A thorough knowledge of structural and chemical properties is essential in the fields of nanotechnology, materials research, and life science, leading to a growing demand for characterization methods for heterogeneous systems on the nanometer scale. However, certain properties are difficult to study with conventional characterization techniques due to either limited resolution or the inability to differentiate materials chemically without inflicting damage or using invasive techniques such as staining. Confocal Raman Imaging can effectively overcome these fundamental obstacles. In Confocal Raman Imaging, the acquisition time for one Raman spectrum is a crucial value, as it determines the acquisition time of the image, which typically consists of tens of thousands of Raman spectra. This article describes how the use of a spectroscopic Electron Multiplying-CCD (EMCCD) as the detector can significantly reduce the acquisition time to a few milliseconds per spectrum, as well as tremendously improve sensitivity.

Metallographic examinations of uranium, as carried out with both optical and orientation contrast microscopies (electron backscatter diffraction or EBSD), typically require a final preparation step that involves acid-etching or electropolishing. Uranium oxidizes relatively rapidly after mechanical polishing, making EBSD impossible without additional preparation steps. Conventional specimen preparation generates a mixture of acids and radioactive waste, so-called “mixed waste” as defined by the Resource Conservation and Recovery Act (RCRA). Mixed waste is more costly to dispose of than either separate component and there is a desire to eliminate mixed waste streams. One simple method to avoid the use of acids in optical metallography is to wait a few minutes for the sample to differentially oxidize to obtain the desired contrast. However, this method is disadvantageous when more than a few micrographs are needed because the sample continues to change with each micrograph.

In photomicrography, two fundamental problems often arise in practice: The higher the magnification and the higher the thickness or three dimensional extension of the specimen, the lower is the depth of focus which can result in parts of the specimen being indistinct. The larger the area of the specimen, the lower is the resolution in images showing the specimen in a total view. Additional problems will occur, when the lowest magnification of the respective microscope is not low enough for inclusion of the specimen’s total size so that only parts of the specimen will be visible in photo micrographic images. Table 1 presents the focal depth and lateral resolution of microscopic objectives with regard to their magnification and numerical aperture (modified from [4] and [7]).

Researchers often examine samples by eye on the microscope — qualitatively scoring each sample for a particular feature of interest. This approach, while suitable for many experiments, sacrifices quantitative results and a permanent record of the experiment. By contrast, if digital images are collected of each sample, software can be used to quantify features of interest. For small experiments, quantitative analysis is often done manually using interactive programs like Adobe Photoshop©. For the large number of images that can be easily collected with automated microscopes, this approach is tedious and time-consuming. NIH Image/ImageJ (http://rsb.info.nih.gov/ij) allows users comfortable writing in a macro language to automate quantitative image analysis. We have developed Cell- Profiler, a free, open-source software package, designed to enable scientists without prior programming experience to quantify relevant features of samples in large numbers of images automatically, in a modular system suitable for processing hundreds of thousands of images.

Micro x-ray fluorescence (MXRF) is a microscopic analysis and imaging technique that is used to characterize the elements in a material non-destructively. Micro XRF instruments use an x-ray source to shine x-rays on a sample, and a detector to detect the characteristic x-rays given off. These fluorescent x-rays have very specific energies corresponding to specific electron energy transitions. Therefore, it is possible to detect and identify all of the elements present in a sample (typically above sodium) as well as measure their concentrations. This technique is widely used for the characterization of materials including polymer and metallic foams, powder samples, forensics applications, geological samples, works of art and nuclear fuels. Commercial MXRF instruments use a fused silica optic (mono or polycapillary) to focus the x-rays on the sample with no optic on the detector (Figure 1a).

Food ingredients are often made by extruding flours and starches derived from cereal grains with proteins. Extrusion is carried out at high temperature, high shear and low water conditions to make low density, foamed pellets that are used as food ingredients. The structure of the crisp matrix varies greatly with the ingredient proportion and is important in forming the organoleptic properties of the crisps. However, it is very difficult to analyse the structure of the matrix with respect to the distribution of the major components, and the authors have been unable to find references for these materials. Furthermore, the materials must be stained without wetting as water will destroy the integrity of the crisps.

