Radiolaria are marine protozoa that have thrived in the world's oceans for millions of years. They are particularly unique among marine plankton in that they build silica skeletons, which have allowed them to be preserved in the fossil record. These skeletons are ornate and complex and often demonstrate perfect geometric form and symmetry. The complex and beautiful glass-like structures are visually interesting when examined with electron microscopy. These attributes, coupled with their availability, size, ease of mounting and preparation make them superb specimens for introducing students to the use of electron microscopy (EM).

Fading of fluorochrome is a significant limitation to fluorescence microscopy. Several anti-fade agents, e.g. n-propyl gallate, are commonly used for glycerol-based mounting media (Longin et al., 1993; Ono et al., 2001). Samples mounted in glycerol must be kept at -20°C for long-term storage to prevent bacterial degradation. In contrast, fluorescent samples cleared and mounted in organic media can be stored indefinitely at room temperature.

Methyl salicylate or oil of wintergreen is an excellent clearing agent (refractive index = 1.53), which works well with a variety of fluorochromes. It has a pleasant aroma but is somewhat difficult to work with since it remains liquid after mounting. It was previously reported that shrimp embryos labeled with tubulin antibody and rhodamine-conjugated secondary antibody maintained their fluorescence after six months (Summers et al. 1993). These same samples, stained in November, 1990 and imaged by confocal microscopy for publication in Hertzler and Clark (1992), are still fluorescent after continuous storage in methyl salicylate at room temperature in the dark (Figure 1). The images of 62-cell stage shrimp embryos taken from these 1990 samples were collected with an Olympus Fluoview 300 laser scanning confocal microscope in January, 2006 in the Dept. of Biology, Central Michigan University.

Freezing damages cell membranes and reduces the histological readability of biological specimens. This is a discussion of why, the consequences, and how to minimize or avoid the damage introduced when using freezing as the means to harden biological tissue in order to cut thin sections for histology.

Pure water can exist in the solid state in three forms. Two forms are crystalline: one with a hexagonal lattice and the other with a cubic lattice. The third form capitalizes on the fact that water can be frozen so rapidly that it does not have time to form a crystal lattice and remains amorphous (vitreous form). Crystal formation is responsible for the expansion of water as it freezes (a property unique of water), which creates specimen preparation artifacts. Vitreous ice does not expand upon solidification. This makes it the only desirable form of ice appropriate for biological specimens.

For years, I have used a hybrid Epon-analog resin to embed in culture dishes. I use a standard Epon formula but utilize the following components: LX-112 and DMP-30 from Ladd Research Industries DDSA and NMA from Electron Microscopy Sciences I know it seems weird, but years ago I tried all sorts things, from the “straight” formulations from each vendor to a bunch of mixtures. This one has never reacted with the plastic of the dishes. Here is how I do the actual embedding of the cell monolayers in the dishes:

1. 1) After the last 100% ethanol, remove the alcohol and cover the bottom of the well with a layer of resin mixture that is about 2 mm deep.

2. 2) Insert embedding tubes that are made by cutting the pyramidal bottoms off of BEEM capsules (just slice them with a fresh razor blade and be sure to insert them so that the manufactured end rather than the cut one is sitting against the dish).

3. 3) After inserting labels into the tubes, put them into the oven at 60° overnight.

4. 4) In the morning, fill just the embedding tubes and return everything to the oven again to finish polymerizing.

5. 5) When the resin is cured, grab the tubes with a pair of needlenosed pliers and snap them out. Sometimes a bit of the bottom of the dish comes away with the block, but often a very smooth block face results. If some of the dish comes up, it is easy to see under a dissecting microscope, and the dish portion comes away easily when trimming the block face.

I often cut away part of the block face with a jeweler's saw, either to keep it in reserve or to re-embed it in order to get cross sections, and then trim the rest into a narrow rectangle.

It is often said that: “…a picture is worth a thousand words… ” This can be certainly true for images from an Atomic Force Microscope.

Although we often know that nanoparticles exist and we have a mental picture of what they look like, it is often helpful to be able to “see” the particles to verify that they are morphologically what we imagine they are. When the particles are much less than a micron in diameter, they cannot be directly visualized with an optical microscope. At best, sub micron particles appear as “diffraction spots” in an optical microscope. It is possible to visualize nanoparticles with SEM or TEM.

