From the nearly mass-less electron to massive ions, charged particle microscopes have diversified over the last few decades. At present, a broad range of available charged particles with varying masses fulfill many applications: from imaging to analysis to nanofabrication.

Nanotechnology represents a major area for research and development and has already led to the introduction of hundreds of products that incorporate nanotechnology-based materials, components, or methods. It is an area of significant investment and economic development globally. NanoInk, Inc. designs and develops instrument systems used for nanotechnology research, education, and manufacturing. Our initial product was built to support sophisticated research programs in academic and government labs. It was based on a complex instrument that was difficult to learn and use: the atomic force microscope (AFM). In order to make our technology accessible and useful to a broader range of potential customers and markets, NanoInk developed a new instrument platform, NLP 2000, that is simpler to learn and operate by users who are not university-trained PhDs. This article will discuss several of the challenges, design goals, and approaches taken in developing this instrument platform.

Compound semiconductors belong to the most important materials for optoelectronic applications. Many of them exhibit favorable optical properties, such as a direct energy band gap (in contrast to silicon) and high-absorption coefficients over a wide spectral range. Moreover, varying the composition of the compound or substituting some of its elements often allows for controlled band gap engineering and optimization for specific applications. Because many compound semiconductors enable efficient conversion of light into electricity and vice versa, they are commonly used materials for optoelectronic devices.

The combination of infrared (IR) spectroscopy and atomic force microscopy (AFM) has produced a technique, called AFM-IR, which is becoming one of the most important recent developments in the field of IR spectroscopy and chemical imaging. Conventional Fourier transform infrared (FT-IR) microspectroscopy is well established as a technique for chemical characterization of small samples down to the 3–10 mm size range. This diffraction-imposed size limit has prevented the application of FT-IR microspectroscopy to smaller analysis regions that are relevant to analysis problems in polymer materials and the life sciences. The nanoIR™ instrument (Anasys Instruments, Santa Barbara, CA) described here uses an AFM probe as the IR absorbance sensor and hence breaks through the diffraction limit to attain spatial resolution improvements of between one and two orders of magnitude beyond previous techniques. Thus, the AFM-IR concept provides chemical information from nanoscale regions of polymers and other organic materials. This article describes the physics behind the technique, followed by results from several applications.

Nano-scale organic/inorganic interfaces are key to a wide range of materials. In many biominerals, for instance bone or teeth, outstanding fracture toughness and wear resistance can be attributed to buried organic/inorganic interfaces. Organic/inorganic interfaces at very small length scales are becoming increasingly important also in nano and electronic materials. For example, functionalized inorganic nanomaterials have great potential in biomedicine or sensing applications. Thin organic films are used to increase the conductivity of LiFePO4 electrodes in lithium ion batteries, and solid electrode interphases (SEI) form by uncontrolled electrolyte decomposition. Organics play a key role in dye-sensitized solar cells, organic photovoltaics, and nano-dielectrics for organic field-effect transistors. The interface between oxide semiconductors and polymer substrates is critical in emergent applications, for example, flexible displays.

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.

Fluorescence microscopy techniques are now prevalent throughout the life sciences and many of the physical sciences. These techniques are often dependent on white light sources that have evolved from the more traditional mercury arc lamp to metal halide sources to the more recent light emitting diodes (LEDs). The newer light sources show more uniform power across the visible light spectrum, allowing for the use of fluorophores and fluorescent proteins outside of the peak wavelengths associated with the more traditional light sources.

Gatan Digital Micrograph (DM) software is considered an industry standard among microscopists. The offline DM application is freely available from Gatan. Unfortunately, DM software has been designed to run only on Microsoft Windows operating systems, thus distancing the microscopy community from popular Unix-based systems, such as Linux or Mac OSX. An ad hoc solution to this problem has required a virtualized Windows operating system running on top of the user's native operating system. This is not only slow, having to emulate each processor instruction, but also requires installation and licensing of Windows and the virtualization software. However, with the aid of open-source resources, it is possible to run DM natively on Linux and Mac OSX (Figure 1). This article was written as a guide with easy-to-follow installation instructions to liberate users from the Windows emulation pidgeonhole and enable them to freely analyze data on Unix-based systems.

