High resolution imaging in electron cryomicroscopy of biological macromolecules is strongly affected by beam-induced charging1. Charging is often expressed in frozen or glucose-embedded specimens as an increase in apparent mass-thickness of the irradiated area. Another obvious effect of charging is blurring of both the unscattered beam and reflections in electron diffraction patterns recorded from crystalline specimens. Coating of ice-embedded specimens with a carbon layer helps to improve the stability of the ice and probably reduce charging of the specimen. Coating in a Gatan ion-beam coater (model 681) of glucose-embedded specimens with thin layers of various conductive materials did reduce charging but the specimens were damaged by the high energy ions used for the coating. In general, coating resulted in much weaker reflections in electron diffraction patterns obtained from coated crystals and faster resolution fall-off.

We modified the Gatan coater by outfitting it with a new chamber that replaced the ion-beam deposition capability for thermal evaporation of carbon rods (Fig. 1).

Endothelial cells (EC) possess receptors for and metabolize low-density lipoprotein (LDL); acetylation of LDL promotes EC metabolism and the intracellular deposition of its cholesteryl ester component. In turn, acetylated LDL linked to the fluorescent dye, Dil (Dil-Ac-LDL), has been used as a marker for endothelial cells, including cerebral microvascular endothelial cells (CEC). Due to the low electron density of fluorescent dyes, however, detection of this marker is not possible ultrastructurally. We demonstrate here the photo-conversion of the chromogen, 3,3'-diaminobenzidine tetrahydrochloride (DAB), into a stable, electron-dense reaction product in subcultured human fetal CEC containing the fluorescent marker, Dil-Ac-LDL, thus promoting identification of these cells via light and electron microscopy. This method, in turn, may be useful in identifying and tracking human fetal CEC grafted into SCID mice as a model for blood-brain barrier angiogenesis.

Isolated human fetal CEC were subcultured and labeled with Dil-Ac-LDL (10 ug/ml).

This tutorial emphasizes the fundamentals of the use of light microscopy in combination with electronic imaging. The goal of the tutorial is to enable the attendees to make informed choices both about the modes of imaging best suited to their application and about the most suitable components to be employed. The didactic portion of the program will consist of five presentations, each dealing with different aspects of modern imaging systems and their applications to problems of biological significance. A laboratory demonstration session, organized to complement the lectures will follow.

The morning session will begin will a brief review by Dr. Spring of the principles of light detection and the formation of the video signal. Dr. Salmon will then describe video-enhanced contrast microscopy. This transmitted light imaging technique, widely utilized to visualize low contrast specimens exploits the wide dynamic range and high gain of video cameras for the capture of images that are virtually invisible through the eyepieces because of high background light.

Light microscopy instrumentation and biological applications continue to expand rapidly, and an important aspect of this expansion is three-dimensional (3-D) imaging and image analysis. For many biological specimens, optical sectioning, i.e. collecting images at a sequence of depths under controlled conditions, provides true 3-D data through the entire specimen. This is especially important for specimens whose thickness exceeds the depth-of-field of the microscope objective lens. This sequence of optical sections is the basis for 3-D image reconstruction providing information from all three specimen dimensions instead of just the traditional two in the image plane of the microscope. The analysis of images in 3-D provides insights into the structure and function of biological specimens that are not available through other means. However, 3-D microscopy also presents additional choices for image collection. The first of which is whether to use widefield or confocal microscopy and the second is whether to utilize digital deblurring or deconvolution methods.

There has been much provocative discussion over the past few years about the advantages and limitations of deconvolution methods. In this tutorial we will provide a reasonable understanding of the underlying theoretical principles of deconvolution, with an aim at understanding expected advantages and limitations. These principles will be presented for a mixed audience composed of biologists, neuroscientists, engineers, physicists and other scientists and graduate students. These principles will be presented in the context of both widefield and confocal microscopy deconvolution.

The basic principles of Fourier optics will be presented. From these principles, we can explain why it is that the confocal microscope provides a superior in-plane and axial (z-axis) resolving power, compared to the widefield microscope. In particular, for example, the Fourier transform of a 3D widefield image contains a missing cone region (to be explained in the tutorial), and this missing cone region effectively spoils the axial resolution of unprocessed widefield data.

