set, and is marked as the specimen plane in the figure). This z-position that the user sets is sometimes also called the eucentric height or eucentric position. The microscope's description of the eucentric height (using its internally defined x/y/z co-ordinate system) will differ both from holder to holder (i.e., the exact location in z of the gird relative to the shaft of a holder can vary by more than 100 μm among the different holders for a single microscope) and on the physical dimensions and orientation of individual grids (i.e., the eucentric height will differ by the grid's thickness depending on whether the specimen is closer to or further from the electron source than the rest of the grid).

 
eucentric height wrong by ~10 μm eucentric height adjusted
using serialEM

If the goniometer were itself perfect and everything else (including the eucentric height and flatness of the grid) were also perfect, it would be possible to set the objective lens conditions to standard focus, tilt the specimen holder to any angle and record an at-focus image of exactly the same part of the specimen as would be seen in images recorded at any other tilt angle. When the eucentric height is incorrect, tilting the specimen holder causes the image of the specimen to sweep in the y-direction (normal to the tilt axis), as shown in the first column of images at the right (where the tilt axis is roughly horizontal). After correcting the z-position, this sweep is much reduced (second column of images), and the residual movement is likely because the tilt and optical axes do not intersect. The image sweep in this case will often happen only in the positive (or negative) tilt direction (and not both).

If this state of perfection were possible, collecting data for tomography would be no more complicated that what was described above: record an image, tilt the specimen, record a new image and simply keep repeating this process. However, goniometers are physical devices and are likely to have imperfections such that even when the goniometer has been carefully "tuned" and even when the specimen is located at the correct position, tilting causes the specimen to move slightly in x, y and z. This movement means that collecting data for tomography really involves recording an image, tilting the specimen, finding the same area that was recorded in the initial image, determining (and adjusting) the defocus, recording a new image and only then repeating these steps over and over.

A human microscope operator can perform these steps, but the process is slow, involves extensive exposure of the sample to the electron beam and (from personal experience) is prone to errors as the number of recorded images increases (and keep in mind both that individual tomographic data sets can have more than 100 images and that a given project could require dozens of data sets). Because of these factors, the integration of computers into (S)TEM instruments has been crucial for ET to become a commonly practiced technique. The current generation of side-entry electron microscopes are all capable of "automated" ET (i.e., the user selects an area for tomography, sets parameters such as the tilt limits, the angular increment between images and the defocus for each image and then lets the computer acquire the images). With a well-behaved specimen, such computer control can record a complete tilt series in significantly less than an hour, allowing the user to collect a dozen or more data sets in a single session at the microscope. When these steps were all done manually, it was seldom possible to acquire more than 3 or perhaps 4 data sets in a single session, and operator error often made the last data sets and last images worse than those from the beginning of the session.

Practical Tomography at IU

We have installed the tomography data collection program serialEM from the Boulder Laboratory for 3-D Electron Microscopy of Cells on the JEOL JEM 3200FS and the new JEOL JEM 1400plus. In addition, we are in constant communication with David Mastronarde (the developer of this program) and Jacob Brink (JEOL USA's tomography guru) with regard to both improvements in the program and its implementation on our instruments. Training to use serialEM requires first that a user be well-trained on the chosen microscope. This is followed by a series of sessions learning both overall concepts related to tomography and the details of serialEM. Once a user has a tomographic tilt series, it becomes necessary to learn to process the images. The EMC maintains an up-to-date version of IMOD (also from the Boulder Laboratory for 3-D Electron Microscopy of Cells) on Karst and a tutorial for its use is available. Examples of data recorded with serialEM and/or processed using IMOD can be found here.

serialEM controls most aspects of the operation of the 3200FS and the 1400plus and allows for automated collection of tomographic tilt series. Tilt range limits are determined by both the specimen holder used and by properties of the grid being examined (the space between grid bars, overall grid thickness, etc.). We currently have three holders (the JEOL high tilt holder , the Fischione dual axis tomography holder and the Gatan 914 cryo-tomography holder) with tilt ranges of ±70° in the JEOL JEM 3200FS when examining an object near the center of a 200 or 300 mesh TEM grid. The 914 cryo-holder should only be used for cryo samples while the other two holders can be used interchangeably for any room temperature specimen. The Fischione holder is designed to make it extremely simple to collect dual tilt-axis data sets, while the JEOL high tilt holder is much easier to use when loading and unloading a grid.

slot and circle grids

Some examples of commercially
available slot and hole grids

As noted above, in addition to the tilt range of a given holder, factors such as the spacing between grid bars and the actual grid thickness also strongly influence the "working tilt range" that can be achieved with any given specimen. The best grids to use for tomography are slot or hole grids which have very large openings that are completely covered by a support film. Since there are no grid bars in these grids, the only regions that become occluded by the grid when highly tilted are those immediately adjacent to the edge of the hole through the grid. Such grids are made by several companies and are available from any vendor of TEM supplies. Although many commercially available slot/hole grids are made from nickel, the EMC does not recommend using nickel grids due to the paramagnetic properties of the metal. Also bear in mind that the material in each of the high tilt holders can potentially block the electron beam for certain areas that can be viewed when the holder is not tilted.