Everyone always wants better resolution from his or her microscopes. With semiconductor manufacturers now shipping product with sub-100 nm gates, measuring features and defects has become a challenge, even for the scanning electron microscope (SEM). For metrology below 100 nm, some manufacturers have begun routinely using TEM (transmission electron microscopy) which is tedious and expensive. As a microscopist, I find this quite disappointing since, in principle, the SEM should be capable of providing more than enough resolution well below 100 nm. Why is it that SEMs with 1 nm spot size can’t provide adequate resolution for 100 nm gates? It turns out that at very high magnification, SEM resolution is limited by how the electron beam interacts with the sample rather than simply the spot size of the beam.

Automated Crystal Orientation Microscopy (ACOM) on a grain specific level has proved to be an invaluable new tool for characterizing polycrystalline materials. It is usually based on scanning facilities using electron diffraction , due to its high sensitivity and spatial resolution, but also attempts have been made which rely upon X-ray or hard synchrotron radiation diffraction. The grain orientations are commonly mapped in pseudo-colors on the scanning grid to construct Crystal Orientation Maps (COM), which represent “images” of the microstructure with the advantage of providing quantitative orientation contrast. In a similar way, misorientations across grain boundaries, Σ values of grain boundaries, or other microstructural characteristics are visualized by mapping the grains in the micrograph with specific colors. The principal objectives are the determination of quantitative, statistically meaningful data sets of crystal orientations, misorientations, the CSL character (Σ) of grain boundaries, local crystal texture (pole figures, ODF, MODF, OCF) and derived entities, phase discrimination and phase identification.

One of the main obstacles to performing electron crystallography analysis in a TEM is that the acquired electron diffraction data often exhibits some form of distortion introduced by the lens system. Recognizing this problem, Capitani et al. has proposed a method to detect such distortion, which is primarily elliptical, by using a single crystal standard. Once such elliptical distortion is characterized, electron diffraction data acquired later can then be corrected by means of image processing. However, it may be desirable to correct such distortion at the instrument level. In this article, a different approach to measuring diffraction elliptical distortion is proposed by characterizing diffraction ring patterns and it is demonstrated that by varying the objective lens stigmation settings, it is possible to eliminate this elliptical distortion completely.

Preparation for TEM of resin-embedded monolayer cell cultures usually requires scraping cells from the culture substrate before or after primary fixation, which disturbs the monolayer and often results in mechanical trauma to the cells. After harvest, the cells are centrifuged and processed as a pellet in a microcentrifuge tube through a prolonged procedure of post-fixation, dehydration, infiltration, and finally embedding to ensure a “well done” sample block for subsequent sectioning, staining, and observing under a transmission electron microscope. Other disadvantages to this method include the loss of cellular orientation and information on cellular interaction, as well as difficulty in collecting a large quantity of cells to form a sizable pellet for processing, which is of special importance when samples are differentiated cells and neurons cultured in low density. In an alternative method, cells can be seeded on either glass or plastic cover slips or Petri dishes, and then processed and embedded directly as a monolayer.

Image acquisition with a CCD camera is a single-press-button activity: after selecting exposure time and adjusting illumination, a button is pressed and the acquired image is perceived as the final, unmodified proof of what was seen in the microscope. Thus it is generally assumed that the image processing steps of e.g., “darkcurrent correction” and “gain normalization” do not alter the information content of the image, but rather eliminate unwanted artifacts.

The intricate relationship between molecular structure and function is a common theme in molecular biology. Visualizing the structure of biological macromolecules through imaging is therefore useful in understanding their varied biological roles. The process is often complex; imaging in a high voltage Transmission Electron Microscope (TEM) involves extensive staining and freezing. The aim of this experiment was to image DNA easily in a close to natural environment in a simple microscope. Samples were imaged using a Leo (Zeiss) 1550 Scanning Electron Microscope (SEM) with a Schottky field emitter in the 15-30kV range to reduce radiation damage. After imaging off-the-shelf DNA, two more DNA samples were dialyzed with RbCl and NaCl and imaged to elucidate what made the DNA visible.

Inspired by architect Frei Otto and design scientist Buckminster Fuller, third year Pratt Institute design students from Jonas Coersmeier’s design studio and research seminar (of Spring 2008) utilized a Table Top SEM to observe micro and nano-scale features produced solely by Mother Nature. After analyzing and documenting the intricacy, beauty and functionality of natural structures, students selected structural entities typically not observed on the macro scale, and utilized the micrograph data to generate analytical drawings followed by generative models for design of a large span structure that would become an aquatic center in the Williamsburg neighborhood of Brooklyn, N.Y.