Three dimensional (3D)microscopy is a powerful toll for the visualization of biological specimens and processes. In 3D microscopy, a 3D image is collected by recording a series of two-dimensional (2D) images focusing the microscope at different planes through the specimen. Each 2D optical slice in this through focus series contains the in-focus information plus contributions from out-of-focus structures that obscure the image and reduce its contrast. There are two complementary approaches to reduce or ameliorate the effects of the out-of-focus contributions, optical and computational. In the optical approach a microscope is used that avoids collecting out-of-focus light, such as a confocal microscope (see and references therein), a two-photon or three-photon fluorescence excitation microscope, or atwo-sided microscope. In the computational approach, the through-focus series is processed in a computer using any of a number of debluring algorithms to reduce or ameliorate the out-of-focus contributions. In the past two decades, several methods for debluring microscopic images have been developed whose common aim is to undo the degradations introduced by the process of image formation and recording

Three-dimensional (3-D) light microscopy has been rapidly expanding for several years, both with respect to instrumentation, and biological applications. This new technology has transformed light microscopy from two-dimensional imaging of 3-D objects to a fully 3-D science. To date, qualitative analysis, reconstruction, and visualization has received far more attention compared to quantitative analysis. The motivations for 3-D image analysis are manifold, and include more accurate morphometry, separation of overlapping objects, and analysis of non-planar regions of interest and of thick specimens without the artifacts arising from physical sectioning. Furthermore, automating this analysis makes it fast, inexpensive, objective, and precise, and the results can be validated by manually confirming the analysis for any selected subset of 3-D images.

Increasingly, quantitative 3-D image analysis is emerging as an attractive technology for many applications. Studies include the testing of human-use products (shampoos, laxatives etc.), drug development, aging and development, cell loss/proliferation, fetal alcohol syndrome, and analysis of medical samples such as Pap smears, breast biopsies and a variety of cancers.

Confocal microscopy is a technique that allows researchers to obtain a highly resolved, high-contrast image of a focal plane in a fluorescent specimen by excluding or rejecting light emanating from out-of-focus planes. However, the basic design of confocals results in limitations for many biologists. It is often difficult to avoid photobleaching of specimens, to visualize fine or faintly-labeled structures, or to acquire high-quality images using short exposure times. The photomultiplier tubes used as the amplification detectors in these systems are restricted to a dynamic range of 8 bits (255 intensity levels), produce noise, and are not quantitatively linear detectors.

Members of the Biomedical Imaging Group at the University of Massachusetts Medical School in Worcester, MA have spent the last fifteen years developing and perfecting a digital imaging system that helps scientists overcome these problems. Scanalytics is the exclusive worldwide licensee of this patented technology which allows researchers to obtain high-resolution, quantitatively accurate three-dimensional images of fluorescent specimens

The possibility of performing scanning electron microscopy under “environmental” conditions (ESEM); where the specimen does not have to be under high-vacuum conditions, or even coated to make it conductive, has existed for several years[l]. Several different flavors of environmental scanning electron microscope now exist, as well as a newer generation of microscope which is capable of imaging at very low accelerating voltages. Techniques for rapidly freezing hydrated specimens and obtaining very high resolution images from them in the SEM have also been improved recently. As a result of all this innovation, it is now possible to examine many classes of specimens in the SEM that were previously thought to be impossible to image (or at least not worthwhile frying).

Among the advantages of the environmental SEM are the fact that images of non-conductive surfaces can be obtained without the necessity for additional sample coating. This can be especially important in dynamic experiments, and hot-stage work, where the presence of a surface coating may substantially affect the samples behavior

The environmental scanning electron microscope (ESEM) first marketed by ElectroScan and now by FEI/Philips has been recognized as the only scanning electron microscope which allows liquid water to be present in the sample chamber at temperatures above freezing. As most biological material contains a high percentage of water it would seem essential to use this technology to examine specimens in their natural state. The purpose of this presentation is to describe in detail the way in which the microscope should be used to maximize the information obtainable from a variety of biological samples. This tutorial will also consider methods which can be applied to “difficult” samples whose structure becomes altered during what would be considered a normal imaging schedule.

The main features of the microscope are the use of the Peltier effect cooling stage and control of chamber water vapor pressure to enable wet specimens to be imaged.