There are relatively few technologies for measurement at the single-molecule scale. Fluorescent imaging, for example, can be used to directly visualize molecules and their interactions, but diffraction limitations and labeling requirements may push the system from its native state. Although recent advances in super-resolution imaging have been able to break this resolution barrier, important challenges remain. Atomic force microscopy (AFM) is capable of imaging molecules at high resolution and at high speed. However, AFM imaging is a surface technique, requiring sample preparation and some immobilization. Other technologies such as optical tweezers and magnetic tweezers are capable of molecular manipulation and spectroscopy to great effect but require a significant apparatus and have limited inherent analytical capabilities.

Both atomic force microscopy (AFM) and Raman spectroscopy are techniques used to gather information about the surface properties of a sample. There are many reasons to combine these two technologies, and this article looks both at the complementary information gained from the techniques and how a researcher having access to a combined system can benefit from the additional information available.

Many industrial companies do not have the expertise, experience, or in-house analytical instrumentation to conduct routine laboratory testing. As such, selecting a contract analytical services provider is a common task. Outsourcing is a cost-effective alternative that can allow companies to increase analytical information available for timely decisions regarding product development and production (see Figure 1).

Sample preparation is a critical step in scanning electron microscopy (SEM) imaging. This is especially true for biological samples because of charge build-up and sensitivity to vacuum and electron beam damage. In terms of ultrastructure imaging, a variety of advancements in detectors and approaches have improved biological imaging such that fewer steps are required for sample preparation. However, the conventional approach incorporating osmium tetroxide fixing, ethanol dehydrating, critical-point drying, and coating still finds useful application. This paper evaluates three biological sample-preparation methodologies for imaging the ultrastructure of immature porcine retina. The three preparation methods examined are critical-point drying (CPD), hexamethyldisilazane (HMDS) dehydration, and direct imaging by environmental scanning electron microscopy (ESEM). Preparation methodologies were evaluated based on resulting image quality and reduced potential for artifacts.

Biologists using electron microscopy to understand the form and function of biological systems at the nanometer scale have long been confronted by a number of difficulties related to fundamental characteristics shared by most biological samples. First is the complexity and range of scales at which significant information is present, from the mesoscopic level in tissues, through the microscopic level of cells and organelles, to the nanoscopic level of macromolecules. Next is the hydrated state of most biological materials, in which water is essential to maintaining functional structure, but, at the same time, is incompatible with the vacuum environment of the scanning electron microscope (SEM). Finally, because of their fragile nature, biological structures are vulnerable to damage, by the electron beam itself or by procedures used to prepare them for SEM analysis. A new cryogenic scanning electron microscope (the Magellan cryo-SEM) workflow solution from FEI addresses all of these issues. It is an integrated workflow from sample preparation to sub-nanometer imaging that addresses the specific needs of biological research.

Shape and size are among the most basic and obvious characteristics of a cell (or of any physical object, for that matter). When a cell is observed through a microscope, one only sees its projection onto the image plane. Rather paradoxically, there are no easy techniques to visualize and measure cell's third dimension—thickness. For example, confocal microscopy requires fluorescent labeling, multiple scanning with a high-power objective, possibly correction for the refractive index mismatch, and even then, generation of a complete three-dimensional profile is not very straightforward or precise. Other techniques for imaging the cell vertical profile, such as ion conductance, digital holographic, or atomic force microscopy, are rather complex and not available to most users.

Research and development of nanomaterials, especially those made of organic molecules, has rapidly increased in the last couple of years. Novel nanomaterials exhibit extraordinary material properties and will have a huge impact on chemical, pharmaceutical, and microelectronic products of the near future.

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.

