The transmission electron microscope (TEM) was invented in the 1930's, and developments in specimen preparation in the 1950's led to its widespread use as a tool to study structure in biologic systems. Similar in principle to the light microscope, but utilizing a much shorter wavelength for better resolution, the TEM has the image-forming beam pass through the specimen. This results in a two-dimensional image which can be difficult to interpret because features from different depths of the three dimensional specimen are superimposed. Traditionally this was dealt with by cutting sections of plastic-embedded specimens so thin (in the 40 to SO nanometer range) that they effectively had only two dimensions. To allow biologists to examine structures in three dimensions, serial sections are stacked and structures reconstructed. Even though computers have made reconstruction easier, the reality is that resolution in the depth dimension is limited by the section thickness. The technique of electron tomography is emerging as a way to overcome this limitation.

In Ihe October, 2001 issue of Microscopy Today, Randy Tindail described and compared his experiences with service contracts offered by insurance companies versus those offered by equipment manufacturers. As a small business with limited funds, we, too, grappled with the onerous costs of service contracts on our SEM and EDS units. However, we believe that we have found an extremely cost-effective means of protecting our equipment investment against the expense of a major failure.

First, a bit of business philosophy: We believe insurance should be designed to protect the insured from catastrophe, and not be expected to take care of every minor expense or inconvenience that may develop.

This article is the second of a series transcribed from the discussion taped during the Core Facility Management session. Bulleted paragraphs indicate comments by individuals attending the session.

D. Sherman: The next topic was suggested based on the discussion at last year's Core Facility Management session. Those of you who have not been involved in formal discussions on scientific ethics probably are going to have your eyes opened a bit. There is no such thing as a quick answer when it comes to ethics. What was initially brought up last year was the question of what are our responsibilities as facility managers when we see things going on that are perhaps not exactly appropriate as far as interpretation of data and such.

I am posting a summary of responses to my request to the Microscopy Listserver on 3 December 2001, for Information about any roles microscopic artifacts may have played in the history of biology. I posted my own experience regarding Sidney Hickson's description of reproductive events in Miliepora spp. (A copy of my somewhat lengthy post is enclosed at the end of this summary.)

To be fair to Sidney Hickson, who drew and described the incredible, imagined sequence of reproductive events in Miliepora spp. as mentioned in my original, I believe that he did print a retraction of the findings in that paper.

Low Voltage Scanning Electron Microscopy (LVSEM), defined as operation in the energy range below 5 keV, has become perhaps the most important single operational mode of the SEM. This is because the LVSEM offers advantages in the imaging of surfaces, in the observation of poorly conducting and insulating materials, and for high spatial resolution X-ray microanalysis. These benefits all occur because a reduction in the energy Eo of the incident beam leads to a rapid fall in the range R of the electrons since R ∼k.E01.66. The reduction in the penetration of the beam has important consequences.

Many SEM stages prevent effective examination of specimens at extremely high tilt angles. Stage tilt may not be allowed at or near 90 degrees, and the design of the stage may cause the loss of X or Y stage motion at high tilts. We designed an adapter for our microscope that solves both problems by shifting the normal stage rotation motion 90 degrees (Figure 1). Specimen stubs are mounted horizontally in the adapter, and all components of the holder except the base plate are tethered to a stationary portion of the stage. Operation of the stage rotation control then rotates the stub around its horizontal axis (similar to a rotisserie). Specimens can be rotated through 360 degrees along this axis, with a full range of X and Y stage movement. A limited amount of stage tilt is also possible, allowing precise positioning of the specimen.

There are two kinds of conductivity measurements possible with scanning probe microscopy (SPM). In the first case, the specific resistance of material directly below the tip is probed. In the second case, SPM probes local potential induced by the lateral current applied through macroscopic contacts, thus providing the information on the mesoscopic transport properties of the sample.

The first set of techniques is invariably based on measuring tip-surface current in contact or intermittent tapping mode. If the tip-surface contact resistance is small (good contact), the current will be limited by the spreading resistance of the sample from which specific resistance can be calculated, assuming that the contact area is known.

A picture is said to be worth a thousand words; in the different fields of microscopy pictures are often our data as well. We interpret, analyze, and share images with our colleagues, and so image preparation, enhancement and presentation are crucial for this process. As image acquisition in microscopy moves away from capture on photographic film in favor of digital imaging methods, digital manipulation of visual data for presentation requires learning to use computer software tools. Adobe Photoshop, currently the standard in the community of both artists and scientists, is a powerful program, but it is NOT specifically designed for scientific data, and so contains dozens of features and tools that may and/or must be avoided in the preparation of micrographs for scientific publication. The learning curve for this type of software can be steep, but the steps outlined below may help to get you started.

A question was asked on the microscopy listserver about fluorescent probes for studying oil-in-water emulsions trapped in a cellulose membrane, I don't have experience with that exact problem (I have studied chloroform/water emulsions rather than emulsions of triglycerides or alkanes), but I can make some general suggestions. The best type of hydrophobic probe to use depends on whether the probe is to be added to the hydrophobic phase before emulsification or after. If it is to be added to the preexisting emulsion as a dimethylsulfoxide stock solution, a probe with some aqueous solubility is required.

I once had a problem looking for cracks in a graphite coating on quartz fibers. This problem is a good example of the general case of defining visible light microscopy inspection criteria. Since specimen lighting may influence critically what one can see. The answer to the question depends a bit on the size of the crack and the opacity and reflectivity of the oxide coated surface. First insure that the microscope has a lighting set up that allows one to dependably see the cracks before worrying about a camera. Most of the digital cameras that can be used with a microscope would probably be adequate to recording the crack, once the viewing situation is set up.

This article presents ideas on some topics related to electron diffraction in the TEM. These are in regard to topics that I have come to think of as standard parts of what it means to do microscopy. However, they represent insights that not all users share (or even agree with, maybe).

Kikuchi lines are of great use in orienting a sample. Unfortunately, in modern microscopes, Kikuchi lines are not seen in selected-area diffraction (SAD). This is because immersion lenses send parallel electrons, from different parts of the sample (like the Kikuchi lines from a flat specimen), to different places in the diffraction pattern.