The histological processing includes a variety of methods and techniques aimed to study the molecular and morphological features of tissues. They are designed for the study of tissues at light or electron microscopy. Different set of experimental methodology are used depending on what we want to study. Thus, methods and techniques can be used in many different combinatios to achieve the result that better fit our needs. In the diagram below, common methods and techiques for processing tissues are shown.
Histological processing begins with the collection of tissue samples to be studied. In plant histology, pieces of the plant body are collected, whereas in animal histology there are two options: get a portion (samples from biopsy or necropsy) of an organ or tissue and start the histological processing, or start the histological processing with the complete body and then get the sample we are interested in, and then continue with the processing. In any case, samples (or the whole body) are first fixed with solutions known as fixatives. They keep the tissular molecules and structures with similar features as they had in living tissues while going through the successive histological techniques. Fixation is like having a photo of a sample when the organism is alive, its organization and features are unaltered during the processing, and they are still preserved when the sample is observed with the microscope. Another way to fix tissues is by fast freezing. This type of fixation is used when chemical fixatives or other steps of the processing affect the tissular features we are interested in. For example, some molecules are modified by fixatives or in chemical solutions in later steps.
After fixation, the next common step is embedding of the sample for obtaining sections of the tissue. The thinner we want a section, the harder should be the final embedding block. Embedding is the infiltration of the sample with liquid substances that are later solidified by temperature (paraffin) or by polymerization (resins). The same hardening can be got with a quick freezing of the sample. Thicker sections (for example, 40 µm) can be obtained without embedding by using special sectioning devices like vibratome. Usually, embedding compounds are not hydrophilic. Thus, first, the water must be replaced with a lipophilic solvent, which in turn is replaced with the embedding substance.
After embedding, or freezing, samples are cut in very thin sections. There are a number of apparatus for cutting that yield sections with different thickness: ultrathin sections (nonometers), semithin sections (between 0.5 and 2 micrometers), thin sections (between 3 and 10 micrometers) and thick (thicker than 10 micrometers). Sections are later processed for tissue studying, which usually involves hydrophilic dyes. There are several types of microscopy techniques, such as phase contrast microscopy, that allow the observation of tissue features without processing. If hydrophilic dyes are used, the embedding media must be replaced with water, otherwise dyes do not enter the tissue. However, semithin sections (for light microscopy) and ultrathin sections (for electron microscopy) can be stained with dyes or contrasted with heavy metals, respectively, without removing the embedding media, which is resin. In sections obtained from freeze samples, dyes are applied once the tissue is thawed.
Processed tissues are observed with microscopes. There are a variety of types of microscopes, but the more common are light and electron microscopes. Light microscopes relay on visible light and lens. Several light microscopy set-ups are available: clear field, fluorescence, phase contrast, polarization or differential interference contrast. Electron microscopes give a far larger magnification that allows the study of the so-called ultraestructure of the tissues.
This basic histological process is modified for specific needs of the study. For example, non-embedding and non-sectioned samples can be observed with the scanning electron microscopy, but only surfaces can be studied. In the next pages, the most common techniques for histological observation are described.