Light microscopy has many advantages and several disadvantages. One of the advantages is that different wavelengths are perceived as different colors, and with the proper use of dyes, filters, etc. several different structures can be imaged in the same object. One the major disadvantages is that the resolution is limited by the wavelength, a limit known as the diffraction barrier. Several recently-developed techniques have allowed light microscopy to “break” the diffraction barrier, a phenomenon known as “super-resolution.” Lothar Schermelleh, Peter Carlton, Sebastian Haase, Lin Shao, Lukman Winoto, Peter Kner, Brian Burke, Cristina Cardoso, David Agard, Mats Gustafsson, Heinhrich Leonardt, and John Sedat have created a new microscope that not only achieves super-resolution, but creates images in 3 dimensions and multiple colors. Not only that, but preparing specimens to examine with this microscope uses conventional methods, and they claim the microscope is easy to use!

All forms of microscopy are being pushed to the limit by nanotechnology. This is especially true for high-resolution scanning electron and helium ion microscopes, which are proving to be extremely useful for nanometer-scale imaging, characterization, and dimensional measurements. Excellent resolution is essential for imaging nanomaterials. Hence, there is a relentless quest to achieve better and better resolution with various electron and ion microscopes and to monitor and maintain these instruments to achieve the best possible performance levels.

The ability to resolve fine details with a microscope has greatly improved over the past 20 years. The resolution achievable with the newest scanning electron microscopes (SEMs) can now be at or below 0.4 nm and for the scanning helium ion microscope (HeIM), 0.24 nm has been reported. To put this into perspective, the {111} crystal plane in silicon (Si) has 0.32 nm lattice spacing, so one cubic nanometer in the Si crystal contains only a few atoms. Therefore, an instrument must be performing at the highest level possible to resolve routinely the finest structures. Clearly, the instrument operator must also be functioning at a high level as well to achieve the best resolution and to prove that the instrument is capable of doing the work.

CryoEM is a powerful tool in the arsenal of structural biologists and soft polymer chemists. Hydrated specimens require a preservation method that will counteract the effects of the electron beam and the high vacuum environment of the electron microscope. Classical specimen preparation techniques using chemical fixatives are not able to capture the native structure of the once hydrated specimen perfectly. In contrast to classical methods for preserving specimens for electron microscopy, rapid freezing of radiation-sensitive specimens such as dispersed biological macromolecular assemblies, 2D crystals, and colloids allows the structural details of the specimen to be captured in their essentially native state to near atomic resolution.

Nanopatterning refers to the fabrication of nanometer-scale structures, meaning patterns with at least one lateral dimension between the size of an individual atom and approximately 100 nm. Direct Write or Maskless Lithography as discussed in this article refers to the use of a focused beam, either an ion beam or an electron beam, to create a patterned image directly into (etch), or on top of (deposition), the target material. Both electron beams and ion beams can be used together with gas injection technology to deposit three dimensional structures on the nanometer scale through the process of either electron beam assisted or ion beam assisted chemical vapor deposition (CVD). The deposition occurs only in the vicinity where the electron beam or ion beam is being scanned. Therefore, the deposit will follow the form of the scanned beam in two dimensions. This approach can be applied to produce three-dimensional objects by successively layering upon the two-dimensional pattern. In the case of ion beams in particular, the direct write process can also produce an etch pattern on the nanometer scale as the ion beam physically mills away the material via ion bombardment. This process can also be chemically enhanced for certain materials such as simultaneous use of water to selectively etch carbon.

Over the past decade, Atomic Force/Scanning Probe Microscopy (AFM/SPM) has emerged as the leading tool for investigations at the nanoscale – doing everything from imaging, to compositional differentiation, to explorations of molecular forces. However, aside from some interesting tweaks, add-ons and repackaging, the field has seen no fundamentally new instruments for several years. For the extremely high-resolution AFM/SPMs, there has literally been no completely new microscope for well over a decade. We report here on the new CypherTM AFM from Asylum Research (Figure 1). that delivers upgrades from the existing field of older AFM/SPMs on a variety of levels.

Dip Pen Nanolithography (DPN) is a scanning probe lithography technique where an atomic force microscope tip is used to transfer molecules to a surface via a solvent meniscus. This technique allows surface patterning on scales of under 100 nanometres. DPN is the nanotechnology analog of the dip pen (also called the quill pen), where the tip of an atomic force microscope cantilever acts as a “pen,” which is coated with a chemical compound or mixture acting as an “ink,” and put in contact with a substrate, the “paper.”

DPN enables direct deposition of nanoscale materials onto a substrate in a flexible manner. The vehicle for deposition can include pyramidal scanning probe microscope tips, hollow tips, and even tips on thermally actuated cantilevers. Recent advances have demonstrated massively parallel patterning using two-dimensional arrays of 55,000 tips, depicted below. Applications of this technology currently range through chemistry, materials science, and the life sciences, and include such work as ultra high density biological nanoarrays, additive photomask repair, and brand protection for pharmaceuticals.

A disruptive innovation in how scanning electron microscopes (SEMs) are designed and built has produced a low cost, but highperformance compact field emission SEM. By leveraging silicon processing technologies, Novelx has miniaturized the core technology inside a SEM. This miniaturization enabled the design of an all-electrostatic electron beam column that when coupled with a thermal field emission (TFE) electron source is optimized for lowvoltage imaging and sub-10nm resolution. In addition, the mySEM© has a quad-segmented microchannel plate (MCP) detector that has a topographic imaging mode that is capable of electron channeling contrast imaging (ECCI) at low imaging voltages. These capabilities were previously only available in high-end and much larger sized field emission SEMs outfitted with additional detectors. The Novelx mySEM show in Fig. 1 is now being used in material science and life science applications for characterizing a variety of energy sensitive nanomaterials, biomaterials and thin films.

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.

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.

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.

Time of flight secondary ion mass spectrometry (ToF-SIMS) and scanning electron microscopy (SEM) provide valuable complementary information about the molecular composition and morphology of biological samples, but both techniques are performed under high vacuum, which is not compatible with hydrated samples. Designing a suitable method to prepare biological (hydrated) samples for high vacuum conditions is important to obtain reliable and scientifically meaningful results from ToF-SIMS and SEM and to enable the routine use of these techniques for characterization. This article will compare freeze-drying and critical point drying for preparing adherent and nonadherent cells for ToF-SIMS and SEM analyses.

Friedrich Johann Karl Becke (1855-1931) was an Austrian geologist, mineralogist and petrologist from the University of Prague, who developed a method for determining the relationship between light refraction and refractive index differences observed in microscopic specimens. The phenomenon, which is now referred to as the formation of Becke lines, has been named for him. Becke also conducted important work on metamorphic rock recrystallization, and later contributed to the development of a descriptive terminology and classification of mineral assemblages in metamorphic rocks.

In regards to the purchase of a histology diamond knife for preparing light microscopy sections, I was one of those folks, in possession of a nice glass knife maker, who was very comfortable with breaking, making and using glass knives. So the question was—did I really need a costly diamond knife for making light microscopy sections?

Once I finally tried a diamond histo knife, I never went back to glass. There are some very good incentives for this: long lasting cutting surface, much time saved in sample preparation, larger block faces can be prepared, and more consistent cutting results obtained. A histo diamond knife is also a big aid in preparing specimens for diagnostic or biological microscopy. These very important bonuses make a good list to have in hand when justifying the purchase to your organization. However, are they worth the price? Yes, I think so—even so far as providing each tech with their own diamond histo knife.