With advancements in aberration correction, the spatial resolution of scanning transmission electron microscopy (STEM) has been enormously improved. In addition to the reduction of the STEM probe size, a dramatic increase in the STEM probe current has been realized, leading to the routine acquisition of high-resolution elemental and chemical maps using electron energy loss spectrometry (EELS). Using EELS combined with these advanced STEM instruments, atomic-level resolution information can be obtained from various types of materials, revealing the nature of interfaces, elemental distribution, presence of defects, and much more. In addition to simple elemental composition distributions, EELS is capable of delivering information about the chemical bonding, local atomic coordination, oxidation states, band gaps, and chemical phases of a broad range of materials at the fundamental resolution limit of the property being probed. Atomic-level EELS maps of these fundamental material properties can now be obtained with the acquisition time, to a large extent, limited only by the speed of the EELS spectrometer and not by the signal being measured. The availability of fast EELS spectrometers with large angular collection efficiencies has closed the gap between the rate of signal generation in the specimen and the speed at which this signal can be detected. This significantly increases the amount of information that can be acquired using EELS. Using the most recent generation of spectrometers, EELS data can be acquired at well over 1,000 spectra per second with a high-duty cycle. Fifth-order spectral aberration correction in this generation of spectrometers allows the use of the large collection angles needed to match the increased convergence angle that Cs-probe-corrected systems present, improving collection efficiency while maintaining energy resolution. These advances, when taken together, result in a well matched source/detector system capable of recording high-energy EELS edges at atomic resolution at a rate fast enough to limit electron beam damage to the sample.

Serial sectioning of TEM blocks is a common technique. The more sections that are desired, the more complex the procedure becomes. Some of the problems faced are staining without losing or obscuring sections, appropriate block trimming, finding the area of interest in the next section, transferring sections to the grid. Here, I relate a set of techniques that permit the microtomist to regularly get over 100 consecutive serial sections on a single slot grid. Much of what is discussed below is not new, but by putting all of these techniques together I believe that a powerful tool for TEM is presented.

The first issue that must be addressed is how to stain the sections on the grid without losing sections or obscuring sections with staining artifacts (e.g. lead pepper). The simplest solution is to stain the sample with both uranium and lead prior to embedding and not stain the sections on the grid.

This article is the fourth in a series describing the setup and use of NIST DTSA-II. The previous articles may be viewed in the Microscopy Today archives at www.microscopy-today.com. Part I of this article described how to use DTSA-II to quantify an energy dispersive X-ray spectrum from a material of unknown composition. That presentation covered each step and provided background material but lacked a concrete example. This follow-up article provides two such examples.

Atomic Force Microscopy (AFM) has developed into a very powerful tool for characterization of surfaces and nanoscale objects. Many physical properties of an object can be studied by AFM with nanometer-scale resolution. Local stiffness, elasticity, conductivity, capacitance, magnetization, surface potential and work function, friction, piezo response—these and many other physical properties can be studied with over 30 AFM modes. What is typically lacking in information provided by AFM studies is the chemical composition of the sample and information about its crystal structure. To obtain this information other characterization techniques are required, such as Raman and fluorescence microscopy. The Raman effect (inelastic light scattering) provides extensive information about sample chemical composition, quality of crystal structure, crystal orientation, presence of impurities and defects, and so on. Information provided by Raman and fluorescence spectroscopy is complementary to the information obtained by AFM. So it is a natural requirement in many research fields to integrate these techniques in one piece of equipment—to provide comprehensive physical, chemical, and structural characterization of the same object. Of course, for routine studies of various samples, it is important to be able to obtain AFM and Raman/fluorescence images of exactly the same sample area, preferably with the same sample scan.

Combinatorial and high-throughput (C&HT) approaches accelerate research by addressing multiple experimental parameters in a parallel or otherwise highly efficient fashion. First used by the pharmaceutical industry for product discovery, the C&HT paradigm is being extended to the study of complex materials systems that require measurements of properties and phenomena over a huge number of conditions. As with traditional materials science, microscopy and imaging of morphology are essential for C&HT materials research.