Jan Hinsch wrote an excellent review article “About the Use of Digital Single Lens Reflex Cameras on Microscopes” in 2004 for the web journal Modern Microscopy, http://www.modernmicroscopy.com/main.asp?article=33&print=true. The purpose of this article is to demonstrate the capabilities of the new Olympus Evolt E-330 DSLR for use with older design microscopes to capture the full diagonal of an 18 mm field number eyepiece that is not high eyepoint. This camera can be fitted with OM mount film camera lenses using the Olympus MF-1 adapter. A key requirement I had before purchasing a DSLR was that critical focusing must be possible at the full resolution of the sensor. I asked the Olympus Precision Instruments Group whether Olympus would have a DSLR available with this capability, and learned from them that the new E-330 DSLR would allow a selected area to be displayed at full resolution from the main CMOS sensor for critical focusing. This 7.5 megapixel sensor measures 13 by 17 mm.

The stereo dissecting microscope is a widely used instrument for macro-structure observation and documentation. The emergence of digital imaging, along with sophisticated imaging software, makes this macro-imaging more efficient. It also makes possible certain special imaging modes that are difficult to accomplish with traditional (film) imaging, such as extended depth of focus imaging ( EDF), automatic mont aging, etc. These special imaging techniques often require dedicated hardware on the microscope, such as a motorized stage.

A challenge was presented to us to find a way to support Drosophila melanogaster so that both the anterior and posterior scutellars could be easily visualized under a dissecting microscope and photographed for purpose of measurement. The resulting solution was to utilize a standard grid box with the lid removed. Several holes were filled with Gary's Magic Mountant to just above the rim of the hole. Freshly dead fruitflies were gently lifted with forceps. Then each was placed into a filled hole and both the head and abdomen were pushed into the mountant. The wings were arranged on the plastic on either side of the hole as seen in the micrographs.

The best results were obtained if the body of the fly was evenly covered with Gary's Magic Mountant and if the photography was accomplished within a few hours of the death of the fly.

The use of methacrylate monomers for embedding has a venerable history in microscopy. Many formulations have been developed over the years for various purposes, ranging from standard TEM observations to low-temperature embedding. Key parameters include the length of the hydrocarbon chain and the presence and kind of cross linking reagent. In the mixture of butyl and methyl methacrylate (BMM) described here, the monomers are relatively short-chained and there is no cross linker at all. This gives the polymerized material a softness that makes it rather unsuitable for TEM, but on the contrary allows the embedment to be removed after sectioning by a brief incubation in acetone. The latter property is good for immunocytochemistry because loss of the embedment means greater access for the antibody to the antigen. BMM generally preserves structure better than paraffin or glycol-methacrylate, and for this reason is a useful choice for light-level immunocytochemistry, particularly when sub-cellular resolution is desired.

As all microscopists are taught, resolution can be measured by knowing the closest distance between two points that can be discriminated in the image. There are some occasions when we want to measure something in a digital image, and the nominal resolution isn't sufficient for the task. One of these, extracting a contour from the image, was summarized in a previous research report. The principles would be valid for any filled area, such as a particle or structure from an X-ray dot map. The ground rule was set that the outermost pixel would be selected at every point on the contour. It was clear from the geometry shown in Fig. 1 that only values of 0°, 45°, 90°, 135° and 180° could be measured. Because of the stair step defect, tracing the sequence of pixel locations only gave offsets to 8 pixels, one directly below, two on the corners below, three pixels in the same location above, and one pixel on either side of the subject pixel. Therefore it was impossible to measure the true curvature of the contour.

Nano-TA is a local thermal analysis technique that combines the high spatial resolution imaging capabilities of atomic force microscopy (AFM) with the ability to obtain understanding of the thermal behaviour of materials with a spatial resolution of sub-100nm. This breakthrough in spatial resolution of thermal analysis, which is ~50× better than previously reported, has profound implications for the fields of polymers and pharmaceuticals where local thermal understanding is key.

The conventional AFM tip is replaced by a special nano-TA probe that has an embedded miniature heater and is controlled by the specially designed nano-TA hardware and software. The nano-TA probe enables a surface to be visualised at nanoscale resolution using the routine imaging modes of AFM. The user is able to select the spatial locations for the investigation of the thermal properties of the surface. Heat is supplied locally via the probe tip and the thermomechanical response is measured.

