Flax (Linum usitatissimum L.) and kenaf (Hibiscus cannabinus L.) are the sources of fibers used for textiles and other industrial applications. Both flax and kenaf produce fibers in the bast region (Fig. 1, 2) which must be separated from other tissues by retting. Although both flax and kenaf are bast fibers, their properties are vastly different. UV absorption microspectrophotometry and histochemistry elucidate their chemistry and structure related to enzymatic retting.

Aromatics such as lignins are produced by plants for protection and strength, but their presence inhibits microbial degradation, which is necessary in retting. Histochemical tests indicated variations in the site and type of aromatics within these two plants (1,2). In flax, acid phloroglucinol but not chlorine-sulfite gave positive reactions occasionally in fiber cell walls in the bast. The other cell walls in the bast did not contain aromatics by these tests, although aromatics occurred in the cuticle.

Porous nylon fabric is used as a filter in blood devices. This study was undertaken to evaluate the interaction between an experimental nylon filter and the cellular elements which pass through it. Our investigation was mainly centered on determining the deposition of cellular material on the nylon filter using Low Voltage Scanning Electron Microscopy (LVSEM) and Transmission Electron Microscopy (TEM).

The filter was prepared for LVSEM by cutting a 2.5 cm2 of the nylon filter material with a razor and placing it in a nylon mesh bag which was stapled shut. The bag was rinsed three times for ten minutes each in fresh Sorensen's phosphate buffer. A graded alcohol series of 25%, 35%, 50%, 70%, 80%, and 90% was used to dehydrate the filter for ten minutes each. This was followed by dehydration twice for 20 minutes in fresh 100% ETOH. The filter was then Critical Point Dried (CPD) in the Tousimis AUTOSAMDRI-814.

A Cryosorption Freeze Drying (CFD) system was evaluated for its effectiveness in preparing delicate biological materials for both low voltage-field emission scanning electron microscopy (LVFESEM) and imaging liquid metal (Ga) ion beam, static time-of-flight, secondary ion mass spectrometry (TOF-SIMS). The primary goals of these studies were to investigate the retention of both structural and chemical integrity using fresh cryoprepared biological material which had not been exposed to any chemical fixation and which would not be coated by any conductive material in order to obtain information from the native surfaces. Duplicate chemically fixed samples were processed for comparison. LV-FESEM (2-2.5kV) was used to assess the quality of the structural preservation of the freezing and freeze drying (FD) protocols. Imaging static TOF-SIMS was used to investigate the surface chemical compositions of the biological samples.

The extraction of lipids and phospholipids during dehydration and plastic embedding steps results in poor preservation of the phospholipid rich lamellar bodies (LB) in alveolar type II epithelial cells. To achieve better retention of phospholipids, we combined inflation fixation and an en bloc staining protocol using 4% aqueous uranyl acetate (UA), thereby improving the preservation of the LBs for both the wild type and transgenic mice expressing modified pulmonary surfactant protein B (SP-B; Akinbi et al., 1997).

Lungs of 6-8 week-old mice were inflation fixed (Bunkingham and Weyder, 1981) with ice cold 2% paraformaldehye and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (SCB), pH 7.3, postfixed in fresh fixative at 4 °C overnight, incubated with 1% osmium tetroxide in 0.1 M SCB at room temperature for 2 hours, and stained en bloc with 4% aqueous UA overnight.

Increasingly, cells isolated from blood and body fluids and cells grown in culture are becoming the experimental models of choice for biological research. The demand for demonstrating biochemical processes morphologically is also becoming commonplace in the electron microscopy laboratory. Successful fixation, in situ embedment, and ultrathinsectioning of cell monolayers can be difficult to achieve for routine transmission electron microscopy. For postembedding immunocytochemistry, processing becomes more complex due to fixation constraints and the use of acrylic resins. The object of this paper is to present a reliable, rapid method for processing monolayers that preserves both the ultrastructure of the cells and antigenicity.

