within mtjs contains a large number of grain boundaries that may be diminishing the properties of devices. ion milling (fib) scanning electron microscope image of a thin tem sample milled by fib. the thin membrane shown here is suitable for tem examination; however, at ~300-nm thickness, it would not be suitable for high-resolution tem without further milling. more recently focused ion beam methods have been used to prepare samples. fib is a relatively new technique to prepare thin samples for tem examination from larger specimens. because fib can be used to micro-machine samples very precisely, it is possible to mill very thin membranes from a specific area of interest in a sample, such as a semiconductor or metal. unlike inert gas ion sputtering, fib makes use of significantly more energetic gallium ions and may alter the composition or structure of the material through gallium implantation. nanowire assisted transfer for a minimal introduction of stress and bending to transmission electron microscopy (tem) samples (lamellae, thin films, and other mechanically and beam sensitive samples), when transferring inside a focused ion beam (fib), flexible metallic nanowires can be attached to a typically rigid micromanipulator. the main advantages of this method include a significant reduction of sample preparation time (quick welding and cutting of nanowire at low beam current), and minimization of stress-induced bending, pt contamination, and ion beam damage. this technique is particularly suitable for in situ electron microscopy sample preparation. replication staphylococcus aureus platinum replica image shot on a tem at 50,000x magnification. samples may also be replicated using cellulose acetate film, the film subsequently coated with a heavy metal such as platinum, the original film dissolved away, and the replica imaged on the tem. variations of the replica technique are used for both materials and biological samples. in materials science a common use is for examining the fresh fracture surface of metal alloys. modifications the capabilities of the tem can be further extended by additional stages and detectors, sometimes incorporated on the same microscope. scanning tem a tem can be modified into a scanning transmission electron microscope (stem) by the addition of a system that rasters a convergent beam across the sample to form the image, when combined with suitable detectors. scanning coils are used to deflect the beam, such as by an electrostatic shift of the beam, where the beam is then collected using a current detector such as a faraday cup, which acts as a direct electron counter. by correlating the electron count to the position of the scanning beam (known as the "probe"), the transmitted component of the beam may be measured. the non-transmitted components may be obtained either by beam tilting or by the use of annular dark field detectors. schematic ray diagram illustrating the optical reciprocity between tem (left) and stem (right). the convergence angle in tem, α t , becomes the collection angle in stem, β s . image inspired by hren et al. fundamentally, tem and stem are linked via helmholtz reciprocity. a stem is a tem in which the electron source and observation point have been switched relative to the direction of travel of the electron beam. see the ray diagrams in the figure on the right. the stem instrument effectively relies on the same optical set-up as a tem, but operates by flipping the direction of travel of the electrons (or reversing time) during operation of a tem. rather than using an aperture to control detected electrons, as in tem, a stem uses various detectors with collection angles that may be adjusted depending on which electrons the user wants to capture. low-voltage electron microscope a low-voltage electron microscope (lvem) is operated at relatively low electron accelerating voltage between 5–25 kv. some of these can be a combination of sem, tem and stem in a single compact instrument. low voltage increases image contrast which is especially important for biological specimens. this increase in contrast significantly reduces, or even eliminates the need to stain. resolutions of a few nm are possible in tem, sem and stem modes. the low energy of the electron beam means that permanent magnets can be used as lenses and thus a miniature column that does not require cooling can be used. cryo-tem cryogenic transmission electron microscopy (cryo-tem) uses a tem with a specimen holder capable of maintaining the specimen at liquid nitrogen or liquid helium temperatures. this allows imaging specimens prepared in vitreous ice, the preferred preparation technique for imaging individual molecules or macromolecular assemblies, imaging of vitrified solid-electrolye interfaces, and imaging of materials that are volatile in high vacuum at room temperature, such as sulfur. environmental/in-situ tem in-situ experiments may also be conducted in tem using differentially pumped sample chambers, or specialized holders. types of in-situ experiments include studying nanomaterials, biological specimens, chemical reactions of molecules, liquid-phase electron microscopy, and material deformation testing. high temperature in-situ tem many phase transformations occur during heating. additionally, coarsening and grain growth, along with other diffusion-related processes occur more rapidly at elevated temperatures, where kinetics are improved, allowing for the observation of related phenomena under transmission electron microscopy within reasonable time scales. this also allows for the observation of phenomena that occur at elevated temperatures and disappear or are not uniformly preserved in ex-situ samples. high temperature tem introduces various additional challenges which must be addressed in the mechanics of high temperature holders, including but not limited to drift-correction, temperature measurement, and decreased spatial resolution at the expense of more complex holders. sample drift in the tem is linearly proportional to the temperature differential between the room and holder. with temperatures as high as 1500c in modern holders, samples may experience significant drift and vertical displacement (bulging), requiring continuous focus or stage adjustments, inducing resolution loss and mechanical drift. individual labs and manufacturers have developed software coupled with advanced cooling systems to correct for thermal drift based on the predicted temperature in the sample chamber these systems often take 30 min-many hours for sample shifts to stabilize. while significant progress has been made, no universal tem attachment has been made to account for drift at elevated temperatures. an additional challenge of