Traditionally, contamination control in SEMs has focused on pump oils, finger prints, dirty specimens, and good vacuum practice in manufacturing. Now, the use of dry pumps at all stages of the vacuum system of new FE SEMs, and the use of better vacuum practices on the part on users and manufacturers have made environmental hydrocarbons, the hydrocarbon background contamination of our world, a significant source of the remaining hydrocarbons in electron microscope vacuum systems. These environment sources of hydrocarbons (HC) cause a loss of resolution and contrast in imaging at the highest levels of magnification.

In a recent paper on the revised formulation of Spurr low viscosity embedding medium with ERL 4221 the importance of maintaining an appropriate anhydride:epoxide (A:E) ratio was discussed. By understanding a few simple concepts about epoxy resin formulations and setting up a formulation table it is possible to create new resin mixtures with good sectioning properties and other desirable properties such as decreased viscosity and increased beam stability.

Before starting a formulation you need to know the molecular weight of the anhydride and the WPE (weight per epoxide equivalent) of the epoxy resin component. The molecular weights and WPEs are usually printed on the bottle or can be obtained from the vendor. An A:E ratio of 0.7:1.0 -1.0:1.0 is used for most biological specimens. Increasing the A:E ratio results in a harder block; decreasing the A:E ratio results in a softer block. Table 1 shows a basic formulation spreadsheet where the molecular weights of the anhydrides and the WPEs of the epoxy resin components can be entered. The A:E ratio is entered under the anhydride for the molar ratio and the molar ratios of the epoxy components are entered under the epoxy components. The calculations are done as shown in each column and row.

Depending on the nature of the study and what sort of information one is trying to gather through the use of FRET, FLIM has some compelling advantages in certain situations, and can provide a quantitative evaluation of the donor, acceptor and FRET pair stoichiometry. It does require access to specialized equipment and software. Different approaches to FLIM data acquisition have different strengths and weaknesses. For dynamic studies requiring high time resolution, FLIM acquisition times can fall well short of ideal.

If a yes/no answer to whether FRET is occurring is all that is required, then the polarization anisotropy of the acceptor can be used to determine FRET between fluorescent proteins (Rizzo and Piston, 2005). This is a relatively simple and robust method for confirming the presence/absence of FRET.

Before SEMs were invented and when they were still of relatively poor resolution, one way to see the fine details of a sample surface was to prepare an electron-transparent replica of the sample surface and view it in a TEM. The carbon-coated surface of the sample was shadowed with a heavy metal to make a replica that mimicked the topography of the original surface, in a sample that could be viewed in the TEM.

We have found some value in this old technique; to examine second-phase particles freed from the metal matrix for EDX, diffraction, and morphology studies—while preserving the original position and distribution of the particles, historically called “extraction replication.”

Arecent discussion on the Microscopy Listserver on the subject of microtoming Si reminded many of us of this decades-old issue. Silicon is one of the easiest materials to prepare for TEM analysis with dozens of protocols available including chemical etching, electropolishing, mechanical polishing with and without ion milling, microcleaving, and the use of a focused ion beam instrument. The time involved in Si preparation can range from minutes to a few hours. Things become more complex if it is desired to prepare a Si semiconductor device with high specimen preparation spatial resolution i.e. prepping a prespecified, very small, structure.

Now and then a technologist, often times in the biological sciences, is asked to prepare a Si TEM specimen and wonders if Si can be microtomed. The results are almost always a pile of Si dust and frequently a damaged diamond knife.

Precollege science education in the United States is not what it could, and should, be. Major changes are being made in the way science is taught, but delivering those changes to thousands of schools is an enormous task. Scientific societies are a major resource; they can organize and train member-volunteers to help teachers bring “real” science to the classroom. The Microscopy Society of America has become part of the effort with Project MICRO (Microscopy In Curriculum - Research Outreach). MICRO is putting MSA members, teaching materials, and microscopes in middle school classrooms nationwide. The idea began in 1993, but it has taken a lot of time and effort to implement.

MSA's early decision to collaborate with experienced science educators at the Lawrence Hall of Science of the University of California at Berkeley was a wise one; their educational materials have a well-earned national reputation for excellence.