The use of fluorescent probes is becoming more and more common in cell biology. It would be useful if we were able to correlate a fluorescent structure with an electron microscopic image. The ability to definitively identify a fluorescent organelle would be very valuable. Recently, Ying Ren, Michael Kruhlak, and David Bazett-Jones devised a clever technique to correlate a structure visualized in the light microscope, even a fluorescing cell, with transmission electron microscopy (TEM).

Two keys to the technique of Ren et al are the use of grids (as used in the TEM) with widely spaced grid bars and the use of Quetol as the embedding resin. The grids allow for cells to be identified between the grid bars, and in turn the bars are used to keep the cell of interest in register throughout the processing for TEM. Quetol resin was used for embedding because of its low auto fluorescence and sectioning properties. The resin also becomes soft and can be cut and easily peeled from glass coverslips when heated to 70°C.

Once in a while we have the privilege of participating in an activity that promises to radically improve our technical capability to explore materials at the microscopic level. The recent practical demonstration of aberration correction in the SEM, the TEM and the STEM signify nothing less than a revolution in the way that we design and operate electron optics for microscopy.

Scherzer understood from the beginning of electron microscopy that an open magnetic lens having cylindrical symmetry will have positive, non-zero aberration coefficients, and so therefore will always have some minimum aberrations. In practical instruments, it was arranged that the limiting defect would be spherical aberration in the objective lens. This allowed the imaging performance to be largely characterized by a single parameter, spherical aberration, and also allowed a certain amount of image simulation and post-experiment analysis to facilitate interpretation of imaging results.

The use of microscopy and imaging methods to study foods is not a new idea. It has been going on since the first light microscopes were developed (White, 1970; White and Shenton, 1974-1984). Microscopy has been used to determine the quality, purity and safety of foodstuffs by detecting and identifying contaminants in foods. The short article by Stephen Carmichael in the May/June 2002 issue of Microscopy Today has again brought food microscopy into the spotlight. The article provided an opportunity to discuss present applications of food microscopy and to give some projections of where it is headed in the future. The reader may not realize that microscopy and imaging methods are used extensively by most major food companies worldwide for product development, quality control, and trouble shooting (Allan-Wojtas, 1999). Often, this work cannot be published because it contains proprietary information. The application of microscopy to food structure analysis is one of the best kept secrets in microscopy today.

Several images can be stitched together (sometimes referred to as “montaging”) through the use of Photoshop. Edges of the images can be feathered and the process automated to reduce the hours of work that can go into this procedure.

Before going any further, let me be quick to say that more than one company has developed protocols to accomplish image stitching. When your lab is looking at large fields, while needing to resolve fine details, then I would strongly recommend the purchase of a system designed for this purpose. Issues surrounding motorized stage accuracy, problems with tilting when a camera is perched on a long optical tube, and image to image correction for contrast, color and uneven illumination can all contribute to confounding issues when image stitching. These companies have worked through these problems with off-the-shelf systems.

A recent thread on the MSA list server about problems with image formats (in this case TIF or TIFF) showed, that there is a bit of confusion in the microscopy community about the best file format for digital images. I will try to shed a bit of light onto this problem.

Digital images are at the core a large array of numbers. One number per pixel for b/w images, 3 numbers per pixel for color images. The simplest file format consists of 2 numbers that define the width and height of the image, and then just a listing of numbers for each pixel. By using the right conventions, the image can be recreated from a data file like mis. In essence, this is the format of a bitmap image (BMP), and other formats derived from it.

Historically, one of the first jobs performed by the new instrument called a “transmission electron microscope” (TEM) was the determination of particle size and distribution. Particle size is still important in controlling the properties of sub-micron and nanoparticle products. The width and average particle size can determine the light scattering properties of a pigment like TiO2, for example. The cost of installing a TEM facility and TEM sample preparation issues makes TEM analysis expensive and labor intensive. So decades ago, nitrogen BET (Brunauer, Emmett, and Teller) surface area determinations were used on sub-micron powders and pigments to minimize these costs. The BET measurement of the exposed external surface area of a material is made by detecting and measuring the amount of nitrogen given off after absorption, assuming one atomic layer of nitrogen is adsorbed.

After carbon nanotubes (CNT) were discovered in 1991, many applications have been proposed that utilize their extraordinary electrical and mechanical properties. One application is as tips for scanning probe microscopy where CNTs offer several advantages including high resolution and the capability to image deep, narrow structures. A recent study of CNT scanning probes for atomic force microscopy (AFM) in semiconductor surface science concluded that an AFM with CNT tips has immense potential as a surface characterization tool in integrated circuit manufacture. Previously researchers had to construct their own CNT probes, but recently CNT AFM probes have become commercially available.

