The requirements for such a medium are that it won't deposit crystals when it evaporates and that it won't be incapable of evaporating. The second condition probably excludes anything containing glycerol. The first is more difficult. A polymer such as gum, polyvinylpyrollidone or gelatin might fiil the bill, especially in the presence of a sugar that can form a syrup that1 s almost solid. Fructose (levulose) has this reputation, and is a good aqueous mountant. Very cheap, but for some reason somewhat too acidic for basic dyes, but can be buffered. Its not 100% predictable. l've had fructose crystallize under coverslips, but you can quickly remove ail the mess with water and then try again.

An old and certain way to make a solid and perhaps permanent aqueous mount is to use a smaller-than-usual coverslip and as little aqueous mountant as possible.

Three-dimensional images, or anaglyphs, can provide useful information as to height relationships between components of an image. They can also enliven presentations before diverse groups. Imagine a boardroom full of executives having their 5th straight meeting of the day. You are there to present preliminary research information that could lead to a major contract. After requesting that everyone to put on red/blue glasses (not exactly the height of fashion), you proceed to show stereo images of hair samples with obviously different degrees of roughness. Correlation is drawn between the different samples and the nutritional components of the dietary supplement being tested. A picture not only saves a lot of words. It also leaves a lasting image in the minds of the observers that could easily translate into $$$. Besides, it's fun to look at neat 3-D images!

When silicon samples are thinned and backlit, they display a series of colors; from deep red in the thicker (-5-10 μm) regions, to orange and yellow in thinner regions, to effectively transparent in the thinnest regions, This colorful effect can be used as a technique for monitoring the thickness of cross-sectional samples prepared for transmission electron microscopy (TEM).

As seen in Figure 1, a cross-sectional TEM sample of a thin, indium tin oxide (ITO) film on glass was being investigated, A sample was prepared for low-angle ion milling by the Barna method with the addition of an adjacent piece of silicon for a thickness indicator. The colors of silicon could then be monitored to indicate when the ITO/glass sample was thin enough for TEM examination. In the figure, the arrow indicates the region of interest on the ITO/glass sample. This figure was produced by photographing the entire TEM sample through an optical microscope, backlit with a standard quartzhalogen illuminator.

Questions keep arising regarding safety regulations for storage of alcohol and other flammable liquids in a laboratory. I hope what follows sheds some light on this issue.

Ethanol is classified as a Class IB combustible liquid, Class IB liquids have a maximum allowable container capacity of 20 liters in “metal (non-DOT approved) or approved plastic containers, safety cans or DOT specification metal drums.”

The amount of combustible liquids allowable is determined by the size of the “laboratory unit”. For hospitals with sprinkler systems, per 100 square feet of laboratory space, Class I, II and IIIA (including IB) flammable liquids are limited to 4 L (1.1 gallons) not in approved storage containers or safety cans and 7,5 L (2 gallons) total, including quantities in storage cabinets and safety cans.

I have read with great interest the considered article by Dr, Barry Rittman (Microscopy Today#98-8, October, pp 8-9). In describing the difficulties of quantitation in immunohistochemistry, Dr. Rittman has gone to great lengths to give an authoritatively balanced view. It is, therefore, with some trepidation that I raise a few issues which may remain unresolved.

Dr. Rittman provides a list of criteria to be considered before applying quantitative analysis to this method. I would place the question of relating color developed to the amount of substrate present higher than number 7 on the list. To the best of my knowledge, there is little evidence that immunocytochemistry is stoichiometric, i.e.,that there is a relationship between color developed and amount of substrate present, indeed many consider that it is not, which represents a serious drawback.

The purpose of my previous article was to identify many of the potential sources of error to be considered when evaluating quantitative data from immunohistochemistry (IHC) and histochemical reactions on sections.

Factors that may affect the evaluation of quantitative IHC data were listed but not necessarily placed in their order or importance. This would be difficult to do, as the relative importance of factors may differ for different antigens and histochemical procedures.

As far as can be determined, there appear to be no definitive articles that unequivocally show that IHC reactions are stoichiometric. In most laboratories, it is impractical to accurately determine section thickness, and furthermore many of the image analysis systems used may be somewhat rudimentary. Accurate determination of section thickness may therefore be somewhat of a moot point.

The world of scanning electron microscopy seems to spin in such a way that every four years there is a dramatic step forward. Field emission sources (FEG), developed in the late 1960's, started life as a commercial disaster. In spite of the problems, certain manufacturers persisted with the first really user friendly FEG SEM reaching the market in the early 1980s. The FEG has been developed through the 1990's to be, without doubt, the source for SEU. But other advances in SEM performance have added to the instrument's performance, the most recent dramatic step being the introduction of semi-inlens imaging, a technique based upon 1980's dual detector imaging systems.

In 1968, as an undergraduate, I was first taught how to operate a Philips EM300 TEM that, at the time, was a state-of-the-art 100 keV TEM for materials studies. Last year, I spent time evaluating current state-of-the-art microscopes, f.e., intermediate-voltage, field-emission gun TEMs, from all the major manufacturers. In watching the operators running these latest microscopes, I was struck by the similarity between many of their actions and the same steps that I had learned, so laboriously, 30 years previously.

Most of us have been here more than once: a critical specimen, often the only one of its kind, does not embed properly. Usually the problem is incompletely polymerized resin. After the panic subsides, a limited number of options are left, and if the specimen is irreplaceable, there are no options, other than trying to salvage it the best way you can. I've had this problem before with low viscosity resins, but the following suggestions should work for other resins as well.

First of all, realize that reprocessed specimens are rarely as good as they would be if things went fine the first time around.

The acrylic embedding media such as LR White, LR Gold, and others, have long been favored over epoxide embedding media for immunocytochemistry in cases where the antigen is sparse or vulnerable to the chemical activity of epoxy monomers. Since acrylics bond through tissue and not with it as epoxides do (1), Au labeling may increase sharply when acrylics are substituted for epoxides. Unfortunately one serious disadvantage of acrylics is their reduced ability to preserve ultrastructure.

Since we do our paraffin-section DAB immunostaining in an EM lab, we often use osmium to darken the reaction product. We simply put a drop of our standard 1% cacodylate-buffered osmium tetroxide on each section in a hood and rinse it off in running tap water after about 2 seconds. It makes light DAB reaction product pretty dark brown, and adds a light brownish tint to the rest of the section. The reaction increases the contrast quite a bit.

For beautiful colors in near perfectly preserved cells and connective tissue, immerse carefully cut pieces, no more than 2 mm in least dimension, overnight in either Heidenhain's SUSA or Helly's fluid or Zenker's fluid without the acetic acid (which is the same as Helly's without the formalin). Wash thoroughly in water (tap water is OK) for several hours (overnight is sensible) and then dehydrate, clear, infiltrate, embed and cut paraffin sections.

After dewaxing and hydrating the slides, put them in an iodine solution (Lugol's, Gram's or 0.5% I2 in 95% ethanol), then in sodium thiosuifate (2-6%) to remove the brown iodine color. Wash in running tap water for a minute to get rid of the chemicals. (This step Is a must after any mercury-containing fix.) Stain by any good, interesting method (meaning almost anything other than hematoxylin & eosln).