There are several methods or techniques used for the examination of tissues and cells. In this article, we’ll be discussing some of those common Methods, their advantages and disadvantages in histopathology.
Teased specimens are occasionally examined in normal saline or another indifferent medium. The specimen is teased with needles in a watch glass, transferred to a microscope slide, a coverslip is placed on top, and the preparation is examined under a microscope with a reduced cone of illumination.
The examination of dissociated tissues using a phase-contrast microscope is a method that is likely to become more important in the future. This type of examination has the following advantages
- It reveals the structure of the cell while the cell is still alive and without staining.
- With practice, the arrangement and movement of mitochondria can be seen quite clearly, and cells in mitosis, particularly in malignant tissue, can be observed.
- Some cells move, and this movement may be distinctive enough to identify them.
These methods allow for the visualization of individual cell details, but they inevitably have the disadvantage of destroying anatomical relationships.
The material to be examined is either pressed against a slide (impression smear), spread with a platinum loop, or crushed and spread with another slide in the smear method. The preparation can then be fixed wet, stained to show specific structures, and mounted in a medium with a high refractive index after clearing. This technique allows for a much more detailed examination than a wet preparation, but it has the same limitations.
In an emergency, very thin slices of fixed or unfixed material can be cut free with a sharp knife or razor, and an urgently required diagnosis can sometimes be made by limiting staining to the surface of the tissue. Brushing a polychrome stain on the surface of the tissue and quickly washing it off controls the staining. Terry (1929) devised this method, which has been used with great accuracy. A special microscope known as the Ultrapak microscope is available, but it is not required. It should be noted that this technique can only be relied on if the operator has extensive experience.
Some living cells will take up specific dyes (vital stains) that color specific elements in the cells, such as mitochondria. Other cells (phagocytic cells) will engulf microscopic colored particles, which are then visible inside the cells; this can be used to demonstrate reticuloendothelial system elements.
Vital staining techniques are typically classified as (a) intravital (within the body) and (b) supravital (with living cells outside the body). Unfortunately, these techniques are extremely limited, and it appears likely that phase-contrast microscopy will largely replace them.
Sectional methods are used in almost all established techniques because the morbid anatomical relationships of cells are preserved and extremely thin sections can be cut. The finished preparation is mounted in a medium with a high refractive index, allowing for detailed examination.
The medium in which the tissue will be embedded is determined by the technique or techniques to be used as well as the type of material to be sectioned. The most common medium for routine use is paraffin wax, but celloidin and gelatin are used on occasion.
- Paraffin Sections
With this technique, the tissue is finally embedded in paraffin wax, allowing for easy sectioning. The first step is to preserve the tissue with a fixative, usually in aqueous solution, which, because it is not miscible with paraffin wax, must be removed and replaced with a wax solvent. This is accomplished by immersing in increasing concentrations of alcohol to remove the water, and then replacing the alcohol with a wax solvent, which is then replaced by molten paraffin wax.
- Water-soluble Waxes (Carbowax and Aquawax)
These materials are waxes that are miscible with water; they eliminate the stages of dehydration and clearing, saving time. A disadvantage of this technique is that it is difficult to remove creases in the tissue, because this is normally done in warm water: dilute alcohols and similar materials have been used in place of water, but with inconsistent success. Only in rare cases are the results as good as those obtained by paraffin embedding, which, along with the difficulties encountered in the method, accounts for the method’s relative unpopularity. It has, however, been used successfully by some workers following fixation by the freeze-drying technique.
- Celloidin Embedding
Celloidin, because of its rubbery consistency, provides greater support to both mixed tissues (such as skin with subcutaneous fat) and very hard tissues such as bone. It is also used when it is preferable to avoid using heat, such as with the central nervous system.
Celloidin is a nitrocellulose that dissolves in equal parts alcohol and ether. As a result, celloidin embedding avoids the stages of clearing, tissue fixation, dehydration, and transfer from absolute alcohol to thin celloidin (2%), and finally embedding in thick celloidin (8 per cent). Unless otherwise specified, these blocks must be cut and stored in dilute alcohol.
- Giant Gelatin Sections
Gough and Wentworth (1949) developed the technique of giant gelatin sections, specifically for lungs. After fixation, whole lungs are embedded in gelatin, which is formolized after it has set, and sections 300-400 microns thick are cut on a special large microtome. These are then laid on Perspex sheets, covered with filter paper, and allowed to dry; the sections stick to the filter paper and can be filed in a book.
- Frozen Sections
Carbon dioxide is used to freeze unfixed or fixed tissue on a special microtome. It is especially useful when a quick diagnosis is required, or when demonstrating material that is soluble in alcohol or clearing agents. When the material to be cut is friable, it can be embedded in gelatin before cutting.
