Reflection microscopy techniques are typically the easiest to employ for routine sample analysis, as they require little or no sample preparation. Included in these methods are reflection- absorption spectroscopy (RAS), which emulates transmission spectroscopy, diffuse reflection infrared spectroscopy (DRIFT), specular or external reflectance, and attenuated total reflectance (ATR). For RAS, DRIFT, and specular reflection, the nature of the resultant spectra is determined by the nature of the sample. These techniques will be discussed in future articles. ATR is primarily a surface technique which provides a high quality absorbance-like infrared spectrum and can be used to readily analyze almost any liquid or solid sample.

In Medieval Europe, manuscripts and formulas on the preparation of colors and dyes used by chemists and artists contained many references to natural plant extracts, The yellow stigmas of aromatic saffron (Crocus sativus) were crushed and used both as a medicine and colorant as far back as the ninth century BCE. In 1437 CE, a manual was written by Cennini, II Libro del Arte, a collection of recipes on the preparation of tempera and block-printing inks from saffron and brazilwood (Caesalpinia echinata), Finely divided brazil wood was prepared by scraping sticks with a knife.

First, it matters not a bit what others are doing, even if they are doing it with the full knowledge and permission of their wastewater treatment officials. This is because every treatment plant is different, and must set its own limits on chemical waste. Approval must be obtained from local officials.

Getting permission from one's own group of officials may be a pleasant or difficult experience, but the way can be eased a bit by being prepared. Realize that most of them do not know what our histological chemicals are, so provide them with the OSHA mandated hazard codes {e.g., flammable, corrosive, carcinogenic, etc.). They also want to know flash point, pH, miscibility with water and odor (if strong).

The use of traditional optics in microscopy has a well-known resolution barrier first presented by Lord Rayleigh. This is the "Rayleigh diffraction limit." In the usual textbook example, the overlapping diffraction rings from two small objects are set so that the central maximum of one pattern falls on the first minimum of the second, The barrier limits the resolution of conventional microscopes to a significant fraction of the wavelength of light.

In the past decade two methods were discovered for surpassing the Rayleigh diffraction barrier. The first was actually an independent rediscovery by D. Pohl er al. of a concept originally proposed by Synge (in a 1928 paper reviewed by Albert Einstein),

Questions have appeared on the microscopy listserver about how to prepare bacteria for SEM. This is an easy problem to solve, the greatest difficulty being preserving flagellae or pili. There are several references in basic EM texts, but briefly the bacteria can be treated as if they are small tissue samples, or negative staining samples for TEM. Details like of time in solutions, transition series, etc. will depend on the particular bacteria under study, and some trial and error will likely be needed. The procedures given here should give a reasonable starting point.

This laboratory has compared several techniques in our effort to produce artifact-free SEM images in order to compare the effect of different stabilizers on microstructure of soft serve frozen yogurt and stirred yogurt. Such samples are a porous matrix of casein micelles fused to form chains which immobilize the liquid phase. Two sample preparation protocols were used: cryo-SEM for the product sampled at lower temperature, and encapsulation of those semi-liquid or soft-set yogurts within an agar gel tube or in drilled aluminum stubs / molds coated with low melting point 3% agar.

Post-fixing with osmium tetroxide before silver-staining sections causes problems because the OsCU can etch the deposited silver. However, it is not a good idea to perform silver enhancement after osmium tetroxide treatment because the deposited osmium can also act as a nucleating site for silver deposition, and the osmicated regions of the sample will be silver enhanced. If gold-toning is used after OsO4 post-fixation, the OSO4 can also nucleate gold particles. OsO4 should always come after silver enhancement.

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.

Recently, a question surfaced on the Microscopy list server regarding preparation of insect eggs for EM evaluation. While the electron microscopy approach involved considerable sample preparation (mounting, coating, etc.), our experience indicated that, for some of these applications, a combination of reflected and fluorescent confocal microscopy provided valuable information with little or no sample preparation required, provided there are not other considerations such as the extended resolution provided by electron microscopy.

In the biological arena, most confocal work is done in fluorescence mode, There have been some forays into imaging silver-stained structures using reflected light confocal but those applications have not been very prevalent in the literature. With the judicious addition of a quarter wave plate (a polarizing accessory) in the light path, the disruptive artifact from the laser beam can be removed from the image, converting any confocal into a dual purpose instrument.