The equipment used for this procedure was a Pelco Model 3440 MAX laboratory microwave oven equipped with a temperature probe and a maximum power output of 800 watts. Using a neon bulb array, the oven cavity is calibrated to determine the microwave energy distribution.

The focused ion beam (FIB) has become an indispensable tool for a variety of applications in materials science, including that of specimen preparation for the transmission electron microscope (TEM). Several FIB specimen preparation techniques have been developed, but some problems result when FIB specimens are analyzed in the TEM. One of these is X-ray fluorescence from bulk material surrounding the thin membrane in FIB-prepared samples. This paper reports on a new FIB specimen preparation method which was devised for the reduction of X-ray fluorescence during energy dispersive X-ray spectroscopy (EDS) in the TEM.

Figure 1 shows three membrane geometries that were investigated in this study on a single-crystal Si substrate with a RF sputter-deposited 50 nm Ni film. Membrane 1 is the most commonly reported geometry in the literature, with an approximately 20 urn wide trench and a membrane having a single wedge with a 1.5° incline.

The focused ion beam (FIB) lift-out method is a high precision technique by which site-specific cross-section transmission electron microscopy (TEM) specimens may be rapidly prepared from virtually any material. The technique is particularly useful when the sample geometry or composition is complex (e.g., fibers, powders, composites and interfaces). In addition to the preparation of TEM specimens, FIB milling has also found widespread utility in micromachining and microfabrication applications as well as specimen preparation for scanning electron microscopy (SEM) and secondary ion mass spectrometry (SIMS).

As the applications of the FEB instrument continue to become more universally recognized, the need to understand the interrelationships between the target material, processing parameters, and process efficiency of the milling phenomena becomes more critical. Incident ion attack angle, target material stopping efficiency and sputtering yield, Y, are important variables governing the milling process. TRIM, a binary collision approximation Monte Carlo simulation code, is used to physically model variables that influence FIB sputtering behavior.

The results of TEM analyses of materials are critically dependent on the quality of the sample prepared. Although numerous techniques have been developed in the last two decades, differential thinning of inhomogeneous materials remains a serious problem. Recently, focused ion beam (FIB) technique has been introduced for cross-sectional sample preparation for TEM and SEM.

A novel system for depositing a fine-grain (∼ 200 nm) ceramic coating on a metal surface via a patent pending Small-Particle Plasma Spray (SPPS) technique has been developed at the Basic Industry Research Laboratory of Northwestern University. To understand the properties of the coated surface, the ceramic/metal interface and the microstructure of the ceramic coating must be investigated. This paper presents a comparison of the microstructure of an A12O3 coating on a mild steel substrate prepared using conventional and FEB techniques.

Sample preparation for transmission electron microscopy (TEM) has been a source of speculation with regards to potential for the creation of artifacts which may confound data gleaned from TEM analysis. For semiconductor integrated circuit (IC) materials characterization, the most common sample preparatory methods are based on final thinning by ion beam milling. The latest shift towards Copper / low dielectric constant (k) composite systems in the semiconductor IC industry provides several challenges for TEM sample preparation resulting from differences in milling rates and materials properties for neighboring features.

In conjunction with process development for integration of Cu / low-k materials, conducted at SEMATECH, we have systematically studied the effects of TEM sample preparation by ion milling in order to search for artifacts that could result from sample thinning procedures. For this purpose we have studied wafers with patterned copper lines isolated by a low-k polymer. One sample was stressed by thermal and electronic bias, while a second was subjected to only thermal stress.

The cross-sectioning of semiconductor wafers for SEM studies has traditionally been carried out by tedious and laborious mechanical grinding and polishing techniques. The mechanically polished surfaces are treated using a “wet chemical” etching method to enhance and delineate certain features or layers in a given specimen. The etched specimens are then coated by conductive layers to prevent charging during SEM examination. As an alternative to “wet chemical etching”, broad-ion beam etching techniques have been developed for surface treatment of mechanically polished specimens. More specifically, we have reported [1] the utilization of a combined process of broad-ion beam etching and coating of mechanically cross sectioned semiconductors in a single vacuum chamber. As a further progress to that work, we report a rapid and reliable technique for preparing precision SEM cross sections. The technique is based on perpendicular broad-ion beam milling of cleaved wafers to expose any desired cross-section through a given feature of the specimen.

