Short courses in scanning electron microscopy (SEM) can quickly sharpen practical skills for industrial microscopists. The SEM and the energy-dispersive X-ray spectrometer (EDS) together constitute one of the most powerful and versatile instruments available for solving industrial problems, but interpreting images and spectra is not quite as simple as acquiring them. Applications of SEM span many disciplines, and each application may require knowledge of different aspects of the microscope, and of the industrial problem at hand, to successfully interpret the images and data obtained. Regardless of the problem, whether transistors or trachea cells, the interpretation of SEM images relies upon the microscopist's understanding the fundamentals of image formation as well as the practical aspects of specimen preparation and microscope operation. Many people using SEMs today have not taken any courses beyond the on-site and demo-lab instruction provided by SEM vendors. Equipment manufacturers provide excellent training on how to use the knobs and menus on the SEM to produce useful images and data via the embedded software functions. Since there are many options and setup procedures, these instrument-specific courses are valuable for the novice and expert alike.

The most prominent vector-transmitted diseases in the first and third world are Lyme disease and malaria, respectively. In both cases the transmitted agents are introduced into the skin from where they eventually disseminate into the blood using active motility. We are interested in deciphering the molecular mechanisms underlying the motility of these pathogens and how they relate to the ultrastructure of the pathogens. Here we provide an overview of the microscopy techniques that we use to achieve these goals.

The Hague, Netherlands, 1690. Christiaan Huygens and Sir Isaac Newton, at odds with their competing wave/particle theories of light, today reached a compromise with their unveiling of a new multibeam SEM/FIB instrument featuring an SEM column designed by Huygens that uses electromagnetic lenses to focus the beam of electrons and a FIB column employing electrostatic lenses to focus the beam of ions devised by Newton. Onlookers were dazzled by its extensive capabilities and range of applications.

Sample preparation is of central importance for the characterization of materials by transmission electron microscopy (TEM). As a guide to researchers seeking practical help on the use of all types of TEM sample preparation techniques, we have created an Internet website. This website has been designed in French and is now translated into English. The website is accessible free of charge.

The TEMSAMPREP website, figure 1 http://temsamprep.in2p3.fr/, is the result of a synergistic effort of an atypical team of five electron microscopy scientists having different research specialties in physics, mineralogy, material science, and biology. They shared five years of human adventure in creating the website to transmit their 30 years of TEM experience to the international community.

Resonant plasmon excitations at the surface of noble metals can localize and amplify an electromagnetic field in a very small volume and are the enabling element of surface enhanced optical microscopies. Tip enhanced Raman spectroscopy (TERS) combines scanning probe microscopy (SPM) with Raman spectroscopy, taking advantage of this enhancing mechanism. So far a 20 nm lateral resolution in chemical imaging of a surface has been achieved. So far a 20 nm lateral resolution in chemical imaging of a surface has been achieved.

Embrittlement of ancient metals is often complex. The details of embrittlement should be investigated in order to select the best remedial measures for restoration and conservation. This article surveys the techniques used for investigating several ancient embrittled silver artifacts and an iron pile-shoe from a Roman bridge. The possibilities for preserving such objects are also discussed.

Rapid advances in fluorescence probe and imaging technologies now provide easily accessible tools for biologists to perform highly detailed analysis of molecular interactions in living cells. However it can be difficult to extract accurate parameters from these experiments because of the complex interplay of diffusion-reaction events with the morphology of the cell. As a result, only a small fraction of the available spatiotemporal information is utilized, and in many cases analysis remains at a qualitative level. The Virtual Cell (VCell, http://vcell.org) simulation environment is uniquely suited to analyzing these types of fluorescence imaging experiments because it is designed to solve reaction-diffusion equations within any given geometry [1]

A laser-based, single nano-particle tracking analysis system was described in the March issue of Microscopy Today. The LM series of analyzers from NanoSight allows nanoscale particles such as viruses to be directly and individually visualised in liquids in real time, from which high-resolution particle size distribution profiles can be obtained, figure 1. The technique is fast, robust, accurate, and low cost representing an attractive alternative or complement to expensive and more complex methods of nano-particle analysis such as dynamic light scattering (photon correlation spectroscopy) or electron microscopy.

By simultaneously and directly measuring the diffusion coefficient of each and every particle, the dedicated Nanoparticle Tracking Analysis (NTA) software suite allows the user to automatically count and size the viruses in a sample. Results are displayed as graphs of size against count of individual particles (or size versus relative brightness), thus overcoming the limitations inherent in other particle analysis systems that generate only mean particle size distribution data with resolution.

Beam Deceleration is a relatively simple method to reduce electron beam energy and improve imaging parameters such as resolution and contrast. The scanning electron microscope (SEM) uses a sharply focused electron beam to probe the specimen surface. The energy of the electrons forming such a probe is determined by the electrical potential of the electron source, referred to as accelerating voltage or high voltage (HV). No matter how many times the electrons are accelerated or decelerated inside the column, they leave the column with an energy corresponding to the high voltage. The high voltage is usually controllable within a range of 200 V to 30 kV for most commercially available SEMs, allowing the operator to select the electron beam energy suitable for the application. Imaging with very low electron beam energy has great importance, which is illustrated by SEM instrumentation development over the last few decades [1–2]. Low voltage microscopy is a topic discussed at most microscopy-related conferences these days, but generally, it is approached with an immersion lens and field emission gun (FEG) SEM system because of the better beam current densities. However, beam deceleration is also a means to bring low kV improvement to SEMs with thermionic electron sources.

