We do this two different ways, using polyethylene (PE) molds or aluminum weigh boats, depending on the sample, You can either heat- or UV-cure these molds-just change the type of “lid” on the embedding mold.

The PE molds are used when the coverslip can be made to fit in the mold. We use JB-4-type molds, available from your favorite microscopy supplier. The key to using these molds is that they have to be pre-treated...fill the inner cup of the mold with LR White, cover with an aluminum JB-4 chuck (try to have enough resin in the mold to come up around the base of the chuck), and polymerize in a 60° C oven for a day or two, until the resin is hard.

Tens of thousands of Americans suffer from muscular dystrophy (MD), a disease that gradually deteriorates a person's skeletal muscle. While there is no effective cure, scientists know MD is caused by a genetic defect and are searching for treatments that will stop or retard the deterioration of muscle. In June, American and British researchers announced the success of a long-term treatment that repairs the genetic defect in Duchenne muscular dystrophy, the most common childhood form of MD.

Last fall, these researchers injected a short strand of nucleic acid into the shin muscle of a six-week old golden retriever - which had a genetic defect that leads to Duchenne MD in dogs - in order to maintain normal levels of dystrophin, the muscle protein missing in Duchenne MD. Their goal was for the nucleic acid to trigger the dog's system to correct the genetic defect. Eleven months later, the injected muscle continues to show normal levels of dystrophin.

Make sure that gloves are worn for this procedure. All containers and gloves used should be rinsed with water and the water put in the waste mercury container for next time.

All solutions containing mercury salts are collected into a jar labelled “Used Mercury Salts”. We keep this in our grossing room. The jar is actually a clear glass 3 kg sodium phosphate container.

When the jar is about half full, to each 900 mL (estimate, don't measure) add 40 grams of sodium carbonate to raise the pH to 8.0 or higher and mix well.

Leave it for a while, then filter the solution and collect the precipitate into a plastic jar for disposal. Put the filter paper in the jar as well. Seal the jar very well. We coat the lid with several layers of paraffin wax until it is about a quarter inch thick.

Inexpensive fixed-focus digital cameras can be used with homemade lens attachments to obtain good quality close-up images and photomicrographs. The current availability of inexpensive digital cameras with resolutions of 1024 x 768 pixels or better is brought about by technical advance and the continual introduction of competing new models and accessories. The digital camera which I use costs less than $150 now

at a liquidation sale or internet auction, and the price continues to decrease. Although more expensive digital cameras are great to have, their advanced features are not necessary for introducing young students to the digital revolution in photography and for exploring the microscopic world.

Often during light and fluorescent microscopy, one wishes to observe samples of cells (e.g., yeast) grown in liquid culture. This is sometimes difficult as the cells can move around under the coverslip and a wet mount can dry out, killing the cells. Protocols in which the cells are resuspended in molten agarose results in cells that are in many different focal planes and often stressed by the high temperature of the molten agarose. This protocol describes a method of depositing small pads of growth media containing agarose onto slides, which eliminates these problems. Choice of growth media depends upon both the growth requirements of the cells and the requirements of the microscopy method. For example, yeast synthetic media works better for fluorescent microscopy than yeast rich media, because the yeast extract in rich media is autofluorescent in the green (fluorescein/GFP) channel.

Microscope immersion oil is used in light microscopy to improve imaging. The use of microscope immersion oil as part of a microscope lens system will produce a brighter and sharper image than a similar design not using immersion oil. The reason is that this oil replaces the air gaps between the condenser and the bottom of the slide and between the top of the slide or cover glass and the objective lens with a medium that has a refractive index equal to the lowest refractive index of these glass components.

How does this work? There is a cone of light extending from a centered point on the specimen to the diameter of the objective lens. The angle of this cone is the Angular Aperture (A.A.). A.A. ranges from 10° for long focus low powered objectives to 140° for short focus high power objectives.

Control of layer thickness is critically important in the manufacture of semiconductor devices. Cross-sectioning exposes device structures for direct examination but conventional sample preparation procedures are difficult, time consuming, and grossly destructive. Cross sections created by focused ion beam (FIB) milling are easier, faster, and less destructive but have not offered the clear layer delineation provided by etching in the conventional sample preparation process. A new gas etch capability (Delineation Etch™ from FEI Company) offers results that are equivalent to conventional wet-etch preparations in a fraction of the time from a single, automated system in the fab without destroying the wafer. The new etch process also has application in milling high-aspect-ratio holes to create contacts to buried metal layers, and in deprocessing devices to reveal silicon and polysilicon structures.

Computer Microvision is a microscopy technique originally developed to study sound-induced motions of sensory cells in the inner ear - motions that are on the order of picometers-i.e., smaller than the radius of a hydrogen atom. Three-dimensional micrornovies of inner ear cells moving in response to sound were analyzed to quantify motions with nanometer precision. Recently we have adapted the technique for in situ measurement of motions of micro-electron mechanical (MEMS) devices.

