For several months there have been recurring threads on microscopy-related internet listservers concerning health and safety issues that have troubled us greatly at Anatech Ltd. The general theme has been to make light of toxicity claims, provide anecdotal comments about having survived many decades of working in labs, and giving the impression that current health and safety regulations are at best a pain and probably are the cunning products of misguided governmental agencies.

The great hazard of formaldehyde is not its proven carcinogenicity. Anyone interested in formaldehyde's effects on humans should read the preamble to OSHA's Formaldehyde Standard (Federal Register 52(233):46168-46312; December 4, 1987). According to studies cited therein, formaldehyde has been directly implicated in causing tumors in the lungs, nasoand oro-pharynx and nasal passages of humans occupationally exposed to levels of formaldehyde not unlike conditions existing in histology laboratories a few decades ago. Repeated and prolonged exposure increases the risk.

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.

This article is the first of a series transcribed from the discussion taped during this session, it is printed with permission of the Microscopy Society of America. Bulleted paragraphs indicate comments by individuals attending the session. Additional discussion topics will be highlighted in future publications.

There appears to be some confusion as to how materials are impregnated or dried. Without getting into the exciting mathematical details best suited for persons who second language is Greek, I offer the following short explanation as to how materials are impregnated or dried.

If the object is porous, the porosity is described in terms of macropores (>50 nm), mesopores (50 - 2 nm), and micropores (2 - 0.8 nm). These three size classes of pores represent different flow mechanisms for the impregnation of porous materials. Solvent penetration of a non-porous particle by imbibing is similar mathematically to solvent penetration of micropores although the actual mechanism is quite different.

To those scientists and researchers who use microscopes with video or digital cameras for the purpose of capture and display, there has always existed a fundamental problem, namely, imaging large tissue samples at high resolution.

For some time, conventional technology has allowed video cameras to be attached to microscopes. The signal generated by these cameras can be captured and saved to a computer's hard drive using a frame grabber. A typical image capture and display resolution is 640 by 480 (307,200) pixels. Although the image produced is ‘good’, it is by no means considered ‘high resolution’. For the sake of discussion, ‘high resolution’ is defined as greater than 1,000 x 1,000 (1,000,000) pixels. High resolution images are essential for medical applications. Today's modern digital cameras can meet or exceed this value. The human eye, if compared to these examples, has the equivalent resolution of greater than 5,000 x 5,000 (25,000,000) pixels.

The genus Cryptantha Lehm ex G. Don section Oreocarya (E. Greene) Payson of the family Boraginaceae presents some problems to botanists, both professional and amateur, in the keying and identification of species. The genus contains approximately 150 species, the section about 60 perennial and biennial herbs located generally in western North America. Identification has presented some taxonomic difficulty due to the variability and lack of distinctive vegetative characters. Botanists have turned to the nutlet (fruit) and flower morphology to aid in identification for precise specific differentiation. In the past, 10X magnification and a decent botanical illustrator were required to provide the illustrations necessary to assist in this identification. We are in the process of collecting micrographs of nutlets (dorsal, sagital, and ventral views) and developing a webpage containing these micrographs along with descriptions of their morphological variations.

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. The Al chuck can usually be removed by hand, but a flathead screwdriver used as a pry will help pop it off if you have trouble.

Bugscope is a second generation educational project in the World Wide Laboratory that provides web browser based control of scientific imaging instrumentation using the Internet. We had previously demonstrated web based remote access to sophisticated scientific imaging systems several years ago in the Chickscope project. The primary goal of the Bugscope project is to demonstrate that relatively low cost, sustainable access to an environmental scanning electron microscope (ESEM) can be made available to K-12 classrooms.

Electron microscopy is clearly the best way to look at enteric viruses, many of which do not grow in tissue culture, and those that can be so coaxed, do so under special conditions that are not routinely found in the culture lab. Biochemical identification (e.g., immunological kits, PCR, Western Blots) require a specific reagent to recognize the virus, and if the right reagent is not used, the viruses will be missed (e.g., if you run a test for rotavirus, you will miss adenovirus, etc.). Furthermore, there are not biochemical reagents for all viruses. With electron microscopy, one can see a wide variety of viruses by doing a negative stain of an aqueous extract of stool.

It has become increasingly obvious that infrared microspectroscopy can be the analysis tool of choice when determining the chemical composition of biological and biomedical samples. Frequently, fluorescence illumination is required for sample characterization, which previously required the use of a separate optical microscope. There has also been a need in the semiconductor manufacturing industry for a single tool for visualizing particle contaminants on integrated wafers as well as the ability to chemically determine their nature. There is now a single microscope platform for conducting rapid Nomarski differential interference contrast and fluorescence illumination sample visualization as well as infrared analysis. This novel infrared microscope has applicability to many fields of investigation, including pharmacology, forensics, cell biology, histology, gemology, and geology.

The semiconductor industry is committed to introducing copper interconnects in place of aluminum for the latest generation of semiconductor devices. With its greater current carrying capacity, the use of copper should enable further reductions in device geometry, power consumption and heat generation, and lead to higher performance and longer battery life in portable devices.

Grain orientations, crystal log raphic ‘texture’ trends and boundary types are crucial in determining properties such as electrical resistively, strength and corrosion resistance. These parameters may be implicated both in the performance of the device and in failure mechanisms, which are of great importance in determining reliability.

