Electron tomography has proven to be an invaluable tool for studying the 3-dimensional organization of a wide range of structures, from large cellular processes down to individual macromolecular complexes. An important requirement for electron tomography of thick specimens is the need for selective staining to delineate the structure of interest from other cellular constituents. of particular usefulness in this regard is the method of fluorescence photooxidation, whereby the reactive oxygen generated by a fluorescent compound is used to oxidize diaminobenzidine into a reaction product that can then be visualized with the electron microscope. The main advantages of this method are that not only does it allow for excellent correlated light and electron microscopy, but also the relatively small size of the oxidizing agent used allows for excellent 3-D labeling with high resolution.

This method has proven to be a particularly versatile technique.

A number of recent studies of grain boundaries and heterophase interfaces have demonstrated the power of combining Z-contrast STEM imaging, EELS and first-principles theoretical modeling to give an essentially complete atomic scale description of structure, bonding and energetics. Impurity sites and valence can be determined experimentally and configurations determined through calculations.

Here we present an investigation of the Si/SiO2 interface. The Z-contrast image in Fig. la, taken with the VG Microscopes HB603U STEM, shows that the atomic structure of Si is maintained up to the last layers visible. The decrease in intensity near the interface could originate from interfacial roughness of around one unit cell (∼0.5 nm), or may represent dechanneling in the slightly buckled columns induced by the oxide. Fig. lb, taken from a sample with ∼1 nm interface roughness, shows a band of bright contrast near the interface. This is not due to impurities or thickness variation since it disappears on increasing the detector angle from 25 mrad to 45 mrad (Fig. lc), and is therefore due to induced strain.

The tutorial presents 3D visualization as implemented at NCMI: beginning with a brief overview of the history and philosophy of scientific visualization, proceeding to a description of general methodologies used throughout the field of visualization, and concluding with specific applications in electron microscopy, confocal microscopy and x-ray diffraction. The majority of the tutorial uses biological examples of visualization to demonstrate concepts and tools.

History and Philosophy: Effective scientific visualization presents data in a simple, accurate conceptual formulation. Over the last one hundred years there has been a sea change in its economics caused by digital computers: beginning with calculated tables that were manually plotted, proceeding to two dimensional image graphics, and most recently multivariate 3D interactive graphics. The capability of scientific visualization has been greatly facilitated by the evolution of computers; particularly over the last ten years the 3D visualization of biological structures is quickly becoming a necessity for analysis, conceptualization, and presentations.

Variable pressure scanning electron microscopes (VPSEM) differ from conventional SEM by operating at pressures ranging from the ‘high vacuum’ SEM levels of 10-6 torr up to typically around 2 torr. The environmental SEM or ESEM is a commercial variant which employs an unique multistage pressure-limiting aperture (PLA) system to attain specimen chamber operating pressures of up to 50 torr. Early instruments used air or argon as the imaging gas but more commonly today water vapour is used. A wide range of gases have been employed, including potentially explosive hydrogen-methane mixtures. The choice of gas is operator-based and can be varied during the imaging session.

Early VPESM were restricted to backscattered electron imaging (BSE) until the development of the gaseous secondary electron detector in the ESEM. Gaseous secondary electron detectors are now available for all models of VPSEM and together with compatible cathodoluminescence and EDS XRay detectors, the full range of SEM-based imaging options is present.

The principal distinguishing feature of VPSEM is, of course, that samples can be examined uncoated. Gas-electron interaction generates a positive ion supply that can minimise conventional charging artefacts, in a simple imaging model.

Microscopy has always been concerned with the observation of samples in their natural states. The earliest instruments, optical microscopes, did not interfere with the samples which were under observation due to the pervasive presence of visible light in the normal evolution of these entities. Man and science persisted in this direction until Ruska in 1933 invented the electron microscope and the real world changed forever (see MSA Rudenberg for an enlightening description of the early days of man's understanding of the electron and the subsequent invention of the electron microscope). The earliest electron beam instruments were single vacuum chamber entities. As single chamber designs, every element in the optical path was under vacuum. These included the electron gun, the lens and coils, recording film, and of course, the specimen itself. The purpose of the vacuum was quite obvious...to eliminate the scattering of the electrons due to the presence of gas.