The Small Angle Cleavage Technique (SACT) is a relatively simple and inexpensive method of producing superior cross sectional TEM specimens. For speed of preparation, it is unsurpassed; for example, ten samples can easily be prepared in about an hour from a typical material. It is particularly well suited for rapidly examining coatings and thin films very soon after they have been deposited. A major limitation of the technique is that it does require the substrate material to cleave or fracture. For this reason, it has been applied almost exclusively to semiconductor materials, but the technique has been extended quite successfully to other substrates such as glass, silicon carbide, quartz, sapphire, and other hard materials. Several procedures have been added or modified to the original technique developed by McCaffrey that makes it much easier to get started in using the technique. A detailed pictorial outline of the technique has been described elsewhere by the authors.

The low voltage phosphors should be efficiently excited with an electron beam value lower than 500V. Thus the properties of low voltage phosphors are directly affected by the surface states of phosphors. Recently, to improve the properties of these phosphors, much analysis has been carried out focused on the surfaces of phosphors using by FESEM, AES or XPS etc.. Nevertheless, the microstructure of phosphors around the surface has not been made clear due to the resolution limits of the above equipments and the difficulties of sample preparation. In this study, in order to analyze the microstructure of phosphors around the surface, we have prepared the thin sections of the (ZnxCdy)S phosphors by an ultramicrotome and carried out high resolution electron microscope(HREM) study on the thin sections.

To carry out ultramicrotomy, the particles of phophors were embedded with acrylic resin (methyl methacrylate: n-butyl methacrylate= 4:6, Benzoyl peroxide 1.5%) in a capsule.

Further development of Vacuum Fluorescent Displays (Fig.l) for low cost production and high yield requires investigation of all the components on a submicron scale at various processing stages.

A variety of specimen preparation methods have been used for making different types of high quality cross-sectional and plan-view TEM specimens from:

1. 1. Initial phosphor materials - ZnCdS powders admixed with conducting powder of ln2O3;

2. 2. In2O3 mixed ZnCdS phosphor layers of ready-made working VFD;

3. 3. W - filament cathodes coated with (Ba,Sr,Ca) oxides.

4. Rapid sharing of results.

Group 1 specimens were made both by direct dispersion of phosphor powder particles on the carbon coated copper grid from acetone diluted powder suspension, and by preparation of cured bulk material from the powder using Gatan G-l epoxy, followed by cutting, grinding-dimpling and final ion milling in Gatan DuoMill 600 (Fig.2).

Specimen contamination and amorphous irradiation damage severely limit the ability to perform accurate electron microscope analysis of materials, especially as specimen areas of interest decrease in size. To analyze smaller areas of interest, electron probe sizes have decreased, while probe currents have increased. The combination of these two factors results in an increase in the amount of carbonaceous contamination formed on the specimen under the electron beam. Recently, the use of low energy plasmas has been shown to be effective in preventing such contamination from occurring (1-3). For TEM, the simultaneous placement of the specimen holder and specimen into such a plasma for short periods of time (usually less than two minutes) results in the ability to hold a converged probe on the specimen for analysis without contamination becoming an problem. Figure 1 is a TEM micrograph of a 304 stainless steel specimen.

The common theme of most forms of thin specimen preparation for the physical sciences has been the controlled, gradual removal of material, whether by electrolytic dissolution, ion bombardment, careful mechanical dimpling or step-by-step tripod polishing. It is somewhat ironic that a “brute force” method, wherein the material is forced at a high rate against an atomically sharp diamond wedge so that an ultrathin slice, or section, is sheared, or fractured, from a ‘block’ of the material, should be viewed not only as acceptable, but with growing enthusiasm in the last several years. This process, known as ultramicrotomy, originated in life science TEM, but has been modified by materials scientists and applied to an impressive array of materials as illustrated in Table 1, culminating, perhaps, with the recent successful sectioning of diamond film.

