to cause damage by application of excessive heat. for this reason materials with either a high melting point, such as tungsten, or those with a low work function (lab6) are required for the gun filament. furthermore, both lanthanum hexaboride and tungsten thermionic sources must be heated in order to achieve thermionic emission, this can be achieved by the use of a small resistive strip. to prevent thermal shock, there is often a delay enforced in the application of current to the tip, to prevent thermal gradients from damaging the filament, the delay is usually a few seconds for lab6, and significantly lower for tungsten. electron lens diagram of a tem split pole piece design lens electron lenses are designed to act in a manner emulating that of an optical lens, by focusing parallel electrons at some constant focal distance. electron lenses may operate electrostatically or magnetically. the majority of electron lenses for tem use electromagnetic coils to generate a convex lens. the field produced for the lens must be radially symmetrical, as deviation from the radial symmetry of the magnetic lens causes aberrations such as astigmatism, and worsens spherical and chromatic aberration. electron lenses are manufactured from iron, iron-cobalt or nickel cobalt alloys, such as permalloy. these are selected for their magnetic properties, such as magnetic saturation, hysteresis and permeability. the components include the yoke, the magnetic coil, the poles, the polepiece, and the external control circuitry. the pole piece must be manufactured in a very symmetrical manner, as this provides the boundary conditions for the magnetic field that forms the lens. imperfections in the manufacture of the pole piece can induce severe distortions in the magnetic field symmetry, which induce distortions that will ultimately limit the lenses' ability to reproduce the object plane. the exact dimensions of the gap, pole piece internal diameter and taper, as well as the overall design of the lens is often performed by finite element analysis of the magnetic field, whilst considering the thermal and electrical constraints of the design. the coils which produce the magnetic field are located within the lens yoke. the coils can contain a variable current, but typically use high voltages, and therefore require significant insulation in order to prevent short-circuiting the lens components. thermal distributors are placed to ensure the extraction of the heat generated by the energy lost to resistance of the coil windings. the windings may be water-cooled, using a chilled water supply in order to facilitate the removal of the high thermal duty. apertures apertures are annular metallic plates, through which electrons that are further than a fixed distance from the optic axis may be excluded. these consist of a small metallic disc that is sufficiently thick to prevent electrons from passing through the disc, whilst permitting axial electrons. this permission of central electrons in a tem causes two effects simultaneously: firstly, apertures decrease the beam intensity as electrons are filtered from the beam, which may be desired in the case of beam sensitive samples. secondly, this filtering removes electrons that are scattered to high angles, which may be due to unwanted processes such as spherical or chromatic aberration, or due to diffraction from interaction within the sample. apertures are either a fixed aperture within the column, such as at the condenser lens, or are a movable aperture, which can be inserted or withdrawn from the beam path, or moved in the plane perpendicular to the beam path. aperture assemblies are mechanical devices which allow for the selection of different aperture sizes, which may be used by the operator to trade off intensity and the filtering effect of the aperture. aperture assemblies are often equipped with micrometers to move the aperture, required during optical calibration. imaging methods imaging methods in tem use the information contained in the electron waves exiting from the sample to form an image. the projector lenses allow for the correct positioning of this electron wave distribution onto the viewing system. the observed intensity, i, of the image, assuming sufficiently high quality of imaging device, can be approximated as proportional to the time-averaged squared absolute value of the amplitude of the electron wavefunctions, where the wave that forms the exit beam is denoted by ψ. i ( x ) = k t 1 − t 0 ∫ t 0 t 1 ψ ψ ∗ d t -t_\int _^\psi \psi ^ }\,dt} different imaging methods therefore attempt to modify the electron waves exiting the sample in a way that provides information about the sample, or the beam itself. from the previous equation, it can be deduced that the observed image depends not only on the amplitude of beam, but also on the phase of the electrons, although phase effects may often be ignored at lower magnifications. higher resolution imaging requires thinner samples and higher energies of incident electrons, which means that the sample can no longer be considered to be absorbing electrons (i.e., via a beer's law effect). instead, the sample can be modeled as an object that does not change the amplitude of the incoming electron wave function, but instead modifies the phase of the incoming wave; in this model, the sample is known as a pure phase object. for sufficiently thin specimens, phase effects dominate the image, complicating analysis of the observed intensities. to improve the contrast in the image, the tem may be operated at a slight defocus to enhance contrast, owing to convolution by the contrast transfer function of the tem, which would normally decrease contrast if the sample was not a weak phase object. schematic view of imaging and diffraction modes in tem. the figure on the right shows the two basic operation modes of tem – imaging and diffraction modes. in both cases the specimen is illuminated with the parallel beam, formed by electron beam shaping with the system of condenser lenses and condenser aperture. after interaction with the sample, on the exit surface of the specimen two types of electrons exist – unscattered (which will correspond to the