Introduction to microscopy
The study of histology, the subject of this text and atlas, is carried out using microscopes of various types in order to visualise the structure of body tissues. Structure is closely related to function, and much can be deduced about the function of cells and tissues by careful examination of their component parts. Taken together with information gathered from biochemistry, physiology and other basic sciences, this study can provide a powerful tool to understand the normal functioning of the body. In addition, acquiring this knowledge is a necessary first step for the understanding of disease. Histology is about looking at structure, and in this introductory section we aim to provide some guidelines to assist the absolute beginner in examining and interpreting the images in this book.
This book mainly uses photomicrographs taken with the light microscope (LM) (colour images) and the electron microscope (EM) (black and white images). Simply put, the LM and EM differ in optical resolution and available magnification. In practical terms ‘resolution’ refers to the capacity of an optical system to reveal detail in a specimen. The resolution available from a conventional LM is only about 0.2 µm. Thus at distances of less than 0.2 µm, objects that are actually separate from one another will appear to merge. In contrast, EM resolution for biological specimens is as little as 1 nm, so that the resolving power is about 200-fold better than LM. In addition, maximum ‘available magnification’ is limited to about ×1000 in most student LMs, whereas an EM readily achieves 100-fold greater magnification, or about ×100 000. EM images are therefore said to display cell and tissue ultrastructure.
There are two types of electron microscope: scanning EM and transmission EM. Scanning EM produces three-dimensional (3D) images, but these are restricted to the surface of the object, with the internal structure concealed from view. Transmission EM is so named because the electron beam must pass through the specimen to form an image. To achieve this, ultrathin sections (50–100 nm) must be cut. Transmission of the electron beam through the tissue results in a two-dimensional (2D) image of the plane of the section. In practice, transmission EM is more informative of biological ultrastructure, and these images predominate in this book. We have supplemented these with scanning EM images where it helps with 3D conceptualisation (cf. Figs 16.13 and 16.14). As a matter of convention the abbreviation EM can be assumed to be a transmission EM, while we have identified scanning EMs as SEM.
The strengths of LM and EM differ yet complement one another very effectively. With LM one can observe large areas of a specimen (usually several cm2). A wide range of staining methods, some empirical, some specific, are available for LM, permitting identification of cell and tissue features; many of these stains are polychromatic, i.e. they produce multiple colours in the specimen which, besides looking pretty, help to identify different components. For certain specimens, sections slightly thicker than usual may be used to demonstrate 3D features. Thus from LM, students can expect to gain an understanding of overall cell and tissue architecture.
The superior resolution and magnification of EM permit visualisation of many features which simply cannot be seen by LM. Yet in some respects EM is less flexible than LM. For example, the available area in EM specimens is generally less than 1 mm2 and this may make it difficult to obtain representative fields. Few staining methods are available for EM and these produce only monochromatic (black and white) images. EM is also costly and time-consuming and usually not available to the average student.
Interpretation of EMs can be quite challenging due to the wide range of magnifications available (×500–190 000 in this book). In other words an EM image is not necessarily of very high magnification. In fact, there is an overlap in the magnification ranges of EM and LM. It is a good idea to consciously note the magnification and/or scale bar on each image in the book. The terms electron-dense and electron-lucent are used to describe the relative darkness and lightness, respectively, as they appear in transmission EM images. Sections examined by EM are almost featureless unless stained with heavy metals (e.g. uranium and lead salts) that bind to cell and tissue components to varying degrees. Significant binding of metal stain to a particular structure will impede transmission of the electron beam through the specimen at that point; the structure will appear dark grey or black and is said to be electron-dense (really too dense to allow passage of electrons). Other structures with little or no affinity for the stain will appear lighter grey or white and are termed electron-lucent because they permit greater transmission of the electron beam.
