Because of the microscopy requirements, options for preparing specimens are limited to:
· Whole-mounts, where an entire organism or structure is small enough or thin enough to be placed directly onto a microscope slide (e.g. a small unicellular or multicellular organism or a membrane that can be stretched thinly on to a slide)
· “Squash” preparations, where cells are intentionally squashed or crushed onto a slide to reveal their contents (e.g. botanical specimens where cells are disrupted to reveal chromosomes)
· Smears, where the specimen consists of cells suspended in a fluid (e.g. blood, semen, cerebro-spinal fluid, or a culture of microorganisms), or where individual cells have been scraped brushed or aspirated (sucked) from a surface or from within an organ (exfoliative cytology). Smears are the basis of the well-known “Pap test” that is used to screen for cervical cancer in women.
· Sections, where specimens are supported in some way so that very thin slices can be cut from them, mounted on slides, and stained. Sections are prepared using an instrument called a “microtome”.
Of these options only whole-mounts and sections preserve the structural relationships between individual cells and extracellular components. Smears and squash preparations provide detail about individual cells and relative cell numbers, but structural relationships are lost. The preparation of sections is the most technically complicated of these methods as it requires specialized equipment and considerable expertise. The microscopic examination of sections by a pathologist forms the corner stone of cancer diagnosis. Although the methodology for preparing sections from both animal and plant material is similar, the following description relates to animal (human) tissues.
There are many different forms of microscopy but the one most commonly employed is “brightfield” microscopy where the specimen is illuminated with a beam of light that passes through it (as opposed to a beam of electrons as in electron microscopy). The general requirements for a specimen to be successfully examined using brightfield microscopy are:
· That the cells and other elements in the specimen are preserved in a “life-like” state (this process is called “fixation”)
· That the specimen is transparent rather than opaque, so that light can pass through it
· That the specimen is thin and flat so that only a single layer of cells is present
· That some components have been differentially coloured (stained) so that they can be clearly distinguished.