The atomic force microscope (AFM) is proving to be as much a tool as an imaging device. Recently, Stefan Thalhammer, Robert Stark, Stefan Müller, Johannes Wienberg and Wolfgang M. Heckl have added another useful technique to this repertoire. They demonstrated that the AFM can be used to cut out an identified section of DNA and then extract that section for amplification.

They began by flow sorting chromosomes, and then increased the specific amount of the target DNA with a “pre-set” in situ hybridization with chromosome-specific DMA to provide a specimen rich in chromosome 2. To identify the target area, selected chromosomes were examined with the AFM in the non-contact mode using stiff cantilevers.

The answer to this question is about as elusive as predicting which way the Dow Jones average will close tomorrow! But seriously, there are the “ten commandments” for a diamond knife to enjoy a long life, the most important ones being as fallows:

1. 1) Because of the extreme sharpness of a diamond knife edge, it should not be touched with any solid object, even for cleaning, This is controversial since some manufacturers actually recommend that the edges be cleaned with sticks of varying types. We ourselves believe such treatment accelerates the wear of a diamond knife.

2. 2) Don't let sections or the remains of sections or other debris dry down onto the knife edge. Keep the knife edge wet until it is ready for cleaning before being put to bed for the night.

3. 3) Use a diamond knife cleaner sold by several firms (including ours) specifically for this purpose, Some typical laboratory ultrasonic cleaners can have enough power to be damaging to a knife.

4. 4) Wash the knife edge one last time with distilled water, and then dry with some kind of "blast", such as from a clean duster,

5. 5) Avoid conditions of chatter at all times. Reduce chatter by varying the clearance angle or slowing the cutting speed. Other common causes of chatter are insufficient tightening of the boat in the microtome, an insufficiently tightened block, or an incompletely cured block.

6. 6) Final block trimming with a razor blade can ieave metal particles from the blade, which are of course damaging to the knife edge, This can be minimized by using a fresh razor blade each time, Then wash the end of the freshly cut block face with distilled water, followed by drying with a duster blast is the final step before the first cut with the knife, This is a final chance to wash away metal particles that could damage the knife edge.

If you are an optical microscopist, chances are you have sometimes wished for a way to increase the depth of focus of your images. In this article I describe a method that does this using a simple combination of functions built into most image processing software - so it will not cost you very much to try, The method, however, is not universal and some trial-and-error will be required to get it to work in different applications. Finally, this method is not limited to microscopy and will work with any imaging system.

The basic steps for creating an image with a large depth of focus are straight forward:

1. 1. Take a series of images at different focal planes

2. 2. Remove the blurred content from each image

3. 3. Combine the debiurred images into a single image

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.

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.

Liquid propane and similar coolants are used in the rapid freezing of biological specimens. These coolants form explosive gas mixtures with air with a 14,000-fold increase in volume over that of the liquid. The liquefied gases have high vapour pressures and, unless they are maintained below their flashpoint, the vapour above them will reach ignitable concentrations. The flashpoint of liquid propane is -104°C. Ethane has a higher vapour pressure, and vapour mixed with air above liquid ethane can be ignited at a coolant temperature of -130°C. The danger is minimized if the coolant is maintained near its freezing point and under a nitrogen atmosphere, in a fume cupboard. Liquid nitrogen evaporates to a 690-fold increase in volume at room temperature. It is important to ventilate the working area, especially when cryo-sectioning in a small room, otherwise there is a possibility of asphyxiation.

Anybody who has ever worked with cells or tissues in culture knows that the dish in which to grow and look at the specimen can be of crucial importance to the results obtained, With the growing importance of live cell microscopy, be it simple phase contrast or sophisticated multilabel confocal laser scanning microscopy (CLSM), people have been looking for dishes that combine optimal growth conditions with optimal optical properties. As follows, I will describe a solution that has worked for us with a multitude of different cells, tissues and applications. Apart from that, it is cheap and easily self-made.

The bottom of a standard 35 mm plastic ceil culture dish has a hole of ca. 10 mm drilled into it and a coverslip glued to its downside to cover the hole. For a glue we usually use Sylgard, an inert silicone elastomer (Dow Corning) or melted paraffin if we intend to take off the coverslip again.

Neutrophil myeioperoxidase standard 3,3'-Diaminobenzidine Tetrahydrochioride (DAB) procedure:

When staining neutrophils, a cell suspension would be preferred, but a finely minced buffy coat can be used, if you thick section and locate the stained cells before thin sectioning. Staining should be carried out as soon as possible after fixation, The myeioperoxidase (MPO) activity falls off such that we process samples within 2-3 weeks of fixation.

Note that fixation is for 1 hour. Enzyme activity may be destroyed if tixed for longer periods.

Creutzfeldt-Jakob disease (CJD) resembles two other diseases, kuru (found in New Guinea tribesmen) and scrapie (found in sheep). CJD is a slow progressive dementia of the central nervous system. The transmissible agent of CJD is unusual for several reasons: it has a long incubation period, up to eight years in some cases; it does not provoke an inflammatory reaction within infected tissues; it has never been isolated; it is resistant to routine paraffin processing. Histotechnologists need to be informed of the potential hazards and of the correct handling of tissues infected with this unconventional agent, even though it has low transmissibility. Treatment of CJD tissues with long sterilization at high temperatures or prolonged exposure to bleach has proven effective in deactivating the agent. This article outlines the precautionary procedures used in our laboratory when processing CJD tissues. (J Histolechnol 12:214, 1989).