Nanofabrication is upon us. But engineers require measurements of physical properties of their materials in order to use them appropriately. Because the size of the object is in the nanometer scale, tiny measuring devices are required. Furthermore, there is the problem of reading these tiny devices. Zhong Lin Wang, Philippe Poncharal, and Walter de Heer at Georgia Tech have solved this problem by using the transmission electron microscope (TEM) to read off the measurements.

A special device was designed to fit inside the TEM, allowing for simultaneous visualization and manipulation of a nanostmcture. The nanostructure that Wang ef al, used was the carbon nanotube which could be clearly visualized by the TEM and has a well-defined structure. Specifically, they used multiwalled nanotubes with diameters of 5 to 50 nm and lengths of 1 to 20 μm. The device they designed for in situ microscopy was a modified specimen holder that allowed carbon nanotubes to be mounted in a conducting medium and positioned across from a counter electrode, which was a droplet of mercury or gold/ platinum balls.

Strictly speaking, it is impossible to authenticate any painting to the extent of naming the artist who produced that object. One can only increase the likelihood thereof. In one celebrated case I felt I had fully proved that Manets palette had indeed been used to paint a particular painting but a “scholar” on whose reaction the art world depends for final acceptance of authenticity only said “someone” could have borrowed Manets palette one fine day and painted that picture. In a second celebrated case I was able to prove, but not convince, the world that a particular painting (Shroud of Turin) was, in fact, a painting and not an artifact produced by some other mysterious means, e.g., some sort of photographic process or resurrection itself.

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.

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.

In North America, one could very easily get a distorted view of the rest of the world when it comes to internet access. in the USA and Canada, one can stay connected continually, without regard to cost. And the connections tend to be fast and convenient.

But this is not the case in most of the rest of the world. While there are some notable exceptions, Australia and Israel for example, many in the international microscopy and microanalysis community have to struggle with connections that are either very slow, or very expensive, or both, in fact, in many parts of the world, internet content is routinely not with the benefit of graphics. This is really a great disadvantage since, in many instances, the real important information is contained within the graphics. Even in the USA and Canada, because of firewall considerations or other concerns, a surprisingly large number of end users do not have internet access at their main workstations, and they have to suffer the inconvenience of using some kind of shared resource, either “down the hall” or worse yet, in another building.

It can be a problem to get suspended (or plated) cells or particles to settle into the apex of a BEEM capsule for embedding, particularly if the cei is/particles can not be embedding in agar or gelatin for some reason. The following simple procedure solves this problem.

Process the suspensions in polypropylene test tubes or microcentrifugation tubes. These can be spun down after each transfer for 5 minutes at 3000 rpm. Once the cells/particles are fixed, en bloc stained, and infiltrated with either Spurr's or LR White resin, I transfer them to BEEM capsules. I do not add additional resin yet. These are placed piggyback in microcentrifuge tubes that have been slit with wire cutters to make them large enough for the BEEM capsules to fit (see following illustration).

Processing cell suspensions or plated cells can be done in polypropylene test tubes or microcentrifuge tubes. These can be spun down after each transfer for 5 minutes at 3000 rpm. Once the cells are fixed, enbloc stained, and infiltrated with either Spurr or LRWhite resin, I transfer them to BEEM capsules. I do not add additional resin yet.

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.

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.

Computer Microvision is a microscopy technique originally developed to study sound-induced motions of sensory cells in the inner ear - motions that are on the order of picometers-i.e., smaller than the radius of a hydrogen atom. Three-dimensional micrornovies of inner ear cells moving in response to sound were analyzed to quantify motions with nanometer precision. Recently we have adapted the technique for in situ measurement of motions of micro-electron mechanical (MEMS) devices.

In Computer Microvision systems, images of MEMS are magnified with an optical microscope and projected onto a CCD camera. Stroboscopic illumination is used to obtain stopaction images of the moving structures. Stop-action images from multiple focal planes provide information about 3D structure and 3D motion. Image analysis algorithms determine motions of all visible structures with nanometer accuracy. The system can measure motions at frequencies as high as 5 MHz with nanometer resolution, i.e., well below the wavelength of light.

With the upcoming Microscopy & Microanalysis Conference only a few weeks away, the question that is in the mind of many industry experts is: “Will this be the meeting where the first commercial x-ray microcalorimeter is introduced?” If it is, you can bet your bottom dollar that there will be a major upheaval in the high resolution x-ray EDS business because of the ten- to fifty-fold improvement in X-ray resolution promised by this extraordinary device.