Biological materials interest biologists and engineers for their complex interactions among constituents and unique mechanical properties. While the heterogeneous tissue structure and its material properties are responsible for intriguing biomechanics, they pose challenges for sectioning, particularly in regions with stark tissue boundaries. Microscopists level this playing field when sectioning samples by embedding them in paraffin or plastic; but for scanning electron microscopy, natural morphology must be preserved without sacrificing the sample’s surface contours. Here we outline a simple preparation method for visualizing in cryoSEM the calcified cartilage of sharks and rays (elasmobranch fishes), a layered biocomposite that has traditionally been considered difficult to prepare for microscopy.

Since publication of the classic text on the electron microscope laboratory by Anderson, the proliferation of microscopes with field emission guns, imaging filters and hardware spherical aberration correctors (giving higher spatial and energy resolution) has resulted in the need to construct special laboratories. As resolutions iinprovel transmission electron microscopes (TEMs) and scanning transmission electron microscopes (STEMs) become more sensitive to ambient conditions. State-of-the-art electron microscopes require state-of-the-art environments, and this means careful design and implementation of microscope sites, from the microscope room to the building that surrounds it. Laboratories have been constructed to house high-sensitive instruments with resolutions ranging down to sub-Angstrom levels; we present the various design philosophies used for some of these laboratories and our experiences with them. Four facilities are described: the National Center for Electron Microscopy OAM Laboratory at LBNL; the FEGTEM Facility at the University of Sheffield; the Center for Integrative Molecular Biosciences at TSRI; and the Advanced Microscopy Laboratory at ORNL.

Materials research, biomedical research, and semiconductor manufacturing can all benefit from nondestructive, high-resolution methods of analysis. As most materials are heterogeneous, it is important to not only acquire high resolution topographic information, but also to identify the chemical composition of samples. A combination of high resolution microscopy with chemically sensitive spectroscopy combined in one instrument allows the detailed characterization of samples with different analytical techniques. When individual instruments are used, returning to a previously surveyed sample area can be very time consuming if not impossible without surface markers.

Freezing damages cell membranes and reduces the histological readability of biological specimens. This is a discussion of why, the consequences, and how to minimize or avoid the damage introduced when using freezing as the means to harden biological tissue in order to cut thin sections for histology.

Pure water can exist in the solid state in three forms. Two forms are crystalline: one with a hexagonal lattice and the other with a cubic lattice. The third form capitalizes on the fact that water can be frozen so rapidly that it does not have time to form a crystal lattice and remains amorphous (vitreous form). Crystal formation is responsible for the expansion of water as it freezes (a property unique of water), which creates specimen preparation artifacts. Vitreous ice does not expand upon solidification. This makes it the only desirable form of ice appropriate for biological specimens.

Transparent specimens are usually examined by dark field, phase contrast and interference contrast light microscopy. In dark field, specimens are illuminated by oblique light beams that come from the periphery of the illuminating apparatus. Therefore, some transparent objects, e.g. unstained native bacteria, are barely visible and fine structures inside them are often not visible. In phase contrast, the discernment of fine detail can be reduced by halo artifacts. The intensity of contrast, i.e. the difference in brightness between the background and specimen, is constant and not variable; it is determined by the specification of the phase ring within the phase contrast lens and dependent on the specific phase differences between the specimen and its surrounding medium. Interference contrast images are free from halo artifacts, but their contrast may be lower than in corresponding phase contrast or dark field images, especially, when transparent specimens are examined in thinlayer preparations.

TEM sample preparation using Focused Ion Beam (FIB) methods becomes more and more interesting for microscopists because the technique allows for reliable and very efficient sample preparation. The first application of TEM sample preparation by FIB-cutting was reported more than 10 years ago. Meanwhile, a lot of experience has been gathered that allows one to discuss the capabilities and limits of the FIB technique in detail.

Several TEM sample preparation techniques are known that all include FIB-cutting but differ in sample pre-preparation, sample handling, etc. This paper focuses on the actual FIB process, FIB tools are closely related to Scanning Electron Microscopes, but instead of an electron beam an ion beam (mostly Ga+ ions) is used to remove and deposit material.