many of these specialized holders is knowing the local sample temperature. many high temperature holders utilize a tungsten filament to locally heat the sample. ambiguity in temperature in furnace heaters (w wire) with thermocouples arises from the thermal contact between the furnace and the tem grid; complicated by temperature gradients along the sample caused by varying thermal conductivity with different samples and grid materials. with different holders both commercial and lab made, different methods for creating temperature calibration are available. manufacturers like gatan use ir pyrometry to measure temperature gradients over their entire sample. an even better method to calibrate is raman spectroscopy which measures the local temperature of si powder on electron transparent windows and quantitatively calibrates the ir pyrometry. these measurements have guaranteed accuracy within 5%. research laboratories have also performed their own calibrations on commercial holders. researchers at nist utilized raman spectroscopy to map the temperature profile of a sample on a tem grid and achieve very precise measurements to enhance their research. similarly, a research group in germany utilized x-ray diffraction to measure slight shifts in lattice spacing caused by changes in temperature to back calculate the exact temperature in the holder. this process required careful calibration and exact tem optics. other examples include the use of eels to measure local temperature using change of gas density, and resistivity changes. optimal resolution in a tem is achieved when spherical aberrations are corrected with objective lens. however, due to the geometry of most tems, inserting large in-situ holders requires the user to compromise the objective lens and endure spherical aberrations. therefore, there is a compromise between the width of the pole-piece gap and spatial resolution below 0.1 nm. research groups at various institutions have tried to overcome spherical aberrations through use of monochromators to achieve 0.05 nm resolution with a 5 mm pole piece gap. in-situ mechanical tem high resolution of tem allows for monitoring the sample in question on a length scale ranging from hundreds of nanometers to several angstroms. this allows for the visualization of both elastic and plastic deformation via strain fields as well as the motion of crystallographic defects such as lattice distortions and dislocation motion. by simultaneously observing deformation phenomena and measuring mechanical response in situ, it is possible to connect nano-mechanical testing information to models that describe both the subtlety and complexity of how materials respond to stress and strain. the material properties and data accuracy obtained from such nano-mechanical tests is largely determined by the mechanical straining holder being used. current straining holders have the ability to perform tensile tests, nano-indentation, compression tests, shear tests and bending tests on materials. classical mechanical holders one of the pioneers of classical holders was dr. heinz g.f. wilsdorf, who conducted a tensile test inside a tem in 1958. in a typical experiment, electron transparent tem samples are cut to shape and glued to a deformable grid. advances in micromanipulators also have also enabled the tensile testing of nanowires and thin films. the deformable grid attaches to the classical tensile holder which stretches the sample using a long rigid shaft attached to a worm gear box actuated by an electric motor located in a housing outside the tem. typically strain rates range from 10 nm/s to 10 μm/s. custom-made holders expanding simple straining actuation have enabled bending tests using a bending holder and shear tests using a shear sample holder. the typical measured sample properties in these experiments are yield strength, elastic modulus, shear modulus, tensile strength, bending strength, and shear strength. in order to study the temperature-dependent mechanical properties of tem samples, the holder can be cooled through a cold finger connected to a liquid nitrogen reservoir. for high temperature experiments, the tem sample can also be heated through a miniaturized furnace or a laser that can typically reach 1000 °c. nano-indentation holders nano-indentation holders perform a hardness test on the material in question by pressing a hard tip into a polished flat surface and measuring the applied force and the resulting displacement on the tem sample through a change in capacitance between a reference and a movable electrostatic plate attached to the tip. the typical measured sample properties are hardness and elastic modulus. although nano-indentation was possible since early 1980s, its investigation using a tem was first reported in 2001 where an aluminum sample deposited on a silicon wedge was investigated. for nanoindentation experiments, tem samples are typically shaped as wedges using a tripod polisher, h-bar window or a micro-nanopillar using focused ion beam to create enough space for a tip to be pressed at the desired electron transparent location. the indenter tips are typically flat punch-type, pyramidal, or wedge shaped elongated in the z-direction. pyramidal tips offer high precision on the order of 10 nm but suffer from sample slip, while wedge indenters have greater contract to prevent slipping but require finite element analysis to model the transmitted stress since the high contact area with the tem sample makes this almost a compression test. micro electro-mechanical systems (mems) micro electro-mechanical systems (mems) based holders provide a cheap and customizable platform to conduct mechanical tests on previously difficult samples to work with such as micropillars, nanowires, and thin films. passive mems are used as simple push to pull devices for in-situ mechanical tests. typically, a nano-indentation holder is used to apply a pushing force at the indentation site. using a geometry of arms, this pushing force translates to a pulling force on a pair of tensile pads to which the sample is attached. thus, a compression applied on the outside of the mems translates to a tension in the central gap where the tem sample is located. it is worth noting that the resulting force-displacement curve needs to be corrected by performing the same test on an empty mems without the tem sample to account for the stiffness of the empty mems. the dimensions and stiffness of the mems can be modified to perform tensile tests on different sized samples with different loads. to smoothen the actuation process, active mems have been developed with built-in actuators and sensors. these devices work by applying a stress using electrical power and measuring strain