The expanding field of cryobiology, in particular that of the study of vitrification for long term storage of tissues for transplantation, has demonstrated that ice is damaging to smooth muscle tissue. Consequently conventional methods such as ultrarapid freezing and freeze substitution are becoming routine protocols to determine the quality of cryopreservation. This article introduces the scientific community to the PS-1000 cryofixation unit (Delaware Diamond Knives, Wilmington, DE) which provides both ultrarapid freezing and a means of validation of freeze substitution methods. When any tissue is frozen or vitrif ed for clinical use it is imperative to know the structural and functional integrity of these tissues. Ice formation within the extracellular matrix and dehydration of multi-cellular tissues, using conventional cryopreservation, is the principal reason why these methods frequently prove to be ineffective.

The rising demand in the semiconductor industry for higher spatial resolution in the analysis of device defects has focused attention on the use of transmission electron microscopy (TEM). However, conventional TEM sample preparation can be difficult and time-consuming, and, depending on the operator, may result in a low yield of quality specimens. One solution to this problem is the use of focused ion beam (FIB) milling for the final stage of TEM sample preparation. However, specimens have to be mechanically thinned prior to FIB and the need to characterize specific devices requires a pre-FIB preparation method that can target specific features on the wafer. We will discuss an innovative and automated solution that isolates specific devices and prepares TEM specimens for subsequent FIB thinning. The complete pre-FIB preparation takes less than 30 minutes and yields a sample in which the targeted feature is positioned a specific distance from the sample edge, thereby minimizing final FIB milling time. The output specimen is automatically packaged for FIB milling and TEM analysis. We also present drawings of the process flow and examples showing TEM results from tungsten filled vias.

To successfully isolate 1-100 μm samples for microscopical examination or analysis, one needs a good set of microtools, with the most essential being the needle and the micropipet. This paper will describe how to make them and how they are used to solve various sample preparation problems.

A significant percentage of technical experts who employ optical microscopes have had little or no formal training in optical microscope basics. Some, typically, were required to use microscopes during their technical education but, in general, microscope terminology and technology was a sideline to their major training. As a result, many useful basic microscope technical details were not learned because they were not necessary to accomplish what was needed in order to survive their major class work. At Florida State University, we try to make the [earning of microscope technology an inherent part of the students training. An important part of this training is this compendium of 50 of the most frequently asked questions about Optical Microscopy.

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.

The recordable compact disc (CDR) is a popular and inexpensive medium for storing and disseminating digital information, such as micrographs and scanned images. This technical note describes unusual degradation that rendered a group of discs unreadable. The discs were purchased as a set. Each disc was packaged in a jewel box and wrapped in clear plastic. Several of the discs were used within weeks of purchase to store data, which included digital micrographs.

This article is the third of a series transcribed from the discussion taped during the Facility Management session at M&M 2000. it is printed with permission of the Microscopy Society of America, Bulleted paragraphs indicate comments by individuals attending the session.

D. Sherman: I am sure that there are many different schemes to try to recoup some costs in facilities we manage. Probably very few of us are really happy with the schemes we have at present. Hopefully this discussion will provide us with information that will be useful in refining the financial management of our different facilities.

Why is it that tip convolution effects rotate with the scan angle in a Nanoscope-based AFM (and possibly other systems)? The tip certainly doesn't rotate in its holder as the scan angle is changed! I posed this question on the SPM mailing list, and got several answers in return, but I was still puzzled until I put all of them together and thought a while. However, I may not be the only puzzled soul out there, so let me attempt to summarize what I've come to understand:

A good way to think about this is to consider a righttriangle tip approaching a cube on a surface:

If the tip approaches the cube along the plane of the page screen, the left side of the cube will appear sharp in an AFM image and the right side of the cube will have a slant introduced into it due to a tip convolution effect.

The precursor of today's light microscope was invented probably in the Netherlands about 1600. In effect it previously involved a two lens system - objective and the eyepiece. As such it was termed a compound microscope. It differed from the simple one-lens microscope of Anthony Leeuwenhoek in the 1600's. The image was so bad, however, that most microscopists preferred to use the simple one-lens microscope. Leeuwenhoek, with his one-lens microscope, magnifying up to 280X, discovered foraminifera and many other “wee beasties”, even bacteria and spermatozoa.

There was a spate of posts concerning the production and collection of ultrathin serial sections on the microscopy listserver, so I wanted to draw the microscopy community's attention to these instructions from an unfortunately obscure microscopy book. “To obtain quality serial sections for electron microscopy the following procedure must be followed for consistent results.

Microscopists have continually developed new techniques for enhancing specimen contrast since Zacharius first built the first compound light microscope around 1595. His simple two lens microscope became the basis for the evolution of light microscopes until 1828. That date is when the first polarizing light microscope was invented by Nichols. Since then, it has served as the building block for most research microscopes that are fitted for accessories made to enhance contrast or measure specific optical properties.