The combined focused ion beam (FIB) and scanning electron microscope (SEM), known as the DualBeam, is well-known for its unique ability to produce site-specific thin samples starting from bulk and then attaching the section to a transmission electron microscope (TEM) grid, all in-situ. It has been reported that producing a thin sample using a 30 kV gallium FIB creates surface damage several tens of nanometers deep. However, recent DualBeam technology improvements now enable the FIB to produce thin samples with a thickness well below 50 nanometers and deliver a tightly focused ion beam at an energy of 2 kV and below, which dramatically reduces the damage depth to as low as 1 to 2 nanometers in typical materials, such as silicon.

This article contains my personal views and was inspired by the many years of problem solving I have applied to imaging biological samples generated by microscopy. Although I come from a background in electron microscopy, I have for the last eighteen years worked exclusively in light microscopy and more recently in live cell imaging.

Here at Duke, I mentor fellow faculty, postdocs and students in finding the best ways, not only to image their experiments in the right microscope, but to anticipate the kind of data they expect to extract from their images.

Microparticles and exosomes are small vesicular fragments of cell membrane which are released from activated and apoptotic cells. Microparticles (MPs) range in size from 0.5-1.5 μm, and exosomes are 0.5 μm and under. For the purposes of this article we will refer to both categories as microparticles. They differ from apoptotic bodies based on their smaller size, intact structure, and lack of degraded nuclear material. MPs have been shown to be released from a variety of cell types including platelets, endothelium, vascular smooth muscle cells, dendritic cells, and tumor cells. Jimenez and others have shown that based on the stimulus and cell type the MPs released are both quantitatively and phenotypically distinct. More recent data have shown the proteomics of MPs released from human umbilical vein endothelial cells differ dependent on whether they are stimulated with PAI or TNF-α.

Ernst Abbe was a brilliant German mathematician and physicist who made several of the most important contributions to the design of lenses for optical microscopy. As a young boy, Abbe lived in an impoverished family where his father labored 16 hours a day to provide for his wife and children. Abbe worked his way through school by earning scholarships and with the help of his father's employer.

Abbe studied physics and mathematics as an undergraduate at the University of Jena and went to graduate school at the University of Göttingen, where he received a doctorate in thermodynamics. In 1863 Abbe joined the faculty at the University of Jena where he taught physics. He met Carl Zeiss in 1866 and became very interested in the optical problems surrounding mid-nineteenth century microscopy.

The advent of techniques to mechanically manipulate single (bio)molecules has sparked large efforts to precisely study the mechanical and elastic properties of proteins, protein fibers, DNA, RNA, etc. Two widely used techniques in this area are atomic force microscopy (AFM) and optical tweezers. Optical tweezers complement AFM at the lower end of the force regime: forces of typically a few hundred picoNewtons down to fractions of a picoNewton can be assessed using optical tweezers. This has allowed for, among other things, the precise measurement of forces and displacements exerted by individual motor proteins. In this report, we focus on the use of optical tweezers for force spectroscopy on single DNA molecules, and on the range of applications that this technique offers to learn not only about DNA itself, but also about the mechanics and thermodynamics of protein-DNA interaction.

Traditional sample preparation supports for electron microscopy have usually been thin films of amorphous or holey carbon stretched over a metallic grid. More recently introduced formats include organic polymers over metallic grids or silicon nitride and silicon oxide membranes over silicon frames. Here, the application of a nanofabricated silicon membrane technology as a novel sample preparation support for electron microscopy is described. These new supports offer several unique characteristics, including nanoscale thickness and pores, which may improve imaging and analysis of materials and biological molecules.

Recently developed technology permits the fabrication of pure silicon membranes that are among the world's thinnest materials—only 5 to 15 nm thick. See Striemer et al., Nature (2007) 445: 749-751 for more details. These silicon membranes are also among the world's first membrane technologies to offer nanoscale pores of 5 to 50 nm. This combination of characteristics is unique and not shared by the materials currently employed as sample supports. Moreover, it suggests that these silicon membranes could overcome many of the problems associated with current generation materials. For example, the holes of lacey and holey carbon grids (1-5 micron diameter) are incompatible with the size of most nanotubes and nanoparticles (2-40 nm). Obtaining background-free images of the entire nanostructure is rarely possible with this incompatible combination of sizes. Another significant issue is the inability to vigorously plasma clean samples on carbon grids. In some cases, this inability to remove organic contaminants can prevent high-resolution imaging of samples.

Born in Iraq as Abu Ali Hasan Ibn al–Haitham, the great Arab physicist is more often known by the Latinized version of his first name, Alhazen. Although he was to eventually become a profound intellectual specializing in mathematics and optics, Alhazen's early education was geared towards religion and prepared him for a career as a minister. However, apparently unhappy in his religious pursuits, Alhazen later decided to devote himself to the study of science and became particularly intrigued by Aristotle.

Capabilities for in-situ studies of materials at elevated temperatures and under gaseous environments have received increasing attention in recent years [1]. With the advent of electron microscopes that provide routine imaging at the atomic level (e.g. aberration-corrected TEM and STEM instruments), it is of particular interest to be able to record images at high temperatures while retaining the inherent resolution of the microscope; that is, the resolution is not limited by drift in the heating holder or other instabilities associated with its operation. A number of commercial and experimental heating devices have been used over the years; some holders are designed with miniature furnaces that heat entire grids [2], while a more recent development used a tiny spiral filament coated with a carbon film as the heater element [3]. These devices, while very useful for some applications (particularly in “environmental microscopes” that employ differential pumping to allow gases at some elevated pressure to be injected around the specimen), are invariably not as stable as might be desired for sub-Ångström imaging experiments. They are also limited by the speed at which the sample can be heated to temperature for stable operation. In collaboration with Protochips Inc. (Raleigh, NC), our laboratory is developing a novel new technology for in-situ heating experiments that overcomes a number of performance problems associated with standard heating stage technologies [4].