A little-known illumination method for light microscopy goes by several names, the most prominent being “circular oblique lighting” (COL) and “hollow-cone illumination”. Matthews notes that hollow-cone or annular bright field illumination can give contrast and resolution superior to that obtainable with narrow-pencil illumination and under favorable conditions comparable to that obtained with phase optics. He demonstrates this with photomicrographs of the same unstained epithelial cell from the mouth mounted in saliva, imaged with a 0.65 numerical aperture (NA) 40× objective. Matthews also notes that the dot pattern of Pleurosigmaangulatum can be resolved with a 0.50 NA objective using circular oblique lighting. Leitz previously marketed the Heine illuminator for transmitted annular (hollow cone) illumination. The NA of the Heine condenser's annular illumination can be adjusted to match the phase annuli in phase contrast objectives. The NA can be increased to provide dark field illumination or circular oblique illumination in bright field. The instructions for the Heine condenser call for the annular illumination just falling within the NA of the objective, what Paul James calls COL and Frithjof A. S. Sterrenberg calls extreme annular illumination, “bright field with very rich contrast.” H. J. Dethloff published a more recent article describing the need for the increased contrast of hollow cone bright field to help resolve the striae of pores in the diatom Amplipleurapellucida. This diatom has been the traditional test of the resolution limit of the light microscope; it is considered a low-contrast subject because the visibility of pores in the transparent amorphous silica frustules is determined by the refractive index difference between the mountant and the frustules. The low contrast makes this a challenging, perhaps even unsuitable, test object for resolution. Resolution tests of modern objectives are done with high-contrast but costly patterns of chrome on glass obtained by electron lithography.

The promise of stem cell therapy offers hope for the treatment of many human diseases and injuries. Spinal cord and traumatic brain injuries can cause the loss of neurons and glial cells. These lost cells may be partially replaced by transplanting multipotent human neural stem cells (hNSCs), which can differentiate into specialized neural cell types. The potential for these remarkable cells to promote recovery of neural function has made them a strong focus of researchers in this field.

In the last decades, a new interdisciplinary science called biomimetics has emerged that has significantly influenced the design of certain new materials. Nature, as a source of inspiration, can give us ideas and concepts to implement new functional properties. One example is the self-cleaning property (superhydrophobicity) of certain plant leaves, better known as the Lotus Effect. This term comes from the lotus leaf (Nelumbo nucifera), the best-known self-cleaning surface in nature. On this kind of leaf, water droplets roll over the leaf surface and collect dirt and other particles from the surfaces. The superhydrophobicity of these leaves is caused, in general, by a hierarchical surface structure, built by a randomly oriented small hydrophobic wax structure on the top of convex cell papillae. The wetting condition of the solid surface is a particular property of materials and depends on both surface energy and surface topography. Many papers have been written to show how superhydrophic surfaces with periodic and random patterns can be made, but few describe and characterize biological self-cleaning surfaces. These papers usually analyze them by using optical profilers and scanning electron microscopy with specific sample preparation (for example, fixation) and in some cases atomic force microscopy. The main conclusion of those works is that binary structures (hierarchical microstructures and nanostructures) and unitary structures (basically nanostructures) are found in superhydrophobic plant leaves. However, no information regarding the general micro-nano structural pattern of biological surfaces on the x-y plane has been reported. These surfaces seem to have a random pattern in most of the cases or at least a vague arrangement of its constitutive elements whose morphologies can be represented by geometrical figures (for example, hexagonal/pentagonal polygons, circles, and straight lines).

X-ray spectroscopy is a widely used and extremely sensitive analytical technique for qualitative as well as quantitative elemental analysis. Typically, high-energy-resolution X-ray spectrometers are integrated with a high-spatial-resolution scanning electron microscope (SEM) or transmission electron microscope (TEM) for X-ray microanalysis applications. The focused electron beam of the SEM or TEM excites characteristic X rays that are emitted by the sample. The integrated X-ray spectrometer can then be used to identify and quantify the elemental composition of the sample on a sub-micron length scale. This combination of energy resolution and spatial resolution makes X-ray microanalysis of great importance to the semiconductor industry.