Dark-field X-ray microscopy (DFXM [1] or DFXRM [2] ) is an imaging technique used for multiscale structural characterisation. It is capable of mapping deeply embedded structural elements with nm-resolution using synchrotron X-ray diffraction-based imaging. The technique works by using scattered X-rays to create a high degree of contrast, and by measuring the intensity and spatial distribution of the diffracted beams, it is possible to obtain a three-dimensional map of the sample's structure, orientation, and local strain.
The first experimental demonstration of dark-field X-ray microscopy was reported in 2006 by a group at the European Synchrotron Radiation Facility in Grenoble, France. Since then, the technique has been rapidly evolving and has shown great promise in multiscale structural characterization. [1] Its development is largely due to advances in synchrotron X-ray sources, which provide highly collimated and intense beams of X-rays. The development of dark-field X-ray microscopy has been driven by the need for non-destructive imaging of bulk crystalline samples at high resolution, and it continues to be an active area of research today. However, dark-field microscopy, [3] [4] dark-field scanning transmission X-ray microscopy, [5] and soft dark-field X-ray microscopy [6] has long been used to map deeply embedded structural elements.
In this technique, a synchrotron light source is used to generate an intense and coherent X-ray beam, which is then focused onto the sample using a specialized objective lens. The objective lens acts as a collimator to select and focus the scattered light, which is then detected by the 2D detector to create a diffraction pattern. [1] The specialized objective lens in DFXM, referred to as an X-ray objective lens, is a crucial component of the instrumentation required for the technique. It can be made from different materials such as beryllium, silicon, and diamond, depending on the specific requirements of the experiment. [8] The objective enables one to enlarge or reduce the spatial resolution and field of view within the sample by varying the number of individual lenses and adjusting and (as in the figure) correspondingly. The diffraction angle is typically 10–30°. [9] [10]
The sample is positioned at an angle such that the direct beam is blocked by a beam stop or aperture, and the diffracted beams from the sample are allowed to pass through a detector. [11]
An embedded crystalline element (for example, a grain or domain) of choice (green) is aligned such that the detector is positioned at a Bragg angle that corresponds to a particular diffraction peak of interest, which is determined by the crystal structure of the sample. The objective magnifies the diffracted beam by a factor and generates an inverted 2D projection of the grain. Through repeated exposures during a 360° rotation of the element around an axis parallel to the diffraction vector, , several 2D projections of the grain are obtained from various angles. [12] A 3D map is then obtained by combining these projections using reconstruction algorithms [13] similar to those developed for CT scanning. If the lattice of the crystalline element exhibits an internal orientation spread, this procedure is repeated for a number of sample tilts, indicated by the angles and . [1]
The current implementation of DFXM at ID06, ESRF, uses a compound refractive lens (CRL) as the objective, giving spatial resolution of 100 nm and angular resolution of 0.001°. [14] [15]
DFXM has been used for the non-destructive investigation of polycrystalline materials and composites, revealing the 3D microstructure, [16] phases, [17] orientation of individual grains, [18] [19] and local strains. [20] [21] It has also been used for in situ studies of materials recrystallisation, [22] dislocations [23] [24] and other defects, and the deformation [20] and fracture mechanisms in materials, such as metals [11] and composites. [25] DFXM can provide insights into the 3D microstructure and deformation of geological materials such as minerals and rocks, [1] and irradiated materials. [26]
DFXM has the potential to revolutionise the field of nanotechnology by providing non-destructive, high-resolution 3D imaging of nanostructures and nanomaterials. It has been used to investigate the 3D morphology of nanowires and to detect structural defects in nanotubes. [27] [28]
DFXM has shown potential for imaging biological tissues and organs with high contrast and resolution. It has been used to visualize the 3D microstructure of cartilage and bone, as well as to detect early-stage breast cancer in mouse model. [1] [29]
The intense X-ray beams used in DFXM can damage delicate samples, particularly biological specimens. [1] DFXM can suffer from imaging artefacts such as ring artefacts, which can affect image quality and limit interpretation. [11]
The instrumentation required for DFXM is expensive and typically only available at synchrotron facilities, making it inaccessible to many researchers. Although DFXM can achieve high spatial resolution, it is still not as high as the resolution achieved by other imaging techniques such as transmission electron microscopy (TEM) or X-ray crystallography. [11]
Preparation of samples for DFXM imaging can be challenging, especially for samples that are not crystalline. There are also limitations on the sample size that can be imaged as the technique works best with thin samples, typically less than 100 microns thick, due to the attenuation of the X-ray beam by thicker samples. [1] DFXM still suffers from long integration times, which can limit its practical applications. This is due to the low flux density of X-rays emitted by synchrotron sources and the high sensitivity required to detect scattered X-rays. [11]
There are several alternative techniques to DFXM, depending on the application, some of which are:
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that contain information about the surface topography and composition of the sample. The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the intensity of the detected signal to produce an image. In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are detected using a secondary electron detector. The number of secondary electrons that can be detected, and thus the signal intensity, depends, among other things, on specimen topography. Some SEMs can achieve resolutions better than 1 nanometer.
