Artifacts in preparations are sample contaminants and may constitute a nuisance if they are not recognized, or an error if they are mistaken as part of the sample. Most artifacts have two characteristics: they are common substances and they are usually minor or trace components of the sample. Occasionally, artifacts are found as a major part of what is assumed to be the sample; for example, microscopical analysis of a plugged pneumatic valve showed cornstarch as the major component. It was soon discovered that the valve had been sent in a plastic bag dusted with cornstarch. Only one percent of the particles were actual sample.

I use any type of “tin” can, of various sizes, for my isopentane container, and they are free!

Parafilm can be placed between the cork and specimen to prevent it from sticking to the cork. This is helpful when re-embedding for cross-sections. Specimens can also pinned to the cork to maintain correct lengths.

I have frozen large muscle specimens, average size about 5 cm x 2 cm x 1 cm, but they must be left in isopentane for at least one minute or even longer. If the specimen is not completely frozen, it will have “moth-eaten” fiber artifacts in the center.

After freezing large specimens, they can be put in polypropylene centrifuge tubes cooled in liquid nitrogen.

My previous article on digital imaging in the April 1997 issue of Microscopy Today noted the use of an Olympus Auto Bellows with a Kodak MegaPlus 1.6i/AB CCD camera for digital photomacrographs equivalent to 4 x 5 Polaroid prints with magnifications between 10X and 50X, The purpose of this article is to document the high resolution obtainable with the bellows system and the design modifications needed to eliminate the plastic dovetail inserts of the Olympus Auto Bellows, which are susceptible to delayed stress cracking when used with a heavier camera like the MegaPlus, Total failure of the dovetail inserts could cause costly damage to the lens and the camera.

Vibration in microscopes may seem a bit baffling, but we have found that solutions to vibration problems are usually quite simple. Vibration is the result of an object being moved until it collides with another object and bounces back. Yes, it can be as simple as shaking, but the normal definition involves collisions. Some source must continuously supply energy to the moving part(s). As long as energy is supplied to the moving part(s) and the part(s) collide, bouncing can occur at a constant frequency. If the parts can be moved so they do not collide or if the energy can be absorbed, the frequency of the bouncing can be changed or stopped.

Those of us who study the structure and physiological function of ion transport cells in animals or their excised organs (gills, for example) are always interested in “quick and dirty” screening methods to identify ion transport epithelia. Having identified epithelial areas rich in putative “ionocytes”, we are also interested in looking at the mix of non-transport cells and “ionocytes” in the tissue. The so-called “silver staining” method can satisfy both of these desiderata quickly and easily.

Silver staining selectively blackens areas of the organism's body surface which are very permeable to chloride and/or silver ions; such areas are often the sites of ion transport epithelia (Croghan, 1958; Holliday, et al., 1990; Kikuchi & Shirashi, 1997).

The scale of features that control the physical properties of engineered materials is ever decreasing. We can investigate the structure of these materials using several techniques. The techniques are usually selected based on the feature size of interest. Typical scales are the millimeter or optical scale, the micron or microscopic scale, and the nanometer or atomic scale. As the feature size decreases, the capabilities of the technique must increase.

Typical features that require at least microscopic characterization include particles in super alloys, grain misorientations in high temperature superconductors and thin films, inclusions in steels, reaction products in gas chambers.

A typical electron microprobe printout of an oxide analysis shows weight percent concentrations of different elements in terms of their oxides, Fe is usually expressed as FeO. For minerals, it also shows calculated number of cations on the basis of an assumed (theoretical) number of oxygen atoms. For examplejn olivines, (Mg,Fe)2SiO4, the theoretical number of oxygen atoms is four and the sum of cations should add up to three if the analysis is good. However, in case of spinels such as magnetite, Fe3O4, which contain significant amounts of Fe2O3, the analysis total falls below 100 and the cation sum (based on four oxygens) exceeds the theoretical cation sum. in other words, the formula calculation shows an oxygen deficiency. Geologists use this information to calculate the amount of Fe2O3 in the spinel assuming the other cations have only one oxidation state.

Looking through a microscope for extended periods is not what we were designed for - it requires holding our bodies in an unnaturally rigid position. This can cause cramped muscles and strained tendons and ligaments in the head, neck, back, shoulders, arms and wrists, Also, repetitive movements associated with microscope work can cause strain injuries. There are specific health and safety regulations for computer use. Yet, you are tied much more to a microscope than to a computer, because of the eye-piece requirements.

Main problem - microscopists know how to align their microscope but few align themselves! Many users are “slumpers”, and need training to avoid problems later.

Last month I addressed the phenomena of beam scatter in the environmental and low vacuum SEM. In that article I covered how beam scatter is affected by chamber pressure, working distance, and accelerating voltage. To briefly summarize, beam scatter becomes worse at higher chamber pressure, longer working distance, and lower accelerating voltages. As the beam scatters, electrons strike the sample some distance away from the primary beam and as a result, generate X-rays from unwanted areas of the sample. It is advantageous for the analyst to keep beam scatter to a minimum to reduce the generation of these X-ray signals. Under conditions of high chamber pressure, long working distance, and low accelerating voltage, it is possible for electrons from the beam to strike the sample on the order of a millimeter or more from the primary beam.