Atom probe tomography has primarily been used for atomic scale characterization of high electrical conductivity materials. A high electrical field applied to needle-shaped specimens evaporates surface atoms, and a time of flight measurement determines each atom's identity. A 2-dimensional detector determines each atom's original position on the specimen. When repeated successively over many surface monolayers, the original specimen can be reconstructed into a 3-dimensional representation. In order to have an accurate 3-D reconstruction of the original, the field required for atomic evaporation must be known a-priori. For many metallic materials, this evaporation field is well characterized, and 3-D reconstructions can be achieved with reasonable accuracy.

The covering wings (elytra) in the Colorado potato beetle Leptinotarsa decemlineata appear shiny, smooth, water-repellent, and slightly slippery, (Fig. 1). These properties are due to the presence of epicuticle, the outermost layer of the insect integument, covered by a wax-like lipid surface layer called grease. Surface waxes have been previously reported in a variety of conditions, from liquid viscous coatings to crystalline structures in form of plates, rods and filaments from many insects (adults and larvae) and arachnids (Hadley, 1981a,b). Beament (1945) and Wigglesworth (1945) considered an outer thin layer of solid wax, whereas Lewis (1962) rather assumed an oil film on the epicuticle surface to be widespread throughout insects.

The Applied Chemical and Morphological Analysis Laboratory (ACMAL) is a multi-user, multi-disciplinary characterization laboratory. ACMAL houses two scanning electron microscopes (SEM and FE-SEM), a transmission electron microscope (TEM), focused ion beam milling system (FIB), four X-ray diffractometers, and an X-ray fluorescence spectrometer. ACMAL operates as a recharge center where users absorb facility operation cost through an hourly use fee. As such, we are keenly interested in encouraging broad access to the facility by lowering obstacles to users. Facility training enhancements provide the best pathway to productive and responsible facility usage.

The application of nanotechnology in disciplines as varied as medicine and electronics is advancing rapidly with carbon nanoparticles (CNPs) such as fullerenes (C60) and nanotubes at the forefront. However, a lack of understanding of the interaction of such small structures with cellular material has resulted in concerns over their impact on human health and since the individual structures have a diameter of ~1 nm they are potentially small enough to penetrate through ion channels or diffuse through pores in the nuclear membrane. Assessing their toxicity is imperative. In response to these concerns there has been an increase in the number of papers addressing the toxicity of carbon nanoparticles over the last few years but much of this data appears contradictory. It is therefore essential to understand how the human body interacts with CNPs and more specifically to elucidate pathways by which CNPs enter the cell and their distribution within.

The scanning electron microscope (SEM) is commonly used to obtain images of a wide variety of samples within a wide range of magnification factors from the order of 10 up to about 105×. This technique is usually applied, but not limited to, the investigation of conductive samples. This is because the interaction of the scanning beam with the sample generates a net charge on the sample surface. Thus, if the sample is conductive, the charge can be quickly disposed of to ground, away from the beam spot. If the sample in non-conductive, the sample becomes locally charged, giving rise to a distortion of the primary beam. In certain conditions, the charge stored on the sample is able to reflect back the incoming electrons, much like an electrostatic mirror.

This short review describes the relevance of cell adhesion in cell biology. Starting with a short overview of the force range of adhesion related biological events and the current biophysical techniques for investigating these events, it will conclude with a description of the use of single cell force spectroscopy for quantifying mechanical properties such as stiffness, surface tension, and bond disruption forces.

Nature has provided us with a wonderful outer surface, the skin. We humans are fascinated with our epidermis and hair. We spend an exorbitant amount of time and money on skin and hair maintenance. We try hard to keep it soft, smooth, radiant, tan or pale, clean or made-up. We try to remove hair from some areas as hard as we try to keep it on others. Only when our skin is afflicted by disease, burns, wounds, or scratches is it apparent that this amazing outer shell protects us from dehydration and infections. To perform this crucial protective function and to be able to respond to injuries, skin needs to undergo self-renewal to repair damaged tissue and to replace old cells. For this formidable task, skin depends on stem cells. Stem cells have the ability to self-perpetuate and to give rise to differentiating cells that constitute one or more tissue types.