bright central beam on the diffraction pattern) and scattered electrons (which change their trajectories due to interaction with the material). in imaging mode, the objective aperture is inserted in a back focal plane (bfp) of the objective lens (where diffraction spots are formed). if using the objective aperture to select only the central beam, the transmitted electrons are passed through the aperture while all others are blocked, and a bright field image (bf image) is obtained. if we allow the signal from a diffracted beam, a dark field image (df image) is received. the selected signal is magnified and projected on a screen (or on a camera) with the help of intermediate and projector lenses. an image of the sample is thus obtained. in diffraction mode, a selected area aperture may be used to determine more precisely the specimen area from which the signal will be displayed. by changing the strength of current to the intermediate lens, the diffraction pattern is projected on a screen. diffraction is a very powerful tool for doing a cell reconstruction and crystal orientation determination. contrast formation the contrast between two adjacent areas in a tem image can be defined as the difference in the electron densities in image plane. due to the scattering of the incident beam by the sample, the amplitude and phase of the electron wave change, which results in amplitude contrast and phase contrast, correspondingly. most images have both contrast components. amplitude–contrast is obtained due to removal of some electrons before the image plane. during their interaction with the specimen some of electrons will be lost due to absorption, or due to scattering at very high angles beyond the physical limitation of microscope or are blocked by the objective aperture. while the first two losses are due to the specimen and microscope construction, the objective aperture can be used by operator to enhance the contrast. bf and df contrast demonstration. tem image of polycrystalline pt film figure on the right shows a tem image (a) and the corresponding diffraction pattern (b) of pt polycrystalline film taken without an objective aperture. in order to enhance the contrast in the tem image the number of scattered beams as visible in the diffraction pattern should be reduced. this can be done by selecting a certain area in the back focal plane such as only the central beam or a specific diffracted beam (angle), or combinations of such beams. by intentionally selecting an objective aperture which only permits the non-diffracted beam to pass beyond the back focal plane (and onto the image plane): one creates a bright-field (bf) image (c), whereas if the central, non-diffracted beam is blocked: one may obtain dark-field (df) images such as those shown in (d-e). the df images (d-e) were obtained by selecting the diffracted beams indicated in diffraction pattern with circles (b) using an aperture at the back focal plane. grains from which electrons are scattered into these diffraction spots appear brighter. more details about diffraction contrast formation are given further. there are two types of amplitude contrast – mass–thickness and diffraction contrast. first, let's consider mass–thickness contrast. when the beam illuminates two neighbouring areas with low mass (or thickness) and high mass (or thickness), the heavier region scatters electrons at bigger angles. these strongly scattered electrons are blocked in bf tem mode by objective aperture. as a result, heavier regions appear darker in bf images (have low intensity). mass–thickness contrast is most important for non–crystalline, amorphous materials. diffraction contrast occurs due to a specific crystallographic orientation of a grain. in such a case the crystal is oriented in a way that there is a high probability of diffraction. diffraction contrast provides information on the orientation of the crystals in a polycrystalline sample, as well as other information such as defects. note that in case diffraction contrast exists, the contrast cannot be interpreted as due to mass or thickness variations. diffraction contrast transmission electron micrograph of dislocations in steel, which are faults in the structure of the crystal lattice at the atomic scale samples can exhibit diffraction contrast, whereby the electron beam undergoes diffraction which in the case of a crystalline sample, disperses electrons into discrete locations in the back focal plane. by the placement of apertures in the back focal plane, i.e. the objective aperture, the desired reciprocal lattice vectors can be selected (or excluded), thus only parts of the sample that are causing the electrons to scatter to the selected reflections will end up projected onto the imaging apparatus. if the reflections that are selected do not include the unscattered beam (which will appear up at the focal point of the lens), then the image will appear dark wherever no sample scattering to the selected peak is present, as such a region without a specimen will appear dark. this is known as a dark-field image. modern tems are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen. applications for this method include the identification of lattice defects in crystals. by carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the type of defect present. if the sample is oriented so that one particular plane is only slightly tilted away from the strongest diffracting angle (known as the bragg angle), any distortion of the crystal plane that locally tilts the plane to the bragg angle will produce particularly strong contrast variations. however, defects that produce only displacement of atoms that do not tilt the crystal towards the bragg angle (i. e. displacements parallel to the crystal plane) will produce weaker contrast. phase contrast crystal structure can also be investigated by high-resolution transmission electron microscopy (hrtem), also known as phase contrast. when using a field emission source and a specimen of uniform thickness, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. image formation is given by the complex modulus of the incoming electron beams. as such, the image is not only dependent