The environmental scanning electron microscope (ESEM™) and variable pressure electron microscope (VPSEM) have become accepted tools in the contemporary electron microscopy facility. Their flexibility and their ability to image almost any sample with little, and often no, specimen preparation has proved so attractive that each manufacturer of scanning electron microscopes now markets a low vacuum model.

The University of Michigan Electron Microbeam Analysis Laboratory (EMAL) operates two variable pressure instruments, an ElectroScan E3 ESEM and a Hitachi S3200N VPSEM. The E3 ESEM was acquired in the early 1990s with funding from the Amoco Foundation and it has been used to examine an extremely wide variety of different materials. Since EMAL serves the entire university community, and offers support to neighboring institutions and local industry, the types of materials examined span a wide range. There are users from Materials Science & Engineering, Chemical Engineering, Nuclear Engineering, Electrical Engineering, Physics, Chemistry, Geology, Biology, Biophysics, Pharmacy and Pharmacology.

When performing X-Ray microanalysis in the ESEM (Environmental Scanning Electron Microscope) or in the VP-SEM (Variable Pressure Scanning Electron Microscope), the operating conditions of the microscope must be optimized. This is to reduce the beam broadening of the incident electrons when they scatter with the gas molecules before entering into the specimen. As a result of this scattering, the incident beam is composed of two parts. The first part of the beam is the unscattered beam and the second part is the scattered beam, named the skirt. in high pressure and long working distances conditions, the diameter of the skirt may extend to several millimeters. in order to show the effect of the skirt on X-Ray generation, a copper strip was placed .5 mm away of the electron beam on a flat Al specimen. The peak to background ratio of the copper line was measured at different pressure (from 25 to 200 Pa) for Air as gas.

Charge contrast imaging (CCI) is a useful new method for imaging sub-micron features in crystalline materials using the unique gas/ion/electron imaging system of the environmental scanning electron microscope (Griffin, 1997; Doehne, 1998). Crystal growth zoning, microfractures, solution boundaries, and areas of chemical alteration or recrystallization can be imaged in a wide range of materials (Griffin, 2000; Watt, et al. 2000). While not fully understood, charge contrast images reflect differences in the ability of materials to accept, store and discharge deposited electrons from the primary electron beam. These differences are expressed, in turn, as contrasts in secondary electron emission from flat samples (e.g. these contrasts are not related to topography, as is usually the case). Charge contrast appears be related to differences in electronic properties which are often controlled by defect density. CCI is also affected by small-scale physical defects (such as microfractures) which appear to affect the distribution and timing of charge buildup and discharge in the sample (Johansen, et al. 1997).

Of all the commercially available scanning electron microscopes which operate at “low vacuum” the ESEM is the most suitable for examining biological samples. in order to maintain samples with liquid water present the specimen chamber must be capable of operating at a pressure of at least 4.6 Torr (611 pascals) of water vapour pressure (the vapour pressure of water at 0°C). Use of lower pressures or a chamber gas other than water vapour will result in evaporation of water from the sample at a rate dependant on the partial pressure difference between the sample and its surrounding environment. Tables of relative humidity as a function of water vapour pressure and temperature are readily available to calculate desired settings for the microscope.

One of the difficulties associated with examining fresh biological material is the need to have the microscope and sample available in the same location at the same time.If sample collection occurs at a site remote from the microscope inevitable necrotic changes will occur before examination can be carried out.

Environmental SEM has greatly extended the utility of SEM as a result of an ESEM's ability to image volatile and insulating samples. However, many institutions have older high-vacuum SEMs that they cannot afford to replace with an ESEM. For this reason, a simple and inexpensive pressure cell has been developed for performing low-vacuum SEM in a standard microscope. Rather than backfilling the entire analysis chamber with gas, a small volume of higher pressure is confined at the sample surface with the use of a pressure cell that has a small aperture for admitting the primary beam and a gas inlet. Figure 1 is a photograph of such a simple pressure cell.