A useful starting point in interpreting EMs is to select several commonly found structures that you can confidently identify and memorise their dimensions. These can then be used as ‘internal rulers’ to gauge the dimensions of numerous other features in the field. For example, plasma membrane and organelle membranes will be visible at medium magnifications as thin electron-dense lines that measure about 10 nm wide. Thus, structures such as intermediate filaments (10 nm in diameter and solid) and microtubules (20–25 nm in diameter but hollow) can be identified. Similarly, individual ribosomes and glycogen particles are 20 to 30 nm in diameter. Being alert to major size differences between organelle types will instill further confidence. For example, nuclear diameter (5–10 µm in most cells) is up to 10 times greater than the diameter of lysosomes and mitochondria (0.2–1.0 µm) and up to 100 times greater than individual Golgi transport vesicles (50–100 nm). The next step is to actively look for the unique set of features that characterises and distinguishes each organelle and inclusion. For example, only mitochondria and the nucleus possess a double membrane, and in mitochondria the inner of these two membranes is thrown into highly characteristic folds.
High-magnification electron micrographs are often required to demonstrate particular features but usually display only a tiny region of the cell. Therefore, do not be surprised if many of the common organelles are not seen in the field. A reliable indicator of high magnification is if an individual membrane appears trilaminar rather than as a single electron-dense line. At low magnification, EM interpretation can actually be more difficult because membranes and the smallest organelles are no longer clearly visible. Get orientated by looking first for the biggest objects, i.e. nuclei and boundaries of the cells themselves and next for the mid-sized organelles such as mitochondria. Regions of interface between cells and extracellular tissue can give clues about tissue heterogeneity.
The traditional staining techniques of histology, developed in the last two centuries from dyes used in the textile industry, remain valuable and widely used as empirical methods for LM. Subsequently, a range of specific methods was developed, enabling LM visualisation of defined intracellular and extracellular constituents. More recently, technical refinements have allowed conceptually similar specific localisations to be achieved at EM level.
One major group of specific methods known as histochemical techniques employs reagents known to react with defined cellular constituents (e.g. lipids, glycogen and DNA), thereby producing selective colouration recognisable by LM. In a subset known as enzyme histochemistry the activity of enzymes can similarly be demonstrated by staining for their specific substrates or end products; these methods are often applicable for both EM and LM. A further subset termed immunohistochemistry has gained rapid acceptance. Immunologically based, this newer method offers high specificity and sensitivity of localisation. In essence, antibodies are raised against specific cellular components (in this context, the antigen) and then conjugated with a visual marker appropriate for LM or EM (e.g. dyes, enzymes, tiny particles of colloidal gold). When the antibody is then applied to the tissue under study, it binds to the antigen. Hence, the site of antibody-antigen binding becomes flagged by the chosen visual marker (e.g. Fig. 1.14; Appendix 2, Notes on staining techniques).
A problem common both to light and electron microscopy is the need to prevent autolytic degeneration and to preserve cellular ultrastructure. Fixatives such as formaldehyde and glutaraldehyde are used for this purpose. Fixation causes cross-linking of macromolecules, which reduces and often arrests biological activity, at the same time rendering the cells more amenable to staining. Most tissues are too thick to be examined directly in the microscope and must therefore be cut into very thin slices (sections). To facilitate the cutting of thin sections, the tissue is usually embedded in a hard medium such as paraffin wax (LM) or a plastic resin (EM); fixed tissues generally require dehydration with organic solvents before the embedding step. Each stage in the fixation, dehydration, embedding, sectioning and final staining sequence may induce artefacts (distortions in cell and tissue architecture, e.g. shrinkage). In situations where preservation of biological activity of cell constituents (e.g. enzymes) is the major objective, thin sections for histochemistry can be obtained from minimally fixed or unfixed frozen tissue; such frozen sections have their own particular artefactual distortions. As noted above, unstained sections are quite lacking in contrast when viewed by conventional LM or EM. However, special types of LM (phase contrast, interference contrast, confocal microscopy) have been developed to address this limitation and are frequently used, for example, to monitor living tissue cultures.