The environmental SEM is an extremely adaptive instrument, allowing a range of materials to be examined under a wide variety of conditions. The limitations of the instrument lie mainly with the restrictions imposed by the need to maintain a moderate vacuum around the electron gun. The primary effect of this has been, in a practical sense, the limited low magnification available. Recently this has been overcome by modifications to the final pressure limiting aperture and secondary electron detector (Fig.l). The modifications are simple and users should be brave in this regard.

A variety of electron detectors now exist including various secondary, backscattered and cathodoluminescence systems (Figs 2-5). These provide an excellent range of options; the ESEM must be regarded as a conventional SEM in that a range of imaging options should be installed. In some cases, e.g. cathodoluminescence, the lack of coating provides an advantage unique to the low vacuum SEMs.

This tutorial will describe the technique of electron backscattered diffraction (EBSD) in the scanning electron microscope (SEM). To properly exploit EBSD in the SEM it is important to understand how these patterns are formed. This discussion will be followed by a description of the hardware required for the collection of electron backscatter patterns (EBSP). We will then discuss the methods used to extract the appropriate crystallographic information from the patterns for orientation determination and phase identification and how these operations can be automated. Following this, a number of applications of the technique for both orientation studies and phase identification will be discussed.

EBSD in the SEM is a phenomenon that has been known for many years. EBSD in the SEM is a technique that permits the crystallography of sub-micron sized regions to be studied from a bulk specimen. These patterns were first observed over 40 years ago, before the development of the SEM and were recorded using a special chamber and photographic film.

Accurate descriptions of dispersed metals are of central importance in optimizing the activity, selec-tivity and stability of a diverse set of catalysts and chemistries. Improved methods for quantifying metal dispersion, alloying and support interactions help form mechanistic understandings of catalyst function at the atomic scale. In this paper we discuss ideal capabilities of a transmission electron microscope for catalysis, and give an update on the use of a next-generation field-emission TEM/STEM for structural and analytical studies of supported metal catalysts.

Characterizing nanometer-sized metal particles requires high performance imaging, high sensitivity analysis, and minimal disruption of the physical state of the particles. No single microscope combines all key performance factors with ease of use. Schottky field-emitter instruments provide high current-density probes with excellent stage stability (nm drifts in > 10 min. observed), < 0.7 eV energy reso-lution for EELS, and a turbo-pumped vacuum system for minimal carbon contamination while using nanometer probes and nanoamps of current. Scanning TEM performance is also key.

Nano-fabrications are now available for us to produce well-defined nano-structures showing quantum effects, quantized electron conductance at point contact, of phase interference of electron waves at quantum wires, and single electron tunneling at nano-capacitors. It seems near that we can fabricate even such nano-structures that Professor L.Feynman had described in his book “miniaturization”: the smallest memory unit(nano-particle), which contains 5x5x5 atoms. Such nano-particles are expected to have physical and chemical properties depending on their surface nature, since they have rather large volume of surface atoms compared with that of the core atoms. The surface atoms of such nano-particles might be reconstructed to a structure different from their bulk crystal structure, as well the surfaces of silicon and GaAs are reconstructed. It is, therefore, interesting to fablicate such nano-particles and to study their structures.

To study structure and properties of nano-particle surfaces, we developed a UHV high-resolution electron microscope equipped with a field-emission gun (UHV-FE-HR TEM: revised version of JEOL 2000V) at our ERATO project, as shown in fig. 1.

The discovery of carbon nanotubes by Iijima has stimulated considerable research into their structural, chemical, electrical and mechanical properties. Apart from their intrinsic beauty as macromolecules, and their possibilities as one-dimensional conductors, researchers are stimulated by the notion that a structurally perfect nanotube may be one of the strongest known materials.

It is known that when nanotubes are bent through large angles, regions of high curvature buckle to develop crimps, similar to those observed when metal pipes are bent too much. Figure la shows a multi-walled nanotube that has one end embedded in a ceramic matrix. The other end is snagged so that the tube is bent. Several crimps are visible on the nanotube. Figure lb shows the same nanotube imaged a few moments later. Remarkably, the nanotube has sprung straight and the crimps have disappeared. This demonstrates that buckling in nanotubes does not involve plastic deformation, and that nanotubes can maintain their structural integrity even after severe deformation.

