The atomic force microscope (AFM), the workhorse of scanning probe microscopes, has become even more versatile. Anneliese Raab, Wenhai Han, Dirk Badt, Sandra Smith-Gill, Stuart Lindsay, Hansgeorg Schindler, and Peter Hinterdorfer have demonstrated that the AFM, in the dynamic force mode, can use antibodies as a probe. Dynamic force microscopy uses a magnetized tip that is oscillated in an alternating magnetic field as the tip scans the surface. This provides a very gentile interaction that can be recorded as a high resolution topographic image. Raab et al., showed that more information can be obtained from the specimen.

We have recently been shopping for a confocal microscope and have found a simple way to test the capabilities of the various units that have been demonstrated to us. Some of the important aspects that we were looking for in our “ideal” multi-user system were:

1. 1) Ease of use.

2. 2) The ability to obtain good X-Y, X-Z and hence X-Y-Z resolution, and the accurate display of these images.

3. 3) The depth within the sample at which the system can resolve structures (this of course depends mostly on the lenses used), but is still an important criteria as it reflects on the system as a whole.

4. 4) “extras” such as a transmission detector and reconstruction/ analysis/image manipulation software.

Fluorescence is a property of certain fluorophores, which allows them to absorb light at one wavelength and emit the light at another, longer wavelength. This fluorescence can be used as a tool to study the interactions of numerous components within a cell. With the use of fluorescent probes, proteins or other cellular matter can be specifically labeled in a fixed or live cell. This allows scientists to closely monitor microscopic cellular functions in order to gain a greater understanding of living biological processes.

Fluorescence Resonance Energy Transfer (FRET) imaging microscopy is an important and reliable tool for studying minute molecular processes in space and time within a single cell. FRET spectroscopy is a better method for studying the structure and localization of proteins in physiological conditions than are X-ray diffraction, nuclear magnetic resonance, and electron microscopy.

Calcium (Ca++) and other diffusible ions cannot be preserved for x-ray microanalysis if specimens have been preserved by fixation using formaldehyde, glutaraldehyde or other fixatives. Atoms, such as Ca, are not diffusible when they are bound to something insoluble, such as bone or hard granules in cells which are sometimes mechanisms for detoxifying. Ions, loosely bound elements and other components such as sugars and amino acids are leached out during chemical fixation and dehydration by solvents. This problem was recognized over a hundred years ago by Richard Altmann. An alternative to wet chemical processing is the purely physical method that involves rapidly freezing the specimen and then removing the frozen water by the process of freeze drying, which is the subject of this article.

Since the development of ratiometric fluorescent calcium indicators like Fura-2 and lndo-1 in the mid-eighties, calcium imaging has become a major approach to studying life cell physiology. The ability to calibrate the spectral properties of the dye as a function of the calcium concentration led to measurements of nearly absolute intracellular calcium concentrations in a large variety of preparations. However, the use of ratiometric calcium indicators in confocal calcium imaging is limited, since the dyes are excited by UV light while the lasers of most confocal microscopes provide visible light only. In addition, high energy UV light may cause rapid dye bleaching and photodamage. Thus it appears advantageous to reduce the illumination of the preparation to the minimum possible minimum. The most frequently used dual-excitation dye Fura-2 requires rapidly alternating excitation at 340 ran and 380 nm, respectively, to permit ratiometric calcium measurements with a maximal efficacy.

Infusion with buffer/sucrose solutions (up to 30% sucrose) has long been used to ‘cryoprotect’ tissue in an attempt to prevent ice crystal artifact in frozen sections. This is helpful for example in sectioning large, fixed tissue blocks that must be frozen relatively slowly in dry ice to allow sectioning on a sliding microtome. In 1977, De Olmos added ethylene glycol to the mixture (30 g cane sugar/50 ml 0.1 M PO4 buffer at pH 7.2 in 20 ml ethylene glycoi) for -10°C storage of free floating sections from lightly fixed primate brain. Jones and Kane in 78 used this solution for storage of sections at -20°C (standard household freezer temperature) for up to one month before horseradish peroxidase histochemical reaction, in their methods, they cautioned that "sucrose attracts insects" (ants, personal communication). We and others have found the above cryoprotectant solution generally useful for storage of free floating sections from fixed brain. We observed that adjacent sections stored at 4°C in buffer for 2 weeks (common practice for Nissl stained sections) lost their reactivity to antibody labelling.

It is well known that biopsy, surgical and postmortem tissues fixed in formaiin and embedded in paraffin require antigen retrieval for diagnostic pathology. Various methods, including heating, enzymatic digestion, ultrasonication, and detergents, are in use for achieving antigen retrieval. A combination of these methods is also employed. However, the most effective methodology for antigen retrieval in a wide variety of tissues involves heating at high temperature using a microwave oven, an autoclave, a pressure cooker, or a hot plate. Among these treatments, heating the tissue sections (4 urn thick) in 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven is the most widely used technique for antigen retrieval. To obtain efficient and reproducible antigen retrieval, the microwave oven must be used carefully and optimally. The following steps are recommended to achieve these goals (Hayat, 2000 a, b).

The causes of most hazards encountered in using a microwave oven are straight forward and can be avoided by taking necessary precautions. Higher power settings and longer durations of heating than optimal for a given study should be avoided. Because overheating is not uncommon, the time setting should be checked.

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.

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.

Since Blum discovered its hardening properties in 1893, formaldehyde has become the most widely used fixative in the world for specimens to be examined by light microscopy. However, since most commercial preparations of formaldehyde contain methanol, a protein precipitant, formaldehyde has been considered an unsatisfactory fixative for tissues to be examined by electron microscopy.In 1973, Carson et a/, described a parallel study comparing the electron microscopic results of fixation with paraformaldehyde vs. formaldehyde. They found that there was no difference in the preservation of ultrastructural morphology provided that the buffer systems were identical. In 1976, McDowell and Trump described a fixative combining commercial formaldehyde and glutaraldehyde (4CF-1G). Both of these fixatives are dual purpose fixatives and preclude the selection of tissue for electron microscopy prior to fixation. They can both be prepared in large quantities and used for routine surgical specimens.

DRS Hadland, formerly Hadland Photonics, has supplied the Department of Measurement, Control and Microtechnology at the University of Ulm with an ultra-high speed digital imaging system to help them gain a greater understanding of the dynamics of moving parts in microdevices.

The measurement of position, velocity and acceleration of moving parts in microdevices is of great interest in the rapidly growing field of microsystem technology in order to solve the problems needed to allow evolution from laboratory use into industrial mass production. Such research is targeted towards gaining a better understanding of the dynamics of the moving parts in microdevices.

For the visual investigation of dynamics in microdevices high optical magnification is necessary because the dimensions of microsystem components are very small. Therefore, velocities of parts of such microdevices appear to be extremely fast and time constraints are very small.