There are over 100,000 different proteins in the human body. Each of these proteins plays an important role, such as catalyzing reactions and fighting disease. Researchers at the National Aeronautics and Space Administration (NASA), are looking at the make-up of proteins that are linked to diseases, to lead to the formation of effective drugs to help battle AIDS, diabetes and cancer.

"We are studying the structure of the proteins and how the protein's structure affects its function," says Dr. Russell Judge, a researcher at NASA. "This research will eventually lead to the creation of a drug that interrupts harmful protein activities."

In order to determine the protein structure, scientists have to grow large uniform protein crystals, measure and count the crystals.

The temperature of a specimen rises when the electron beam interacts with it, producing ionization and excitation of atoms and breaking molecular bonds. Energy loss in bulk materials is ultimately converted to heat, but for small particles some of the energy escapes. Of the energy lost by the electrons in the incident beam, that which is absorbed by the specimen and degraded to heat includes oscillations of valence electrons, the kinetic energy of low-energy secondary electrons, and radiationless recombination of ionized atoms or moiecuies. Energy not absorbed includes brehmsstrahlung, characteristic x-rays, and the kinetic energy of higher-energy secondary electrons. Glaeser (1979) estimated that 50% of the energy loss is confined to a distance of about 5 nm from the track of the incident electron, while the other 50% is largely due to secondary electrons having 0.5 to 5 keV kinetic energy.

Recording images from the TEM with photographic film has a number of advantages over digital imaging. The first advantage is cost. Most institutions, whether academic or industrial, already have a darkroom so there is little or no start up cost. Even if a new darkroom must be equipped, a professional quality enlarger, lens, timer, paper cutter, paper safe, easel and safelight are easily under $2500 total. Trays and chemicals are inexpensive while an automatic print processor, which speeds up work significantly, while reducing exposure to chemicals, is about $2500. Additional lenses, so the same enlarger can be used for other film formats (35 mm), are about $200 each.

As humans, we are very sight oriented. As microscopists, we usually acquire data that is in the form of images. Since micrographs, or pictures, are what we deal with, we tend to become involved with the presentation of the data and its visual impact. When making a figure plate for publication, do you often find yourself moving images around, adjusting sizes, tonal gradations, and positions to achieve a more visually pleasing arrangement? Microscopy must be the most artistic of the sciences. We might like to have fun with our images, as well.

Images formed by electrons are collected on media that sees only if the electrons were there or not. Traditional electron micrographs are black and white images; photons are needed for the colors that we can detect.

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.

A selection of the more common questions arising during a typical lab course for materials majors learning to use a TEM is as follows

1. 1) Why must I insert the objective aperture, which governs image contrast, in the diffraction plane?

   2) Why must I insert the selected area diffraction aperture, which governs the diffraction pattern contrast in the image plane?

   3) Why is the TEM relatively useless if both apertures are in at the same time or both are out?

   4) Why is the contrast in my images always greatest when the beam is parallel - which makes the screen intensity so weak, I sometimes can't see the contrast anyhow?

   5) Why, when I need to record a diffraction pattern, do I have to learn to estimate exposure time from the analog screen, rather than just push a button and get a correct exposure, as in most other optical instruments and cameras?

Geoff eloquently makes a case for film as opposed to digital image acquisition and processing. And while I agree with many of his points, I also have a slightly different point of view on several aspects.

Let's start with the darkroom. While Geoff is correct in saying that many institutions already have a darkroom or have access to one at no cost, this only tends to hide the cost. Somebody has to build the darkroom and somebody has to pay for building maintainingand equipping the darkroom. If this is paid out of some overhead and not paid for directly, then the cost is hidden in that overhead, which would be less without the darkroom.

In the course of both physical and failure analysis of semiconductor chips (i.e., verifying what you actually deposited as a layer, vs, what caused the circuit to fail), it is essential to have appropriate deprocessing tools at your disposal in order to evaluate complex semiconductor structures, Deprocessing techniques are developed for each product manufactured and involve multi-step procedures that reveal the layer-by-layer secrets of the chip, These techniques require constant tweaking in duration and procedure as the manufacturing process imposes changes and as the architecture of the semiconductor changes. While there are many tools that assist in these analytical pursuits, such as RIE (reactive ion etching - a dry etching technique), ion milling, and microcleaving, the wet chemical etching of tungsten is sometimes more reproducible than RIE techniques.