on the number of electrons hitting the screen, making direct interpretation of phase contrast images slightly more complex. however this effect can be used to an advantage, as it can be manipulated to provide more information about the sample, such as in complex phase retrieval techniques. diffraction crystalline diffraction pattern from a twinned grain of fcc austenitic steel as previously stated, by adjusting the magnetic lenses such that the back focal plane of the lens rather than the imaging plane is placed on the imaging apparatus a diffraction pattern can be generated. for thin crystalline samples, this produces an image that consists of a pattern of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline or amorphous solid material. for the single crystal case the diffraction pattern is dependent upon the orientation of the specimen and the structure of the sample illuminated by the electron beam. this image provides the investigator with information about the space group symmetries in the crystal and the crystal's orientation to the beam path. this is typically done without using any information but the position at which the diffraction spots appear and the observed image symmetries. diffraction patterns can have a large dynamic range, and for crystalline samples, may have intensities greater than those recordable by ccd. as such, tems may still be equipped with film cartridges for the purpose of obtaining these images, as the film is a single use detector. convergent-beam kikuchi lines from silicon, near the zone axis analysis of diffraction patterns beyond point-position can be complex, as the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, and spherical and chromatic aberration. although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and can require extensive computer simulation and analysis, such as electron multislice analysis. more complex behavior in the diffraction plane is also possible, with phenomena such as kikuchi lines arising from multiple diffraction within the crystalline lattice. in convergent beam electron diffraction (cbed) where a non-parallel, i.e. converging, electron wavefront is produced by concentrating the electron beam into a fine probe at the sample surface, the interaction of the convergent beam can provide information beyond structural data such as sample thickness. electron energy loss spectroscopy (eels) using the advanced technique of electron energy loss spectroscopy (eels), for tems appropriately equipped, electrons can be separated into a spectrum based upon their velocity (which is closely related to their kinetic energy, and thus energy loss from the beam energy), using magnetic sector based devices known as eel spectrometers. these devices allow for the selection of particular energy values, which can be associated with the way the electron has interacted with the sample. for example, different elements in a sample result in different electron energies in the beam after the sample. this normally results in chromatic aberration – however this effect can, for example, be used to generate an image which provides information on elemental composition, based upon the atomic transition during electron-electron interaction. eels spectrometers can often be operated in both spectroscopic and imaging modes, allowing for isolation or rejection of elastically scattered beams. as for many images inelastic scattering will include information that may not be of interest to the investigator thus reducing observable signals of interest, eels imaging can be used to enhance contrast in observed images, including both bright field and diffraction, by rejecting unwanted components. three-dimensional imaging 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 a sample of cells (black) stained with osmium tetroxide and uranyl acetate embedded in epoxy resin (amber) ready for sectioning. sample preparation in tem can be a complex procedure. tem specimens should be less than 100 nanometers 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 nanometers. 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 a diamond knife blade used for cutting ultrathin sections (typically 70 to 350 nm) for transmission electron microscopy. biological tissue is often embedded in a resin block then thinned to less than 100 nm on an ultramicrotome. the resin block is fractured as it passes over a glass or diamond knife edge. this method is used to obtain thin, minimally deformed samples that allow for the observation of tissue ultrastructure. inorganic samples, such as aluminium, may also be embedded in resins and ultrathin sectioned in this way, using either coated glass, sapphire or larger angle diamond knives. to prevent charge build-up at the sample surface when viewing in the tem, tissue samples need to be coated with a thin layer of conducting material, such as carbon. sample staining a section of a cell of bacillus subtilis, taken with a tecnai t-12 tem. the scale bar is 200 nm. main article: staining tem samples of biological tissues need high atomic number stains to enhance contrast. the stain absorbs the beam electrons or scatters part of the electron beam which otherwise is projected onto the imaging system. compounds of heavy metals such as osmium, lead, uranium or gold (in immunogold labelling) may be used prior to tem observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein region. this process requires an understanding of how heavy metals bind to specific biological tissues and cellular structures. mechanical milling mechanical polishing is also used to prepare samples for imaging on the tem. polishing needs to be done to a high quality, to ensure constant sample thickness across the region of interest. a diamond, or cubic boron nitride polishing compound may be used in the final stages of polishing to remove any scratches that may cause contrast fluctuations due to varying sample thickness. even after careful mechanical milling, additional fine methods such as ion etching may be required to perform final stage thinning. chemical etching certain samples may be prepared by chemical etching, particularly metallic specimens. these samples are thinned using a chemical etchant, such as an acid, to prepare the sample for tem observation. devices to control the thinning process may allow the operator to control either the voltage or current passing through the specimen, and may include systems to detect when the sample has been thinned to a sufficient level of optical transparency. ion etching ion etching is a sputtering process that can remove very fine quantities of material. this is used to perform a finishing polish of specimens polished by other means. ion etching uses an inert gas passed through an electric field to generate a plasma stream that is directed to the sample surface. acceleration energies for gases such as argon are typically a few kilovolts. the sample may be rotated to promote even polishing of the sample surface. the sputtering rate of such methods is on the order of tens of micrometers per hour, limiting the method to only extremely fine polishing. ion etching by argon gas has been recently shown to be able to file down mtj stack structures to a specific layer which has then been atomically resolved. the tem images taken in plan view rather than cross-section reveal that the mgo layer