Our work demonstrates that high-resolution electron backscatter diffraction (EBSD) is now both possible and routinely achievable using both hot and cold field emission gun scanning electron microscopes (FEGSEMs). Our results demonstrate that EBSD can be successfully used to map grain orientation and to reveal and classify grain boundaries for this application.

Monte Carlo simulation of electron scattering in solids has proven valuable to electron microscopists for many years. The electron trajectories, x-ray generation volumes, and scattered electron signals produced by these simulations are used in quantitative x-ray microanalysis, image interpretation, experimental design, and hypothesis testing. Unfortunately, these simulations are often computationally expensive, especially when used to simulate an image or survey a multidimensional region of parameter space.

Here we present techniques for performing Monte Carlo simulations in parallel on a cluster of existing desktop computers. The simulation of multiple, independent electron trajectories in a sample and the collateral calculation of detected xray and electron signals fall into a class of computational problems termed “embarrassingly parallel”, since no information needs to be exchanged between parallel threads of execution during the calculation.

A scanning chemical microscope utilizing a flat pH sensor has proven effective for analyzing a number of biological specimens including human teeth surfaces, plant leafs, and rice.

The key to this microscope, the “pH imaging sensor”, is an electrochemical semiconductor that can be made to function as an array of point sensors when scanned by backside laser illumination. Measured pH values at each measurement point can be processed into a pH image. So far a resolution of 5 μm with a 0.01 pH sensitivity has been attained by this imaging technology, although most images are made using 100 μm resolution. The time to acquire an image is, of course, dependent on the size of the measured area. For example, a 6.4 mm x 6.4 mm image with a resolution of 100 urn can be acquired in 25 s. Linear pH profiles can also be obtained at a speed of 1 cm/s.

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 al., described a parallel study comparing the electron microscopic results of fixation with paraformaidehyde 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. The fixative containing formaldehyde alone does not need to be refrigerated and is stable for months; whereas, the formaldehyde-glutaraldehyde mixture should be refrigerated.

Sections of formalin-fixed and paraffin-embedded tissues are commonly used to detect antigens of diagnostic, therapeutic, or prognostic importance in patients with many types of cancers. Sections (- 5 μm thick) cut from paraffin-embedded tissues are mounted on glass slides usually one or two days before immunostaining. In some cases, paraffin blocks are no longer available, and so immunohistochemistry must be performed on unstained slides that have been prepared sometime ago. it is not uncommon in both clinical and research laboratories to store unstained paraffin-embedded sections on glass slides at room temperature. Also, research laboratories that serve as reference centers for multi-institutional studies store slides for future use in order to rapidly return the tissue blocks to the originating institution. Such institutions want tissue blocks returned to them as soon as possible, as this may have medicolegal implications.

Storage of sections also becomes necessary when one marker study is proceeding and future marker studies need to be followed.

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.

On the Microscopy ListServer, a thread came up about problems with safety when loading cold traps from ladders. Working from a ladder is not a very safe proposition and being up there with a Dewar full of liquid nitrogen brings the safety officer to attention.

An alternative to trying to climb a ladder with a Dewar flask and pour the liquid nitrogen into the trap would be to have a pump for the liquid nitrogen in a Dewar flask on the ground that could be controlled from the top of a ladder.

In my research, I am interested in understanding cellular morphogenetic events in early somitic myogenesis in the chicken embryo, and much of this work involves viewing fluorescently labeled cells in embryonic tissues. For this work, I am using 0.2% (weight/volume in 100% tetraglycol (Sigma)) concentrations of dil (1,1', di-octadecyl-3,3,3',3',-tetramethylindocarbocyanine perchlorate) and diO (3, 3'-dioctadecyloxacarbocyanine perchlorate) to focally label small areas (approximately 10 cells) of the epithelial somite known as the dermomyotome.

This effect can be understood by considering what happens to the cantilever and tip as the base of the vibrating cantilever is brought closer to the sample (like in a force curve). When the vibrating cantilever first approaches the surface, the tip senses attractive forces. In standard imaging conditions, these are usually due to meniscus forces between a layer of adsorbed liquid (often water) on the surface of the sample and the tip. However, even in clean conditions, other attractive forces like Van der Waals will generally be present. These attractive forces move the resonance of the cantilever downward in frequency. Since the cantilever is being driven at the original resonance frequency of the cantilever, this will cause the cantilever amplitude to decrease as the attractive forces puli the resonance peak out from under the drive frequency. This decrease is often quite linear with distance, but doesn't have to be. In this case the tip is actually turning around some distance (this could typically be a few nanometers) before it touches the surface. This regime is commonly called the “attractive” regime because attractive forces dominate the behavior of the cantilever.

A very basic point that is almost always overlooked when discussing SEM “resolution,” is the difference between “resolution” and “resolving power.”

An SEM might have a resolving power of 1 nm or so based on the observation of gaps between gold particles on a featureless carbon film. Fine—if ones job is to look at gold on C.

For the actual 3-D samples most everyone looks at, EVERY micrograph has a DIFFERENT resolution, closer to 10 nm, because of secondaries generated by back scattered electrons, edge effects, …etc. If you want resolutions closer to the instrument's resolving power you have to look at thin TEM specimens without sharp edges and angles.

In our semiconductor lab we have several FESEMs advertised to have resolutions of 1 nm that can not resolve 4 and 5 nm layers in a film stack.