Julius Rheinberg devised a simple contrast enhancement method in 1896. He published his findings in the journal of the Royal Microscopical Society for the benefit of others. Today, Rheinberg illumination (RHI) has given way to phase contrast, differential interference contrast, confocal, laser microscopy, scanning probe and atomic force microscopy. The latter four are very recent technological advances.

Most objects in our world are 3-dimensionai (3-D), and this is certainly true of cellular ultrastructure. The challenge in microscopy has always been how to analyze this 3-D information. Traditional thin-section microscopy has a minimal ability to view 3-D structure, because it is limited to viewing thin, almost twodimensional (2-D) planes taken from the specimen. The development of high and intermediate voltage electron microscopes (HVEM and IVEM) provides the ability to investigate the ultrastructure of thick biological samples—allowing a unique view of the 3-D interrelationships of cells and organelles.

After an inquiry from the Microscopy Listserver, I went back to my 1980 copy of Scanning Electron Microscopy, volume I. Several authors had investigated the structure of thin metal films by depositing the films onto carbon-film-covered TEM grids and imaging the films at high magnification. There were several proposals for new devices that have since become standards for high-resolution coaters, but the Listserver inquiry was for a fine conducting film suitabie for high-resolution SEM from an existing sputter coater.There were several factors studied that influenced the fine structure of the films. The first was the materials sputtered: for a given set of conditions of voltage, current and time, platinum gave the finest film, 60% gold-40% palladium (Au/Pd) the next finest and pure gold the least fine.

Several summers ago, while working at the Marine Biological Laboratory in Woods Hole, Massachusetts, George Augustine (Duke University) and I wished to look at the circulation in the giant synapse of the squid, Loligo pealei. At that time, Dr. Eugene Copeland suggested the use of India ink in order to see the circulatory system. The ink contains carbon particles that are visible under the EM. In addition, it is visible to the naked eye.

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.

Traditionally the field of microscopy has centered on the creation of highly magnified images of things too small to be seen with the eye alone. This was certainly the case with scanning probe microscopy, which burst on to the scene some 15 years ago with dramatic images of individual atoms. We are visual beings and the rapid acceptance of SPM followed directly from its ability to represent physical interactions in an intuitive image format. However, much of the vaiue of SPM today derives from its ability detect and measure a wide variety of specific interactions between its scanning probe and the sample. As the technology continues to mature, emphasis is shifting from its ability to display these signals as images to its ability to quantify and measure them.

Fluorescence microscopy has proved to be an invaluable tool for biomedical science since it is possible to visualise small quantities of labelled materials (such as intracellular ions and proteins) in both fixed and living cells. However, the conventional wide field fluorescence microscope suffers from the disadvantage that objects outside the focal plane also fluoresce (in response to the excitation light) and this leads to a marked loss of contrast for objects in the focal plane. This is especially a problem when the fluorescent probe is distributed throughout the thickness of the cell and the cell is thicker than about 1 μm. The confocal microscope overcomes this problem by illuminating the preparation with a point source of excitation light and limiting the collection of light with a pinhole that is confocal with the illumination source. This converts the microscope from an imaging system to a point detector and images are produced by scanning the illuminating and detecting point over the specimen to build an image (in much the same way that a television set produces an image). The basic idea behind the confocal system is shown in Figure 1, and it should be noted that light from points outside the focal plane is defocused at the pinhole and so does not pass through the pinhole efficiently.

This article is the final of a 4-part series transcribed from the discussion taped during the Facility Management session at M&M 2000. It is printed with permission of the Microscopy Society of America. Bulleted paragraphs indicate comments by individuals attending the session.

To get back to billing and cost recovery, one of the problems we continually face in our lab is that people can come through the door with any kind of problem under the face of the earth. We are wide open to any sort of EM research. This entails an awful lot of protocol development.

Using illustrations from published technical articles is part of many lectures and presentation citations, and given the availability of flat bed scanners, should be easy to accomplish. The problem that remains is removing halftone patterns and other periodic noise that result from printing and scanning technology. Practically all magazines and newspapers are printed using a regular halftone pattern that uses an array of dots varied in size to produce the visual illusion of continuous gray scale. Color images are usually printed with three, four or even more such patterns using different colored inks and different pattern orientations. Scanning such images into a computer can introduce a further pattern due to the moire interference between the printed pattern and the spacing of the sensors in the scanner. Such patterns are also characteristic of images obtained from single-chip video cameras because of the color filters present in front of the light sensors on the chip.

Over the several years that imaging energy filters have been available commercially, numerous and wide-ranging applications have demonstrated elemental mapping with a resolution approaching 1 nm. A few reports have even shown resolutions <0.4 nm. Elemental mapping by energy-filtered transmission electron microscopy (Eftem) is clearly an attractive and powerful tool, but some aspects of the techniques can be complex, with many pitfalls awaiting the unwary. This tutorial aims to cover some practical aspects of elemental mapping by Eftem. It is based largely on the author's work at the ORNL Share User Facility, where Eftem research has been performed since 1994 with a Gatan imaging filter (GIF) interfaced to a Philips CM30T operated at 300 kV with a LaB6 cathode. Most of the applications have been to metals and ceramics, emphasizing interfacial segregation and precipitation.