The current imaging trend in optical microscopy, scanning electron microscopy (SEM) or transmission electron microscopy (TEM) is to record all data digitally. Most manufacturers currently market digital acquisition systems with their microscope packages. The advantages of digital acquisition include: almost instant viewing of the data as a high-quality positive image (a major benefit when compared to TEM images recorded onto film, where one must wait until after the microscope session to develop the images); the ability to readily quantify features in the images and measure intensities; and extremely compact storage (removable 5.25” storage devices which now can hold up to several gigabytes of data).

Advances in computer processing power have enabled new threedimensional image enhancement and visualization techniques. Data storage capacities of a gigabyte or more are affordable and commonplace. The Power Macintosh computers are RISC based processors which perform graphical and computational tasks many times faster than the older 68000 based systems. These new systems are ideal platforms for threedimensional deconvolution of microscope data sets.

Ever since electron microscopes were developed, it has been the goal of microscopists to observe specimens in their natural state, free from artefacts which can often be introduced through specimen preparation. For most biological specimens, that includes the presence of water. With a pressure of 10-4 torr or lower required to operate a scanning electron microscope (SEM), liquid water, which required a pressure of above 5 torr, was clearly a problem.

Although several attempts had been made to examine hydrated specimens in a SEM, the first published results of water imaged in a stable and reproducible manner in the SEM, were presented at the Eighth International Congress on Electron Microscopy in Canberra in 1974 (Robinson, 1974).

In this note we show the potential usefulness of glutaraldehyde (GA) in confocai microscopic studies of wood-fungal interaction. We are presently developing methods to examine by the confocai laser scanning microscope (CLSM), the pattern of distribution of fungal hyphae within wood in relation to fungal degradation and sapstaining of wood. CLSM has the potential to be a very useful tool in such studies for a variety of reasons, including its use for optical sectioning to produce computer assisted 3-D images.

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For numerous reasons a closed-circuit water cooling system is the preferred option for providing cooling water to the electron microscope. Cooling water is required by the electron microscope to cool the diffusion pumps and to keep the electronics' and coiumn temperature stable.

A closed-circuit water cooling system is essential if the local water supply has a high chloride concentration, has floating particles, is acidic, has a water temperature that fluctuates and is uncontrollable. This potentially leads to specimen drift problems in the TEW, and/or has a water temperature that is very cold. This potentially leads to condensation problems or diffusion pumps not functioning properly in the TEM.

Instructions are given as follows for making five aqueous mounting media: glycerol jelly, buffered glycerol with anti-fade, fructose syrup, Apathy's gum-syrup, and a polyvinylpyrrolidone medium whose composition can be varied to suit the user's needs, For each, hints are given on usage and the preservation of the coverslipped preparations. Media made in the laboratory are cheaper than commercial aqueous mountants, and do not contain any secret ingredients.

These notes address conventional specimen preparation for higher plant tissues. While in many cases freeze-substitution has been shown to be a superior method, the equipment required is not generally available in most electron microscopic laboratories, Thus, the more conventional procedures outlined herein have been found to work satisfactorily for most leaf material, and have been most successful in the fixation and embedment of small root slices (from near the root tip), An adjustment of the osmotic pressure may be necessary if the plant material used is highly vacuolated and if there is an exceptionally high water content.

Immunohistochemical analysis was required to identity lymphoid cell subtypes in mouse appendage joint synovium tissue. Hamster and rat monoclonal antibodies directed toward lymphoid cell subtypes were found to be nonreactive in formalin-fixed decalcified appendages, and this necessitated the use of cryostat sections for immunohistochemical procedures. Initial attempts to section mouse joints using conventional cryostat techniques did not yield adequate sections, and it was clear that a different approach was necessary.

I use immunocytochemical detection schemes that employ the use of primary and secondary antibodies for either tissue sections or whole mount preparations in order to study the in situ distribution of proteins and labeled nucleic acids. As the antibodies are either laboriously generated, or represent costly purchases. I have sought a protocol that minimizes the volume of antibody solution needed. Additionally I need a protocol that involves a quick, thorough, and gentle application and removal of the antibody solutions (mm the specimen with no danger of the specimen drying out during the one or two hour incubations. After trying many various schemes 1 have settled into the use of a procedure which very effectively retains the antibody solution on the specimen without damaging the delicate sections or whole mounts, and which is easy to add and remove.

The underlying mechanisms whereby cells sense and respond to external stimuli are not well understood, but for some time it has been known that the extracellular matrix has an influence on the behavior of cells. For example, changing the flexibility and/or adhesiveness of the matrix can change the shape of cells, and determine whether or not they continue to divide or begin to differentiate. Recently, in a series of elegant experiments, a group of cell biologists in Boston have demonstrated the presence of an interconnected molecular network pervading the entire living cell, extending from its outer surroundings into the genes in its nucleus.

Ready or not, digital imaging in the microscope lab Is here to stay. While I am one of those fogies who insist that digital images are nowhere near as good as photographic prints, I've also bowed to the inevitable, and am beginning to find ways to assist the clients in our multi-user facility with their digital imaging needs, We can acquire digital images from our FESEM, from our light microscope with a CCD, and from a flatbed scanner. Clients use their images for image analysis, transport to others scientists via the Internet or World Wide Web, for reports, slides, overhead transparencies, and publication, Generally, these images need to be manipulated for use, just as one would manipulate a photographic print to correct any deficiencies in exposure or area of view. Adobe PhotoShop is the software of choice for image adjustment, accounting for more than 80% of the digital image-editing software market.

Hexamethyltiisilizane (HMDS) is an excellent method of chemical drying of hydrated specimens. There are several variables involved in its use, the most easily controlled being the number of transitional steps from 100% eihanol (EtOH) to 100% HMDS and the drying temperature.

Fixation and dehydration are the same for both HMDS and CPD. Once thespecimenisinthefinal 100% ethanol, it must then be transferred to 100% HMDS through a graded series of ethanol-HMDS mixtures.