In Computer Microvision systems, images of MEMS are magnified with an optical microscope and projected onto a CCD camera. Stroboscopic illumination is used to obtain stopaction images of the moving structures. Stop-action images from multiple focal planes provide information about 3D structure and 3D motion. Image analysis algorithms determine motions of all visible structures with nanometer accuracy. The system can measure motions at frequencies as high as 5 MHz with nanometer resolution, i.e., well below the wavelength of light.

The performance of an electron lens depends solely on the size of the space between the pole pieces. This may be a simplification, but not much of one.

In this article, I will describe the operation of electron lenses in general but with specific reference to how they relate to working with SEM long working distances. The theme is related to EBSD performance when long working distances are needed with steeply tilted samples, and where large beam currents are needed to reduce noise in the EBSD pattern.

The lenses in electron microscopes are made of magnetic fields. The field bends the path of the electrons as they travel through it and so focuses them. The magnetic fields are themselves produced by an electric current flowing through a coil of wire. As the current is increased, the field gets stronger and so the lens focuses more strongly - up to a point.

Poorly conducting specimens can be examined without coating by using a variable pressure SEM. However, many labs may only have a high vacuum SEM, or for other reasons, choose a high vacuum mode. In order to examine insulating specimens in a high vacuum SEM (using operating conditions conducive to BSE and X-ray analysis) specimens must be coated with a conductive thin film.

The perspective of this article is from a materials point of view, but the principles remain the same for biological examinations requiring similar information.

Back Scattered Electron (BSE) image contrast is primarily a function of the average atomic number of an imaged area. This is particularly true for polished specimens where there is no topography to contribute to contrast. The BSE coefficient is the ratio of back scattered electrons to incident (beam) electrons.

With the upcoming Microscopy & Microanalysis Conference only a few weeks away, the question that is in the mind of many industry experts is: “Will this be the meeting where the first commercial x-ray microcalorimeter is introduced?” If it is, you can bet your bottom dollar that there will be a major upheaval in the high resolution x-ray EDS business because of the ten- to fifty-fold improvement in X-ray resolution promised by this extraordinary device.

This is not that uncommon of a question. The first rule of thumb is that if it does not look like gold, then it probably is not gold. However, there is one exception and that is when the sputtering process creates some kind of surface structure that leads to an optical effect, there may be a nongold looking gold color (more like gray). But this typically does not adversely effect the sputtering rate.

Other cases would be when either a) contamination from external sources (e.g., fnger prints) or b) build up of contaminants from the use of gold with insufficient purity. If a), then the “problem” is solved by a good solvent washing and scrubbing with something like acetone. If b), then it will not rub off with solvent and it would be something building up in the way of impurities from within the gold foil itself.

I did serial sectioning for years on large single hole grids using a very simple technique that made the potential problems of film thickness, wrinkles and section loss very minor. I was not the original developer of the method and do not remember who originally gave it to me. It goes as follows:

• 1) Have your machine shop cut some thin pieces of Plexiglas into the size of glass slides. At one end, drill about a dozen holes, roughly 5 mm in diameter, in an area about the size of a formvar film cast on glass slides. These slides will serve as your template for holding your films.

Many years ago we regularly used platinum crucibles in many analytical chemistry procedures, and according to my analytical chemistry book, platinum can be cleaned by fusion with potassium or sodium bisulfite. I later adopted this procedure with good success to clean the platinum apertures for an RCA EML electron microscope.

Get asmall ceramic crucible, put a bit of the bisulfite in it, heat it until the bisulfite melts, then drop the apertures into the melt. After a few minutes allow the melt to solidify, dissolve the bisulfite with hot water, and your apertures should be as bright and shiny as new.

It is not safeto store osmium in ordinary laboratory plastic containers. Osmium penetrates polyolefin plastics (polyethylene, polypropylene) to some depth, and reacts with them, so containers made from these plastics or of polystyrene or polycarbonate with polyolefin closures are not recommended.

Osmium is usually supplied with some protective packaging and, should further security be desired, I would consider placing the glass ampoules inside polyethylene or polypropylene tubes. This would allow considerable shock protection and, if the ampoule did break, the outer container would protect against leakage for the short time required to get it to a fume cupboard. The same principle can be applied to osmium solutions prepared in glass bottles: enclosure in an outer polyethylene bottle will give shock protection and temporary containment. Also, its blackening indicates how much leakage has occurred from the supposedly closed glass container.

In this regard, we have a rule that osmium solutions are never stored in the laboratory refrigerator.

A central theme of modern materials science has been the exploration of the relationship between the microstructure of a material and its macroscopic properties. Beginning in the late 19th century, the developing field of metallography permitted scientists to view the microstructure of metal alloys. Mechanical polishing followed by selective chemical etching produced differential relief on chemically distinct phases or at grain boundaries. With such specimens, reflection optical microscopy revealed structures with micrometer and even finer dimensions. The microstructural world that was found proved to be highly complex, and most alloys were observed to be chemically differentiated into two or more distinct phases.