Over the past few decades, the demands of modern analytical electron microscopy have increased the need for TEM specimen preparation techniques with a minimum of misleading artifacts in terms of chemical microanalysis. At the same time, the demands of modern industrial materials, be they semiconductor, polymeric or composite in nature, call for speed, flexibility and high spatial resolution as well. The response from the electron microscopy community, especially that portion in the private sector, have been to devise (or advocate) radically different forms of TEM thin specimen preparation from that of classic replication, electropolishing and ion thinning.

This tutorial sets forth the goals of TEM specimen preparation, and the requirements for a "good" TEM specimen. The strategic choices governing which technique to use for preparing a wide variety of specimens will be covered. A TEM Specimen Preparation Flow Chart will be used to plot a course that makes optimum use of the preparation techniques available as a function of the type of specimen to be prepared.

While the techniques offer high spatial resolution in some cases down to the atomic scale and three dimensional mapping of surface topography, there still remain issues related to quantitative interpretation of scanned probe data, particularly in the recently developed phase contrast imaging modes. In this talk, the various modes of force microscopy will be introduced, along with examples. New techniques are starting to emerge which allow us to use the scanned probe microscope to measure properties such as local adhesion and local elastic and plastic deformation of samples.

Each scanned probe technique relies on a very sharp probe positioned within a few nanometers of the surface of interest. Some combination of probe and/or substrate positioning is required to provide sub-nm-resolution, three-dimensional motion of the probe relative to the substrate. When the probe translates laterally (horizontally) relative to the sample, any change in the height of the surface causes the detected probe signal to change.

Many imaging processes, including both conventional wide-field and confocal fluorescence microscopy, can be described to a good approximation as linear and spatially-invariant. Linearity means that the image of an extended specimen is simply the sum of the images of the parts of the specimen - in the limit the specimen can be regarded as a collection of points, and its image is the sum of the images of the points - i.e. weighted instances of the imaging system's point spread function (psf). In the case of spatially invariant imaging the psf is the same over the entire field of imaging. Mathematically, the image is the convolution of the specimen with the system psf. In principle, this convolution can be reversed to remove degradation introduced by the imaging process, a computational procedure often called deconvolution or, more generally, restoration.

This report summarizes our experiences gained with deconvolving 3-d images of various types of biological samples - animal and plant cells; live and fixed. We compared raw, unmodified images with images processed with standard filtering methods and images improved by using deconvolution. The blind decovolution software package from AutoQuant, Inc. was used. All images were acquired using a BioRad MRC 1024 confocal microscope (attached to a Nikon Diaphot microscope), using fluorescence or reflected light. For thin (up to 25 μm) flat specimens a 60x PlanApo NA1.4 lens was used while for thick (25-200 μm) water containing samples a 40x Fluor NA1.15 water immersion lens was used.

In an attempt to test the usefulness and validity of deconvolution for various types of biological samples we adopted the following approaches:

1. collect images of structures that are known to be both fluorescent and reflective - compare the results of deconvolution of fluorescence images with reflected light images that are not deconvolved.

We are using wide-field deconvolution microscopy to study the locomotion of cells within a three-dimensional cell mass. How cells move within such an environment is poorly understood, even though such movement occurs widely, for example, during embryonic development or tumor cell invasion. In these and other cases, cells moving within a cell mass face challenges not encountered by isolated cells crawling on a flat substrate. Cells within a mass must clear a path for themselves, and also gain traction on other cells which themselves may be moving in the same or another direction. To understand the mechanisms of such 3D movement, we observe and analyze it in both normal and mutant cells. The mutant analysis sheds light on which molecules are important for 3D locomotion.

To visualize either normal or mutant cell locomotion within a three-dimensional cell mass, we require time-lapse 3D imaging at light levels low enough to maintain specimen viability.