Rapid freezing of biological samples for subsequent processing and examination in the TEM has been around since the 1960's. Freezing in general, is advantageous because it can provide an instantaneous cessation of all biological activity and hall ultrastructural rearrangement The freezing ‘fixes” biological structures in water ice until such time that the ice can be substituted for a chemical fixative. Freezing is typically conducted at liquid nitrogen temperatures (-196°C), and freeze substitution (FS) is then carried out over days to weeks on dry ice (-80°C) (Severs el. al., 1995).

Fifteen years ago, I learned from a neurologist how to embed perfused, glutaraldehyde-fixed rat brain in egg yolk (from store-bought, separated chicken eggs). The original technical note by Adoff (reference below) for whole dog brain tissue was used, and we adapted the procedure for rat brain.

1) Fix the whole brain, minus the meningas, in 10% formalin or glutaraldehydesucrose (1 % glutaraldehyde + 30% sucrose), then place in a specimen container of 30% sucrose, 10% formalin and 0.9% NaCl, The brain is left in this solution until it sinks (less than a week). This step prevents formation of ice crystals when freezing (The meninges must be removed, or the brain will not adhere to the egg yolk.).

Cellulose nitrate is also known as celloidin, collodion, low viscosity nitrocellulose (LVN) necoloidine, nitrocellulose, parlodion, pyroxylin, and soluble gun cotton. (Some of these are trade-names; for even more synonyms, see the Merck Index, under pyroxylin.) It is made by treating cellulose with a mixture of concentrated nitric and sulfuric acids, This converts some of the hydroxyl (-OH) groups of cellulose to nitrate ester (-ONO2) groups. The molecular size and physical form of the original cellulose and the proportion of nitrated hydroxyl groups determine the properties of the product. It is useful in microtechnique because of its physical properties and its unusual responses to solvents and other liquids.

A major goal of Atomic Force Microscopy (AFM) is to achieve nanometer resolution on surface topography, Vibrating cantilever mode (VCM) is an important configuration of an AFU instrument, It was proposed in the first AFM paper.

VCM in ultra-high vacuum (UHV) results in true AFM atomic resolution, which reveals atomic scale surface defects such as a single missing atom in a lattice. However, the VCM operation in air has many difficulties due to the surface contamination on the sample and the AFM tip. The most popular operation modes of the VCM are the non-contact mode and the Tapping mode. Both of these have limited lateral resolution in air.

Reagents available for laboratory, research, and clinical use are often acquired with a specified expiration date. Yet, many are not, depending on the manufacturer and reagent type. What a manufacturer does is frequency determined by how they label the product.

The U.S. Ricd and Drug Administration [FDA] has defined categories by which reagents can he labeled based on their “intended use”. individual reagents (e.g., antibodies) intended for clinical diagnostic laboratory use will be labeled as an in Vitro Diagnostic [IVD] or Analyte Specific Reagent [ASR]. Under these categories, specified expiration dates must be supported by stability testing which establishes the product shelf life.

Complete casts of the vasculature of organs and tissues are obtained by infusing low viscosity resins into the vasculature and allowing the resin to polymerize. Dissolving away the surrounding tissue with alkali leaves a model of the intricate, three-dimensional distribution of vessels in that tissue, which is not easily obtainable by any other means, and which can then be studied with scanning electron microscopy (SEM), Because well prepared casts appear to faithfully replicate the true vascular anatomy of organs including the dimensions of vessels and details of imprints of the endothelial cells lining their lumens, they must also contain quantitative information about that vasculature.

Depending on the quality of the glass slide (not all brands seem to be the same quality and therefore display variable physical properties), post-polymerized specimen blocks can be separated from object slides. These can be of varying area size, and can include a selected area of your former semi-thin section or the whole area (as used in re-embedding techniques).

It is well known to electron microscopists working in the field of materials structure research that the importance of transmission electron diffraction patterns (TED) is: (i) establishing the crystallographic orientation correlations revealed during the practical observation of multiphase specimens and (ii) the knowledge of exact crystallographic orientation of a single crystal area in the specimen relating to image contrast interpretation.

In a recent microscopy discussion group, a correspondent questioned the use of a contrast enhancement method applied to a microscopy image for publication. What kinds of image processing, folk asked, are “acceptable” in a general sense? What kinds of image processing should be noted in the text or caption and what kind of images should be archived?

It is an interesting set of questions, and I face them wearing three hats, I am a forensic pathologist who performs forensic image interpretation in the investigation of homicide and assault, an anatomic pathologist with an interest in confocal microscopy, and a computer scientist with training in image processing and computer vision. Image processing is part and parcel of my everyday work. I use tools such as contrast enhancement, debarring, and photogrammetry for image interpretation. I build tools for visualization in my confocal work. I collaborate in the design of data acquisition devices for the evaluation of crime scenes and bodily injury.

When the workload is light, we like to hone our skills by performing analysis on difficult samples (or at least that's the justification for this particular diversion). During the long winter of 1996-97, we took up the challenge of imaging snowflakes using our scanning electron microscope (SEM). Snow, being both nonconductive and liquid at room temperature, provides some challenges to the microscopist.

We have a JEOL 5800LV, which when operating in “Low Vacuum” mode, can image nonconductive samples without charging. This is achieved by allowing a relatively high pressure in the sample chamber. The air in the chamber ionizes in the beam and neutralizes the charge buildup on the sample.