It is certainly possible to use mesh EM grids for tomography. However, as the mesh gets finer, there is less and less useful area when the grid is tilted to higher and higher angles (see the example images for different mesh grids), and the EMC recommends using 200 mesh grids for specimens where tomography might be an option.

diagram showing increase in apparent thickness as tilt angle increasesIn addition, as the specimen tilt angle goes higher and higher, the apparent thickness (the distance that the electron beam must travel when passing through the specimen) increases for any specimen that can be described as a "slab" (i.e., much thinner in the z-dimension than in the x- and y-dimensions). This means that even in cases where the geometry of the specimen holder and the grid allow tilting as high as 70° or 80°, the specimen behaves as if it were much thicker (3x just above 70° and well over 5x at 80°). Even for relatively thin specimens, the image quality degrades significantly when electrons pass through this much material, and data collection must often be stopped due to this issue before the physical geometry prevents the user from acquiring additional images.

All these factors (the holder itself, blocking the electron beam by the grid that supports the specimen and apparent increases in specimen thickness as the tilt angle increases) limit the tilt range over which a series of images can be collected. This results in systematically limited sampling of the specimen which has well understood effects on the 3d reconstruction generated from the image series: information in the z-direction is broadened or smeared (more rigorously put, the point spread function for the reconstruction is much worse in the z direction than it is in x or y). This effect is present regardless of whether the reconstruction is performed using actual Radon transforms, weighted back-projection or filling 3d Fourier space with 2d Fourier transforms, though some of the computationally-intensive iterative methods can be used to make the reconstructions look better.

part of Figure 1 from McIntosh et al. (2005) Trends in Cell Bio. 15, 43-51Another issue that arises due to the limited tilt range of the input projection images is harder to describe but easy to illustrate (also see the page on dual tilt-axis tomography). The issue is that features in the image which run perpendicular to the tilt axis are poorly resolved in the reconstructed volume. In the figure at the right (from McIntosh et al. (2005) Trends in Cell Bio 15, 43-15), the left panel shows the original image of Goethe while the other panels are reconstructions of the original (2d) image based on back-projecting different sets of 1-dimensional (1d) projections generated at 1° increments. The middle panel uses a tilt range of ±90°. The detail in this reconstruction is quite good though this middle image is clearly different from the original. The wavy lines in the background that radiate from the center of the image are the result of sampling the original image using an increment of 1° and could be reduced by sampling more finely. On the other hand, the right panel uses 1° projections that only cover ±60° (i.e., a restricted tilt range that would be commonly encountered in electron tomography). Horizontal features in the original image such as Goethe's mouth and eyebrows are entirely absent from the reconstruction. This 2d example shows the sorts of reconstruction artifacts that can be encountered in ET, and much of the current work in ET revolves around methods to minimize such artifacts.

serialEM can record a finely sampled tilt series using the full size of the UltraScan CCD camera on the 3200FS and the OneView camera on the 1400plus in considerably less than 1 hour for well-behaved samples. On the 3200FS, serialEM has the ability to use the in-column energy filter to record zero-loss images, plasmon images and/or energy filtered images of defined energy loss (EFTEM) and to record STEM images. The program also has built-in low dose capabilities for either microscope, which can be coupled to the energy filter for cryo electron tomography (cryoET) on the 3200FS.

People who want to use either instrument for tomography should contact the David Morgan to discuss a particular project. Users will in most cases need to dedicate additional time both for sample preparation and for training in the use of serialEM.


Advanced Concepts and Techniques

This discussion has dealt with the most simple aspects of tomography: different ways to formalize the conversion of 2d projection images into 3d reconstructions, the tilt geometry of a side-entry electron microscope and factors that influence it, practical aspects of tomography in general and a few details about tomogrpahy in the EMC. There are many additional aspects of tomography that are important both when thinking about how to design a tomography experiment and when dealing with real tomographic data. The following topics are addressed elsewhere in the EMC website: