Laura E. Reuss

Specimen Preparation for Electron Microscopy

The purpose of fixation is twofold: to bring about the rapid cessation of biological activity and to preserve the structure of the cell. Ideally, the colloidal Suspension of the cytoplasm and organelles within a cell is turned into a gel that maintains the spatial relationship of the components while providing sufficient stability for them to survive the solvent action of aqueous buffers, dehydration agents, and plastic resins. The objective is to process tissues and cells without significant changes in size, shape, and positional relationships of the cellular components and to preserve as much of the biological activity and chemical nature of cellular constituents, such as enzymes and antigenic proteins.

Staining Methods for Semithins and Ultrathins

When we speak of staining semithins, we are generally referring to 0.25- to 0.5-μm-thick sections cut from blocks of epoxide-embedded tissues. There are other resins such as the acrylic resins (Lowicryls, LR White and LR Gold) in common use that will have different staining responses from epoxides because of their partial miscibility with water. In addition, semithin frozen sections have still other staining characteristics resulting from their significant hydrophilicity. For the purposes of this chapter, however, we will limit our discussion almost exclusively to the epoxide sections, since these are the most commonly used resins for biological work. Any of the stains for these resins will work with much reduced staining time on more water-miscible sections.

Morphometry and Stereology

Most biologists interested in morphometric analysis and stereology purchase programs to do the work, and a specific knowledge of all the algorithms and Statistical calculations behind their Operation is not critical. The programs are booted up, the menus are consulted to see what functions are possible, and the Operator instructs the Computer to perform certain Operations from the menu. At the same time, if the Operator does not possess knowledge concerning the types of questions that may be asked, the limitations inherent in the sampling techniques being used, and the general underlying assumptions necessary for morphometric analyses, the Software may not be used to its ultimate extent. The purpose of this chapter is to give an overview of the capabilities and a few of the shortcomings of morphometric analysis programs used today. Several texts (Baidock and Graham, 2000; Hader, 1991; Howard and Reed, 1998; Mouton, 2002; Russ, 1986, 1990, 2002; Russ and Dehoff, 2000) cover most aspects of the mechanics behind morphometric analysis and stereology and address specific applications in great detail. They are recommended reading prior to embarking upon a project requiring morphometric or stereologic analysis methods. The text by Misell (1978) was written prior to the currently available Software, but deals extensively with the principles and methods for image enhancement and analysis not confined to Computers.

Transmission Electron Microscopy

Microscopy tools for direct visualization of cytological details include the light microscope, the confocal scanning microscope, and the transmission electron microscope (TEM). The scanning electron microscope involves circuitry that processes electronic Signals into an indirect image seen on a cathode ray tube or digital screen. All of the direct visualization methods involve light or electrons interacting with specimens and utilize either glass or magnetic lenses to project an image to the eye, to a screen, or to film. When considering the development of electron microscopes, it is useful to recognize the antecedents from the realm of light microscopy. The concepts of electron optics underlying electron microscopes are primarily extrapolations from the physics of light optics.

Digital Imaging and Telemedicine

Digital imaging has become an important aspect of image acquisition, storage, and Output in all arenas of cytological investigation and analysis, particularly since the late 1980s, when Computer storage media and computational speed began their rapid improvement that continues through the present time. Even though Photographie materials still hold more pixels, or picture elements, than digital media, the ease with which images can be captured, manipulated, and Output to Microsoft Powerpoint® presentations, emails, web pages, and photographic-quality Computer prints has led to the widespread aeeeptance of digital images for scientific presentation. Some Journals currently request electronic Submission of papers, including illustrations, in digital format, totally bypassing hard copy of either text or images. Finally, telemedicine (Weinstein et al, 2001; Williams et al, 2001) allows pathologists and other medical personnel to send images quickly to other scientists and doctors for examination to help determine the cause of pathology and the treatment that should follow for the patient. This subdiseipline of digital imaging is guiding the way for biologists to share images and the information contained within them quickly with colleagues throughout the world.

General Sources for Information Concerning Microscopy

Throughout the text, various books and Journal articles are listed. There are atlases on ultrastructure for insects, protozoans, fungi, mammals, and other organisms. The list below of Journals and general books is not exhaustive but offers a starting point to begin researching questions about biological ultrastructure.

High-Voltage Transmission Electron Microscopes (HVEM)

The first high-voltage electron microscope (HVEM) capable of generating 1,000 kV was put into Operation in Toulouse, France in the laboratory of Dr. G. Dupouy in 1960 (Dupouy, 1985). In 1969, Dupouy installed a 3,000-kV instrument, which offered improved specimen penetration and reduced chromatic aberration, but did not offer significant advantages over the original 1,000-kV instrument.

Electron Microscopy Equipment and Supplies

Listed below are some of the suppliers, primarily in the United States, who can provide the products necessary for light and electron microscopy work. There are other suppliers, but this list will provide a starting point when in need of specific items.

Replicas, Shadowing, and Negative Staining

Contrast in transmission electron microscopic (TEM) specimens occurs when there are specimen areas that stop (scatter) electrons and also areas that let most of the electrons pass through. Thus, the image results from subtractive contrast. We impregnate tissues and sections with a variety of heavy metals to scatter electrons as discussed in Chapter 4, but we can also Surround particulates with heavy metals (negative staining) or cover particulates, cells, and tissues with thin metal films that have areas of differential beam-stopping capability (replicas produced by shadowing). This chapter will discuss these two added techniques for building subtractive contrast, pointing out the commonly used techniques and a few remedies to specific problems that may be encountered.

Intermediate Voltage Electron Microscopes (IVEM), Electron Tomography, and Single-Particle Electron Microscopy

Contemporary IVEMs as typified by those from JEOL (JEM-2010 and JEM-3010) and FEICO/Philips (Tecnai T20 and Tecnai T30) possess most of the advantages of beam penetration of HVEMs without most of the disadvantages associated with HVEM cost, stability, and Space requirements. The original IVEM units did not provide images at low accelerating voltages (100-120 kV) that were comparable with conventional TEM images, but modern IVEMs are considerably more flexible. It is now possible to achieve high resolution for both ultrathin sections and semithin sections in excess of 3 p,m with IVEMs, particularly if equipped with energy filtration systems.