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 large number of natural and synthetic species ranging from virus particles to small polymer beads have been employed over trie years as labels in electron microscopic applications. Perhaps the most useful and versatile of these labels are the colloidal heavy metals. The colloidal labels can be synthesized as round regular spheres in sizes from 1 to 150 nanometers in diameter and thus can be used where molecular and sub-molecular ranges of spatial resolution are required. They are electron “dense” and good emitters of secondary and back-scattered electrons and hence are readily detectable in both scanning electron microscopic imaging systems and transmission electron imaging systems. Larger particles, 10nm and above, can be identified via their shape in force based imaging systems.

“Low Voltage Scanning Electron Microscopy (LVSEM), defined as operation in the energy range below 5 keV” was described by David Joy and Dale Newbury (1) in the previous issue of this journal as being “perhaps the most important single operational mode of the SEM.” In their paper they describe both the advantages and disadvantages of this method.

Ramsey's principle, which was established by Jack Ramsey over many years as a microscopist and microanalyst, is to say: “There is no best way of doing ANYTHING.”

SEM Instrument calibration is something most people don't always realize they need to do. Images look about right so the magnification seems close enough. Today, measurements are being done in specialized scanned beam metrology instruments everyday, so correct magnification calibration is very important. Calibration is probably more acknowledged from the point of the transmission electron microscope than for the scanning microscope. But, it still needs to be done. Today, people can buy SEM's that cost around $100,000 (refurbished ones somewhat less) to $2.5M with one instrument model from one manufacturer costing about $8M. Believe it or not, often the instrument buyer thinks that as soon as they get the instrument into their facility (since it is new and they paid a lot of money for it) that it is perfect and everything is correct.

First, the OEM is obligated to service contract customers first. Without a could go to the bottom of the list. This would not be all that different with a third party firm—although they might try a little harder to be timely. With service contracts, payment has been made up front (or at least committed up front) for a entire year. This is guaranteed income and includes a guaranteed liability (for the OEM) “to maintain the instrument/system to its original specifications”. Service organizations must and do take this responsibility seriously. This liability doesn't exist with biliable customers, and work will be done to the best of the OEM's ability and only to the limit of the specific purchase order.

Formaldehyde is one of the most rapidly penetrating fixatives used. Unfortunately, it is one of the slowest to fix tissue. This paradox was finally explained by Burnett in 1982. An fine description of the properties of formaldehyde may be found in John Kiernan's book.

The penetration rate of formaldehyde in mm/hr is a variable thing. It depends on how the data is obtained. It may also vary slightly depending on tissue type. The penetration rate of formaldehyde fixatives has been extensively studied, often with conflicting results. The penetration of non-coagulating fixatives is difficult to measure.

Microscopy labs have very quickly moved from keeping track of and storing thousands of samples and negatives “atoms,” to having to now deal with the same number of samples, and a few negatives, but in addition, we may now have thousands of digital images “bits.” In a two-year microscopy teaching program, which includes 18 microscopy courses, there are thousands of images, which must be collected from a variety of input devices running on various platforms. These must then be associated with different types of data files that need to be tagged to reports as well as being tagged to projects and courses. All of this information must be accessible, first to all those involved with doing the project, as well as to anyone associated with needing the results.

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.

There was a recent thread on the microscopy listserver by John McCaffrey (who hosted the discussion on TEM calibration at the 2001 M&M facility managers session) and Richard Beanland, dealing with the calibration of TEMs. This discussion was prompted by a calibration question from John Basgen, who was looking for more precision and more long-lived calibration specimens. The discussion complements and extends the one of the M&M 2001 managers meeting on EM calibration (Microscopy Today, January/February 2002, issue #02-1), and we are running this separately from that meeting discussion. (MT-ed.)

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.

All laboratory workers face the ongoing requirement that all chemicals and solutions used in the lab be labeled with the date of receipt or preparation, together with the expiration date of the chemical and the initials of the preparer, as well as any known hazards associated with that chemical. Officials from accrediting agencies, sometimes unannounced, pick and poke around laboratories looking for outdated chemicals and solutions, including biological stains left unsuspectingly at the back of a fridge or on a seldom used shelf.

First, it is important to know whether the objective of the analysis is to measure the total amount of the two metals in the assembly as a whole, or to measure their proportions in each of the different areas; the tin solder, the intermetallic layers and so on. The purpose to which the results are to be put is also important. To some extent the answer depends on knowing this, as a biased but highly repeatable measure of something is often useful for internal purposes.

For Au/Sn intermetallic compounds which have quite specific compositions, the absorption/enhancement effects will be different than for a low-concentration solid solution of either of the metals in the other.

K-12 instruction in biology has traditionally taken a very descriptive approach. This is in marked contrast to quantitative as well as qualitative way of looking at things in physics and chemistry. This qualitative/descriptive approach even extends into the iaboratory portion of the biological course. One way to introduce a more quantitative approach is in the microscopy portion of the biology curriculum. Because cellular structure is primarily a microscopic province It makes sense to introduce students to the different microscopic tools such as TEM and SEM, as well as the light microscope that are used to investigate cell structure.

Two particular questions keep coming up regarding Atomic Force Microscopy (AFM), and we would like to address them here.

The lateral resolution of AFM is a parameter that currently has no accepted definition. The definition used in our lab is “the minimum distance between objects at which you can distinguish distinct objects.” In other words, imagine a surface with two sharp features: upon bringing the two features closer and closer together, at some point only one object is seen. That distance is the resolution limit. It is useful to define “see” as having a dip in height greater than the noise level in the image.

According to the International Standards Organization (ISO), for companies that are in compliance with ISO-9000 or QS-9000, traceable measurements shall be made when products or processes require dimensional measurements be made to a known uncertainty. These measurements are often made with a traceable ruler or micrometer. For magnification (the ratio of object size to image size) to be traceable, both the image and object size must be measured with calibration standards that have traceable dimensions. In the current ISO jargon the “uncertainty” of the instruments used to make the measurements must be known. The word “accuracy” is now only considered to be a subjective term and shall remain dimensionless.

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.

Designing a microscopy and/or analytical instrumentation facility can be completed in a step by step approach, whether it is a completely new facility or an existing one moving into expanded quarters. The procedure is independent of size. The same basic procedure is required for one room or an entire building. There are several major factors that must be taken into consideration. Check lists can be compiled by the microscopist/analyst. One should easily be able to start at any point along the way. The time and effort Initially put into the design will pay for itself many times over in both the convenience and efficiency of the resulting laboratory.