Way back in 1966, when I first started my industrial career at U.S. Steel's Edgar C, Bain Laboratory for Fundamental Research (which is what it was called before it became a grease spot in Corporate America) Gene Fischione and Dick Glenn were engaged in a "moon race" of sorts to bring out some of the earliest electrolytic jet polishers. Remy Schoone helped Gene build his model; and Bob Sober helped Dick make the competition, I haven't used either one of these fine instruments in nearly thirty years, so the only connection is historical and the good feelings I have about the four nice guys who made them.

Questions have arisen recently on the Histonet listserver about the properties of zinc formalin fixative. The advantage of zinc formalin over the usual formalin fixatives appears to be that zinc ions hold macromolecules in their native conformation via coordinate bonds, preventing the damaging crosslinkages that formaldehyde alone would create. The result is greatly enhanced immunopreservation: rarely is antigen retrieval necessary, and primary antibodies can be diluted 2 to 10 fold greater than usual.

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.

Today, charge coupled devices (CCDs) have become the de-facto standard for scientific imaging, In fact, their potential for high resolution, sensitivity, wide dynamic range and low noise has made them the device of choice for most fluorescent microscopy imaging applications, This is especially true for quantitative microscopy applications such as ratio imaging, fluorescent in-situ hybridization and image deconvolution.

However, choosing the most appropriate CCD camera for each application can be quite challenging given the broad range of features and performance specifications. This month's article focuses on a single characteristic of the CCD: the chip grade.

During a recent one week "Intensive SEM" course that took place in Johannesburg, South Africa, the students came up with a superb cleaning technique that they wished to report upon. The results are, or should be, interesting to everyone who has to clean a cathode assembly! This note was originally posted on MSA's microscopy listserver and generated several replies. I have included more information below in response to these replies.

Often the most time consuming operation during the routine use of a SEM or TEM is the cleaning of a cathode assembly. The procedure, outlined as follows, requires little operator intervention, is free from possible cathode contamination by the cleaning media, and takes comparatively very iittle time

There are two approaches to determining the thickness of a carbon film evaporated onto a specimen. One approach is to do the evaporation first and to measure the result afterwards. There are several ways to do this. The second approach is to put down a determined amount of carbon in the first place. This clearly has advantages in many cases. The method described here is of this second kind. The thickness of the carbon coating is not measured, but by a predetermined recipe, the amount put on is controlled at the time of evaporation, In principle, the thickness could be measured during the evaporation with a quartz-crystal thickness monitor. However, we find that the monitor we have does not work well with carbon.

One conventional method for picking up ultrathin sections for transmission electron microscopy (TEM) is to prepare the mesh or slot grids with a formvar support film and then pick up your sections as they are floating in the knife boat. An alternative method is to first pick up sections on a naked grid and then lay them down on formvar film suspended over holes drilled in an aluminum bridge. I use this method when picking up serial sections on slot grids. A description of the method follows. The bridges I have were manufactured at the University of Georgia instrumentation Shop. They can also be purchased pre-made (for example, from SPI, Inc. They list them as Domino Racks), or one can make them from aluminum with a vise and drill. The sample bridge in the figure is similar to what I use. It is 5 cm long, 2.5 cm wide.

FT-IR (Fourier Transform Infrared) spectroscopy is an instrumental technique for measuring the infrared spectrum of materials. Many organic compounds have characteristic spectra that can be used for their identification, and specific functional groups (hydroxyl, carbonyl. amine, etc.) have characteristic bands at certain frequencies.

Microscopes have been coupled to FT-IR instruments and allow the IR spectrum determination of single particles. This greatly increases the identification possibilities of small particles and contaminants. These particles can be mounted on sodium chloride or potassium bromide salt crystals, since these materials are transparent to infrared radiation.

Microscopy is unique among its analytical counterparts: it requires a hand-eye-brain coordination not needed in any other type of analytical instrumentation. On one hand, manufacturers of microscopes and auxiliary technologies are working to make systems seamless, transparent, and easier to use. On the other hand, microscopists, even those who will use the microscope as routine tools, need to be knowledgeable in their core disciplines as well as fluent in microscopy and sample preparation. It is not enough that a the person running a microscope be trained to "push this button; turn this knob". Especially as we move into the next millennium, it will become increasingly important to understand the light/matter, electron/matter, or probe/matter interactions which occur when examining a specimen. Microscopists of all descriptions will need to know how to optimize both the sample and the technology, how to interpret the information presented by the image, and how to separate artifact from true information. To compound the problem, the convergence of computer, diverse software packages, microscope, camera, and analytical techniques is producing a new type of multiple level information which, in turn, requires expertise in multiple disciplines.

A perennial favorite debate among TEM users is which side of the grid to use, the dull side or the shiny side. This question popped up again on the Microscopy tistserver, and received several welt-informed replies, tn the past, the answer has typically been "whichever works" or "Welt, I learned it this way". Now opinion seems to be leaning towards a particular side. Read on for the answers

From John Bozzola, Director, Center for Electron Microscopy, Southern Illinois University:

This is a good basic question - one that many microscopists have asked when coating grids with various plastic substrates.

Measuring electron intensity is sometimes a useful thing to do as a part of an electron microscope study. The electron intensity recorded through a sample is one of the important pieces of information needed in determining the thickness of a thin film or the molecular weight of biological structures by electron scattering methods. Modern digital imaging systems in transmission electron microscopes allow relatively easy determination of electron intensities, but some labs don't have the expensive digital systems. Photographic film has been used traditionally to record electron microscope pictures, and film can record a very high density of spatial information.

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.

Acetone, alcohol, and propylene oxide have different mechanical and chemical properties. They should not be chosen haphazardly but should be used with care to suit the particular demands of the embedding conditions.

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).