Synthesis of mesoscopic silicates has stimulated considerable interest as a biomimetic approach to the fabrication of organic/inorganic nanocomposites. However, for future applications of these materials, controlled shapes and patterns, particularly as continuous thin films, rather than the microscopic particulates, need to be processed. Recently, we have described a synthesis scheme, using the supramolecular assemblies of surfactant molecules as a template for the condensation of inorganic species, to prepare silica mesoscopic thin films at a solid interface from a bulk solution. Here, we concentrate on the growth of such thin films and the effect of the template on the nanostructure formation.

We employed tetraethoxy silane (TEOS), dissolved in acidic solution, as a silicate source and cetyltrimethyl ammonium chloride (CTAC) as the templating surfactant. Typical molar ratio was 1 TEOS : 1.2 CTAC : 9.2 HC1 : 1000 H2O. Concentrations of TEOS and HCl in the mixture were approximately l/7th of those for typical particle synthesis scheme, in order to sufficiently slow down the rate of TEOS hydration such that homogeneous nucleation is effectively switched off.

One technology for high-density information storage employs magneto-optic thin films. Key material properties include a large magneto-optic Kerr rotation of polarized light (especially in the blue wavelength range), high perpendicular magnetic anisotropy, good reflectivity and a large magnetic coercivity. These can be achieved in nano-scale magnetic multilayers (e.g., Co-Pt) in which one of the constituent layers is ferromagnetic. Naturally the magnetic properties can be manipulated to a great extent by the preparation conditions, and the microstructure is most readily revealed by cross-section high-resolution electron microscopy (HREM). In our work, extremely high quality multilayers were fabricated by sputtering using a variety of inert gases, and most notably the influence of interface sharpness on properties was assessed. Intermetallic compounds, which are essentially “natural” multilayers, were also extensively studied.

Figure 1 shows magneto-optic Kerr rotation hysteresis loops for multilayers sputtered in 5 mtorr of either argon or the heavier xenon gas.

Thin film multilayers have been the focus of extensive studies recently due to the interesting properties they exhibit. Since the improvement in properties can be attributed directly to the unique nanoscale microstructures, it is essential to understand the factors affecting the microstructural stability in these nanolayer structures. The intermetallic compound, MoSi2, despite its superior oxidation resistance and high melting point, suffers from inadequate high temperature strength and low temperature ductility, properties which hinder its high temperature structural applications [1]. SiC is a potential second phase reinforcement due to its high temperature strength and thermal compatibility with MoSi2. The addition of SiC in a nanolayered configuration has been shown to exhibit significant increase in hardness after annealing [2]. It has also been shown that when annealed above 900°C, the layers break down and grain growth sets in, with a significant decrease in hardness and. Due to the lack of a thermochemical driving force, the two phases remain separate at all temperatures investigated. In this study, the stability of the MoSi2/SiC nanolayers structure under ion irradiation has been investigated.

The large surface area: volume ratios and fine grain size of nanophase materials give rise to novel and exciting structural and electrical properties that are of considerable scientific and technological interest. Using copper as a model system we have investigated the sintering of sputtered copper nanoparticles (4-20nm diameter) with a copper substrate in a novel UHV in-situ TEM.

The nanoparticles were generated in a UHV chamber built into the side of the column by sputtering in 1.5Torr Ar. They were transported into the microscope in the gas phase and deposited on an electron transparent (001) copper foil mounted on a heated support. A typical bright-field (BF) image of the sample immediately after deposition at room temperature is shown in Fig. 1. The particles have assumed a random orientation on the substrate and remain stable for many hours at room temperature. The presence of both single particles and agglomerates of particles is evident in this image and examples are labelled ‘P’ and ‘A’, respectively

The ordering of size selected nanocrystaline particles into large, well ordered supercrystals has been under recent study. Here we describe the ordering of silver nanocrystals, of core diameter ca. 4.5nm, passivated with alkythiolate self-assembled monolayers. The faceted nanocrystals are found to size selectively condense into ordered supercrystals with edge lengths on the order of 0.1 to 0.5 microns. Further, evidence has been found in High Resolution TEM micrographs of packed monolayers of these same nanocrystals implying an orientational ordering of the silver nanocrystals’ atomic lattices within and co-aligning with the supercrystal’s “superlattice”. A three dimensional model using a truncated octahedron for the nanocrystal core morphology is constructed from this evidence into an FCC superlattice which fits the remaining experimental observations.

Micrographs of the same region of a nanocrystal Ag supercrystal (NCASX) are shown in figure 1 under differing defocus conditions.