The aperture for admitting the primary beam needs to be small enough to limit the escaping gas to an amount below what the standard SEM can pump and still maintain high vacuum. On the other hand, the aperture should be as large as possible to provide a useful field of view.

In a conventional scanning electron microscope, a thin, grounded conductive coating is applied to specimens that are poor electrical conductors to prevent retarding and deflection of the incident electron beam. in a variable pressure or environmental scanning electron microscope (ESEM), excess charge on the surface of uncoated poorly conducting specimens is balanced using ionized environmental gas. Ionized gas in environmental mode and grounded conductive coatings in conventional or high vacuum mode minimize charging at the specimen surface, however significant charge trapping may still occur in the implanted sub-surface regions of poorly conducting materials. A small fraction (<10-6) of the incident electrons are trapped at irradiation induced or pre-existing defects within the irradiated specimen. The trapped charge induces a highly localized electric field which can result in electro-migration and micro-segregation of charged mobile defect species within the irradiated micro-volume of specimen.

The Environmental Scanning Electron Microscope (ESEM) is capable of image generation in a gaseous environment at sample chamber pressures of up to 20 torr. in an ESEM, low energy secondary electrons emitted from a sample surface, by virtue of the primary electron beam, are accelerated towards the positively biased metallic ring (typically +30 to +550V) Gaseous Secondary Electron Detector (GSED). As these electrons accelerate towards the ring they undergo ionizing collisions with gas molecules producing positive ions and additional electrons known as environmental secondary electrons. The environmental electrons further ionize the gas on their way to the ring producing a cascade amplification of the original signal. The amplified signal induced in the ring is used to form an image. The electric field generated between the GSED ring and the grounded stage causes the positive ions produced in the cascade to drift towards the sample, effectively neutralizing negative charge build up on the surface of a non-conducting sample.

An increasing body of experimental evidence indicates that the flux and distribution of gaseous ions significantly affects secondary electron contrast of dielectric materials imaged in low vacuum and environmental scanning electron microscopes. Unusual contrast has been identified in some insulating materials, and attributed to charging effects (1-3). However, as these contrast mechanisms have not been reported in conventional microscopes, it would seem that the ions play an important role in making the contrast manifest. The effects of ions can include the creation of space charge in the gas above the specimen, influencing the charge state of the specimen surface, and electron-ion recombination (possibly leading to energy filtering of emitted secondary electrons). Space charge and surface potential can influence the gain realised in the gas amplification cascade by altering the detector field (4). However, surface potential, and recombination affect the number of electrons injected into the amplification cascade, to give rise to contrast variations in the final image.

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 [1]. in particular it has been possible to image in conditions that allow the samples to remain fully hydrated during observation. This unique feature of the ESEM has been exploited to image the dehydration of aqueous protein gel materials which have been created under controlled conditions to produce a range of Gel types.

Drops of several μl of the Gels were placed in the Peltier cooling stage of the FEI-Philips Electroscan E3, and allowed to stabilize at 4.1°C. Several drops of water were placed on the stagesurround to maintain a high vapor pressure of water around the sample. The chamber was then pumped down to the working pressure of 6 torr and then back-filled with pure water vapor following the technique of Cameron et.al. [2].

We present experimental evidence for the effects of electron-ion recombination on contrast formation in secondary electron (SE) images obtained using an environmental scanning electron microscope (ESEM). Inclusion of the effects of SE-ion recombination in the theory of ESEM image formation accounts for contrast reversal observed, under appropriate conditions, in SE images of conductors (ie, in the absence of localized specimen charging) and of insulators that exhibit localized charging. Previously unexplained dynamic contrast caused by temporal charging can be understood if both conventional models of the effects of charging on SE emission, and the proposed role of SEion recombination in ESEM image formation are accounted for.