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a detector such as a scintillator attached to a charge-coupled device or a direct electron detector.
A synchrotron light source is a source of electromagnetic radiation (EM) usually produced by a storage ring, for scientific and technical purposes. First observed in synchrotrons, synchrotron light is now produced by storage rings and other specialized particle accelerators, typically accelerating electrons. Once the high-energy electron beam has been generated, it is directed into auxiliary components such as bending magnets and insertion devices in storage rings and free electron lasers. These supply the strong magnetic fields perpendicular to the beam that are needed to stimulate the high energy electrons to emit photons.
An X-ray microscope uses electromagnetic radiation in the X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations.
Electron backscatter diffraction (EBSD) is a scanning electron microscopy (SEM) technique used to study the crystallographic structure of materials. EBSD is carried out in a scanning electron microscope equipped with an EBSD detector comprising at least a phosphorescent screen, a compact lens and a low-light camera. In the microscope an incident beam of electrons hits a tilted sample. As backscattered electrons leave the sample, they interact with the atoms and are both elastically diffracted and lose energy, leaving the sample at various scattering angles before reaching the phosphor screen forming Kikuchi patterns (EBSPs). The EBSD spatial resolution depends on many factors, including the nature of the material under study and the sample preparation. They can be indexed to provide information about the material's grain structure, grain orientation, and phase at the micro-scale. EBSD is used for impurities and defect studies, plastic deformation, and statistical analysis for average misorientation, grain size, and crystallographic texture. EBSD can also be combined with energy-dispersive X-ray spectroscopy (EDS), cathodoluminescence (CL), and wavelength-dispersive X-ray spectroscopy (WDS) for advanced phase identification and materials discovery.
Electron crystallography is a subset of methods in electron diffraction focusing just upon detailed determination of the positions of atoms in solids using a transmission electron microscope (TEM). It can involve the use of high-resolution transmission electron microscopy images, electron diffraction patterns including convergent-beam electron diffraction or combinations of these. It has been successful in determining some bulk structures, and also surface structures. Two related methods are low-energy electron diffraction which has solved the structure of many surfaces, and reflection high-energy electron diffraction which is used to monitor surfaces often during growth.
A scanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). Pronunciation is [stɛm] or [ɛsti:i:ɛm]. As with a conventional transmission electron microscope (CTEM), images are formed by electrons passing through a sufficiently thin specimen. However, unlike CTEM, in STEM the electron beam is focused to a fine spot which is then scanned over the sample in a raster illumination system constructed so that the sample is illuminated at each point with the beam parallel to the optical axis. The rastering of the beam across the sample makes STEM suitable for analytical techniques such as Z-contrast annular dark-field imaging, and spectroscopic mapping by energy dispersive X-ray (EDX) spectroscopy, or electron energy loss spectroscopy (EELS). These signals can be obtained simultaneously, allowing direct correlation of images and spectroscopic data.
Phase-contrast imaging is a method of imaging that has a range of different applications. It measures differences in the refractive index of different materials to differentiate between structures under analysis. In conventional light microscopy, phase contrast can be employed to distinguish between structures of similar transparency, and to examine crystals on the basis of their double refraction. This has uses in biological, medical and geological science. In X-ray tomography, the same physical principles can be used to increase image contrast by highlighting small details of differing refractive index within structures that are otherwise uniform. In transmission electron microscopy (TEM), phase contrast enables very high resolution (HR) imaging, making it possible to distinguish features a few Angstrom apart.
Diffraction topography is a imaging technique based on Bragg diffraction. Diffraction topographic images ("topographies") record the intensity profile of a beam of X-rays diffracted by a crystal. A topography thus represents a two-dimensional spatial intensity mapping (image) of the X-rays diffracted in a specific direction, so regions which diffract substantially will appear brighter than those which do not. This is equivalent to the spatial fine structure of a Laue reflection. Topographs often reveal the irregularities in a non-ideal crystal lattice. X-ray diffraction topography is one variant of X-ray imaging, making use of diffraction contrast rather than absorption contrast which is usually used in radiography and computed tomography (CT). Topography is exploited to a lesser extends with neutrons, and is the same concept as dark field imaging in an electron microscope.
Electron tomography (ET) is a tomography technique for obtaining detailed 3D structures of sub-cellular, macro-molecular, or materials specimens. Electron tomography is an extension of traditional transmission electron microscopy and uses a transmission electron microscope to collect the data. In the process, a beam of electrons is passed through the sample at incremental degrees of rotation around the center of the target sample. This information is collected and used to assemble a three-dimensional image of the target. For biological applications, the typical resolution of ET systems are in the 5–20 nm range, suitable for examining supra-molecular multi-protein structures, although not the secondary and tertiary structure of an individual protein or polypeptide. Recently, atomic resolution in 3D electron tomography reconstructions has been demonstrated.