- Cryostat Cut Sections
The use of a deep freeze type cabinet to house the microtome, knife, tissue, etc. and to keep them at a low temperature (— 10° to — 20° C) during the actual section cutting process has resulted in a series of new methods allowing the rapid sectioning of fresh unfixed tissues that have been quickly frozen at a very low temperature (— 160° C). These sections are ideal for histochemical analysis as well as the newer fluorescent antigen—antibody techniques. It has the distinct advantage over freeze drying of being faster and posing fewer technical challenges. It is increasingly being used as an alternative to the frozen section technique for rapid diagnosis.
Fluorescent Antigen-Antibody Techniques
This technique takes advantage of the fact that fluorescent dyes like fluorescein isothiocyanate can be attached to proteins like gamma globulins without interfering with their function as an antibody or antigen. Fluorescence microscopy can reveal the precise location of a specific antigen or antibody after dye attachment to protein (conjugation). This technique has resulted in a much better understanding of immunity at the cellular level, particularly in diseases with an auto-immune pathogenesis.
- Polarizing Microscope
A polarizing microscope is especially useful for detecting and partially identifying birefringent or anisotropie objects such as foreign bodies, crystals, specific lipids, cross-striated muscle, and myelinated nerve fibers. When these are placed between crossed Nicol prisms or Polaroid material sheets, they rotate the plane of polarized light and appear bright against a dark background.
- Dark Ground Illumination
Dark ground illumination is obtained by fitting a special type of condenser that produces a hollow cone of light with a one-seventh inch or a one-twelfth inch oil immersion objective fitted with a funnel stop. The numerical aperture of the objective cannot be greater than 1.0. Objects are seen by the light they scatter, with no direct light entering the objective. This technique is used to demonstrate Treponema pallidum, blood flagellates, and other objects in wet preparations. It takes a lot of practice to interpret the appearance of cells using this technique, because internal structures tend to be hidden by the refracted light that forms a halo.
- Phase-Contrast Microscopy
Zernicke invented this type of microscope, which converts slight differences in refractive index into degrees of light and shade. It has only been commercially produced since 1946. This allows for detailed examination of unstained objects, and because cell constituents such as mitochondria, granules, nucleoli, and so on can be seen clearly, it represents a significant advancement in microscopy. During division, living cells and organisms can be studied and photographed, and researchers can observe the effects of foreign substances on them. While it is still primarily used for research, it is increasingly being used in routine laboratory work. The current development has significant advantages in that a routine instrument can be quickly adapted for phase microscopy, and the phase-contrast objectives can be used for routine work with no discernible loss of definition.
- Interference Microscopy
The interference microscope combines some of the principles of both phase-contrast and polarizing microscopes, allowing it to see unstained objects in great detail. It has an advantage over the phase-contrast microscope in that it has no halo. Objects are either differentially colored or viewed against a background of colored wavebands, depending on the type of interference microscope used. The weight of a cell can be calculated to 10″14 grammes by measuring the difference in color between cell components or the change in wave front caused by specific objects. Because it is essentially a microscopical balance, the weight of objects such as single mitochondria can be estimated with great precision.
Although some workers consider this microscope to be the successor to the phase-contrast microscope, the adjustments are critical and complicated, and it is unlikely that it will become a routine instrument in its current form.
- Ultra-Violet Light Microscopy
The normal compound microscope’s resolution is limited by two factors: the numerical aperture of the objective and the wavelength of the light source used. Using ultra-violet light, which has a shorter wavelength than white light, allows for an increase in resolution. Because ultraviolet light does not belong to the visible spectrum, the image must be captured photographically, and the lenses used must be made of quartz.
- Fluorescent Microscopy
Fluorescent materials have the property of converting light rays from the invisible spectrum (ultraviolet) to the visible spectrum. On a dark background, fluorescent material examined with this technique will appear bright. Although some elements are naturally fluorescent (innate fluorescence), the use of fluorescent dyes allows for the specific demonstration of many tissue elements and bacteria.
- Electron Microscopy
The use of electrons instead of light rays, and electrical lenses instead of glass lenses, has greatly increased the magnification and resolution previously available. This technique revealed many ultra-microscopic particles, such as smaller viruses, for the first time.
One disadvantage of this method is that the material must be dried and examined in vacuum, which introduces artifacts that detract from the method’s value.
Because micro-incineration is used to determine the presence of mineral elements, fixation and processing of the tissue must not increase or decrease its mineral content.
Duplicate sections are prepared, with one incinerated at a controlled high temperature and the other stained using a standard method as a control.
Following incineration, a coverslip is placed on the section as soon as possible to prevent both moisture absorption from the air and ash movement. Normal microscopic methods, dark ground illumination, polarizing microscopy, and, if possible, electron microscopy are then used to examine this section. The interpretation of results may necessitate considerable experience.