In an ESEM, the extent of charging exhibited by insulating specimens can be controlled by varying the type and pressure (P) of the imaging gas, and operating parameters that determine the extent to which the gas is ionized (eg, detector bias, VGSED, and working distance, WD).

In a focused ion beam (FIB) instrument, ions (typically Ga+) obtained from a liquid metal ion source are accelerated down a column at energies up to ∽ 50 keV. The beam of ions is focused by electrostatic and octopole lens systems and the ion dose (and beam diameter) is controlled using real and/or virtual apertures. Beam sizes in FIB instruments on the order of 5-7 nm may be achieved.

The versatility of the FIB instrument enables large regions of material (e.g., 500 μm3) to be removed at high beam currents in just a couple of minutes. Lower beam currents (i.e., beam diameters) are usually used to remove smaller amounts of material within the same time frame (e.g., ∽ 5μm3). The introduction of an organometallic gas in close proximity to the target allows for the deposition of metals, SiO2, and other materials, by an ion beam assisted chemical vapor deposition process.

A microscope facility at teaching institutions needs to be more than an isolated refuge of researchers. Students must be given easier access to the equipment and an increased awareness of the power of these tools for understanding the scientific principles taught in their lecture classes. The San Diego State College of Sciences supports a shared instrumentation Electron Microscope Facility, a suite of seven rooms that occupies 1500 square feet. The laboratory is fully equipped for all aspects of sample preparation for examination by confocal scanning light microscopy, scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Historically, specialists with many years training in equipment operation, sample preparation, and research protocols have had exclusive access to EM facilities. At SDSU, however, every effort is made to encourage inclusivity via familiarity with and hands-on access to equipment in the EM Facility. in all, over 1000 SDSU students and several hundred community members participate each year in opportunities to access the SDSU EM Facility.

The Biosciences Electron Microscopy Facility at UBC has at present two TEMs, one SEM, two confocals. several fluorescent light microscopes and an above average amount of digital imaging equipment used p by graduate students. The facility supports the Faculty of Medicine and the Faculty of Science and is run by a Director (myself) with one assistant. It is busy having an average of 600 individual users per year. The only way this number of users can be accommodated is by making them independent of staff. Training students had been previously on a one-to-one basis. This is very time consuming. Four years ago with no full time assistant, I decided to approach training in a different way. We started a confocal microscopy workshop aided by one PhD student.

We run a 1.5 day practical confocal workshop. It is one day as a group, giving the basic concept of confocal microscopy, and using the instrument from “this is how you switch the machine on” to “this is how you deal with the stack of images.”

For many years the means of communicating electron microscopy techniques had been through either the spoken or the written word. with the advent of the computer and in particular the notebook computer more exotic methods of information presentation were made available. Now with the compact disk or the DVD, with their ability to store very large quantities of data, even more new avenues have been opened up for trainers.

There are a number of programs by small organisations, like QM Slide Show and EZ Slide, that enable the trainer to take the simple approach to presentation preparation. Using a Windows Notepad interface or similar the trainer gathers bitmap files in a simple sequence, referencing the file source to activate each part of the presentation. This type of presentation is extremely flexible, so much so that the trainer, with a large selection of bitmap files and a good filing system, may develop a completely new or modified presentation in just a few minutes.

Two of the greatest challenges facing any educator are deciding what information will be included in a syllabus and how to present it for maximum learning. Distilling the experience derived from a quarter of a century teaching microscopy exposes one driving concept: the importance of education, not just training.

The difference between these two approaches is fundamental. Education involves laying a foundation on good science, building a vocabulary for better communication and future growth, and engendering a questioning attitude about the probe-matter interactions which, in turn, leads to valid interpretation of the information in an image. Training is a matter of learning what knob to turn, which button to push: an approach which overlooks the sample itself; how the light, electron, or scanning force is interacting with that sample; and what can be learned as a result.