Low-energy electron microscopy, or LEEM, is an analytical surface science technique used to image atomically clean surfaces, atom-surface interactions, and thin (crystalline) films. In LEEM, high-energy electrons are emitted from an electron gun, focused using a set of condenser optics, and sent through a magnetic beam deflector. The “fast” electrons travel through an objective lens and begin decelerating to low energies near the sample surface because the sample is held at a potential near that of the gun. The low-energy electrons are now termed “surface-sensitive” and the near-surface sampling depth can be varied by tuning the energy of the incident electrons. The low-energy elastically backscattered electrons travel back through the objective lens, reaccelerate to the gun voltage, and pass through the beam separator again. However, now the electrons travel away from the condenser optics and into the projector lenses. Imaging of the back focal plane of the objective lens into the object plane of the projector lens produces a diffraction pattern at the imaging plane and recorded in a number of different ways. The intensity distribution of the diffraction pattern will depend on the periodicity at the sample surface and is a direct result of the wave nature of the electrons. One can produce individual images of the diffraction pattern spot intensities by turning off the intermediate lens and inserting a contrast aperture in the back focal plane of the objective lens, thus allowing for real-time observations of dynamic processes at surfaces. Such phenomena include : tomography, phase transitions, adsorption, reaction, segregation, thin film growth, etching, strain relief, sublimation, and magnetic microstructure. These investigations are only possible because of the accessibility of the sample; allowing for a wide variety of in situ studies over a wide temperature range. LEEM was invented by Ernst Bauer in 1962; however, not fully developed until 1985.
Coherent diffractive imaging (CDI) is a "lensless" technique for 2D or 3D reconstruction of the image of nanoscale structures such as nanotubes, nanocrystals, porous nanocrystalline layers, defects, potentially proteins, and more. In CDI, a highly coherent beam of X-rays, electrons or other wavelike particle or photon is incident on an object.
Ptychography is a computational method of microscopic imaging. It generates images by processing many coherent interference patterns that have been scattered from an object of interest. Its defining characteristic is translational invariance, which means that the interference patterns are generated by one constant function moving laterally by a known amount with respect to another constant function. The interference patterns occur some distance away from these two components, so that the scattered waves spread out and "fold" into one another as shown in the figure.
Three-dimensional X-ray diffraction (3DXRD) is a microscopy technique using hard X-rays to investigate the internal structure of polycrystalline materials in three dimensions. For a given sample, 3DXRD returns the shape, juxtaposition, and orientation of the crystallites ("grains") it is made of. 3DXRD allows investigating micrometer- to millimetre-sized samples with resolution ranging from hundreds of nanometers to micrometers. Other techniques employing X-rays to investigate the internal structure of polycrystalline materials include X-ray diffraction contrast tomography (DCT) and high energy X-ray diffraction (HEDM).
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution. This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for macromolecular structure determination without the need for crystallization.
4D scanning transmission electron microscopy is a subset of scanning transmission electron microscopy (STEM) which utilizes a pixelated electron detector to capture a convergent beam electron diffraction (CBED) pattern at each scan location. This technique captures a 2 dimensional reciprocal space image associated with each scan point as the beam rasters across a 2 dimensional region in real space, hence the name 4D STEM. Its development was enabled by evolution in STEM detectors and improvements computational power. The technique has applications in visual diffraction imaging, phase orientation and strain mapping, phase contrast analysis, among others.
Angus J. Wilkinson is a professor of materials science based at the Department of Materials, University of Oxford. He is a specialist in micromechanics, electron microscopy and crystal plasticity. He assists in overseeing the MicroMechanics group while focusing on the fundamentals of material deformation. He developed the HR-EBSD method for mapping stress and dislocation density at high spatial resolution used at the micron scale in mechanical testing and micro-cantilevers to extract data on mechanical properties that are relevant to materials engineering.
Transmission Kikuchi Diffraction (TKD), also sometimes called transmission-electron backscatter diffraction (t-EBSD), is a method for orientation mapping at the nanoscale. It’s used for analysing the microstructures of thin transmission electron microscopy (TEM) specimens in the scanning electron microscope (SEM). This technique has been widely utilised in the characterization of nano-crystalline materials, including oxides, superconductors, and metallic alloys.
X-ray diffraction computed tomography is an experimental technique that combines X-ray diffraction with the computed tomography data acquisition approach. X-ray diffraction (XRD) computed tomography (CT) was first introduced in 1987 by Harding et al. using a laboratory diffractometer and a monochromatic X-ray pencil beam. The first implementation of the technique at synchrotron facilities was performed in 1998 by Kleuker et al.