Carbon nanotubes (sometimes called buckytubes) are closed seamless shells ot graphitic carbon typically one to tens of nanometers in diameter and several micrometers long. The structure of a closed-dome single-walled nanotube is illustrated in Figure 1. Carbon nanotube probe tips offer several advantages.

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. When tests are made to evaluate the performance of a CLSM, the usual subjective assessment is accomplished by using a histological test slide to create a “pretty picture.” Without the use of functional tests many of the machines may be working at sub-optimal performance levels, delivering sub optimum performance, and possibly misleading data. In order to replace the subjectivity in evaluating a confocal microscope, tests were derived or perfected that measure field illumination, lens clarity, laser power, laser stability, dkhroic functionality, spectral registration, axial resolution, scanning stability, PMT quality, overall machine stability, and system noise (1-3). It is anticipated by using this type of test data, performance standards for confocal microscopes will be obtained and the current subjectivity in evaluating CLSM performance will be eliminated.

Modern microelectronics have rapidly decreased in geometry to enhance the speed and processing power of computers. Advanced devices are approaching design rules of sub 0.13 micron in size, and the trend continues at the rate dictated by Moore's Law, Coupled with this reduction in device size, is a change in materials used for producing these devices. Traditional aluminum interconnect metallurgy and oxide dielectric materials are being replaced with copper and low-k materials in an effort to continue the trend of shrinking device sizes and higher processing capacities.

These changes in materials and device sizes have provided the impetus for alternative methods for producing cross sections. Although focused ion beam instrumentation has been successfully used for preparing cross sections, a combinatorial approach using polishing and argon ion milling has been found to dramatically enhance the ability to produce high quality cross sectional samples in a reasonably short amount of time.

If we look back at the year 2003 it becomes quite clear that the South African microscopy year was quite unique. …Of course, I can't speak for all the companies and laboratories in the country, but from my personal view it was definitely interesting.

FEI definitely had a great year in that they managed to sell 5 systems. Then LEO added to that another 3, and we have heard that there is also a Cameca probe being bought. This may not sound like a lot, but if you take into consideration that in the last few years an average of 4-5 EM columns per year would register as a banner year. Given that we have seen our currency improve over the past 2 years, it does imply that buying imported equipment is getting cheaper. However as this effect has also negatively affected exports, we can't say that we have become richer, and so could afford more systems.

Stephen Carmichael wrote about Cryoelectron Tomography in the May 2003 issue of Microscopy Today. Citing new preparation methods, small cells can be vitrified, observed frozen in the TEM and a series of digital images captured while the specimen is being rotated around the axis perpendicular to the electron beam producing a 3-D tomogram. Gina Sosinski and Maryann Martone wrote about imaging big and messy biological structures using cryo-electron Tomography in the July issue of Microscopy Today. Cryo-HRSEM now also seeks to provide 3-D information approaching the molecular level from frozen hydrated cell and molecular systems. Vitrification procedures for small specimens such as platelets and biomolecules on grids are accomplished by plunge freezing in liquefied etiiane as is done with cryo-TEM procedures. Bulk specimens such as organic hydrogels and tissues are routinely high pressure frozen (HPF) in 3mm gold planchets. Employing an in-lens cryostage, identical to those used in cryo-TEM, cryo-HRSEM provides 3-D high-resolution images because secondary electrons are efficiently collected above the lens in a single scan thus minimizing specimen irradiation.

Propylene oxide (1,2-epoxy propane) has long been utilized as a transitional solvent in the preparation of biological tissues. This compound compound is completely miscible with the various epoxy resins utilized for infiltrating tissues. Ethanol, perhaps the most widely used dehydration agent, does not possess this characteristic and must be removed before the process of infiltration commences. It is common practice amongst biological microscopists to pass tissues for a few minutes through pure propylene oxide (PO) immediately prior to infiltration. The tissues then are pre-infiltrated with various proportions of PO and the embedding medium of choice. For example, one traditional recipe takes the tissues progressively from pure PO to 2:1 PO/resin, 1:1 PO/resin, and 1:2 PO/resin mixtures, with stays of a few minutes at eacil stage. A lengthy procedure such as tills probably is not necessary and has never been favored by this worker.

Low accelerating voltage operation is an excellent mode of scanning electron microscopy and it is extensively used for measurements in semiconductor production. The beam penetration is small, and if properly applied, the specimen charging is kept at acceptable levels. But, is this always enough? Today, the scanning electron microscope (SEM) is being used in photomask metrology and imaging where charging is excessive. Charging is difficult to quantify and control as it varies greatly with instruments, operating conditions and sample. Therefore, it is also very difficult to model accurately. For accurate metrology charging must be overcome because the dynamic charging of the sample deflects the electron beam from its intended position and the intensity of the induced signal may vary uncontrollably. Deflection of the electron beam of even a few nanometers potentially results in a measurement error that is significant to modern semiconductor production.