In histology and organography, it is a good practice to study tissular structures in their context, which is sometimes difficult to do it in thin sections. For example, the capillary net organization and connectivity, the branching pattern of an axon, the spatial organization of nephrons in the kidney, or the expression of a particular gen in the embryo. The study of thick tissue samples faces several handicaps. The quality of light microscopy images depends on the penetration of light through the tissues, which are mostly translucent or opaques. In addition, tissues have sometimes molecules and cellular structures with different light refraction index, which causes light dispersion. Finally, there are tissular substances that absorb the light, such as heme groups, melanin and other pigments.
If we need to study cellular details in thick biological samples, there are two ways to prevent the absorption and dispersion of the light when it crosses the tissues.
Tissue sections provide a bidimensional view of a tridimensional structure. In addition, the rule is that the more magnification we want, the thinner have to be the sections if sharp images are needed. In thin and semithin sections, the light has to cross a thin layer of tissues. There are apparatuses to obtain thick sections (>30-50 µm), thin sections (2-10 µm) and semithin sections (< 1 µm) to be visualized at light microscopy. Nowadays, there are computer programs that are able to render tridimensional images from many consecutive sections. Although this is tedious and time-consuming, it has been used in a variety studies. However, a rather complex and expensive setup is needed, and not all labs can not afford it. That is why is not a widespread technique in the histology labs. Very thick sections can be studied with multiphoton microscopes, with can get sharp images of tissues located as deep as several hundred µm. However, is sometimes necessary to get a tridimensional image of larger samples.
Tissue clearing is a procedure to change the optical properties of tissues, and it allows getting sharp images from deep tissues in thick samples. The main effect is a decrease of the absorption and dispersion of light as it crosses the sample. Commonly, clearing removes lipids, pigments and other substances that hinder the light. In addition, the water is substituted by a medium that gives a homogeneous light refraction index along the sample so that light dispersion is minimized. By tissue clearing, it is possible to study very thick samples, from several mm to cm in thickness.
The general protocol includes fixation, permeabilization, decoloration of pigmented structures, and homogenization of the light refraction index. Fixation is essential and should be stronger than usual because the tissues undergo a higher chemical stress. Formaldehyde, glutaraldehyde and acrylamide derivatives are commonly used. Permeabilization is necessary to allow substances, such as antibodies and RNA probes, to reach the deepest regions of the sample. This is convenient also for a good penetration of the substances that will homogenize the light refraction index. Organic solvents, substances that remove lipids, and hyperhydrated substances, are used as substances for permeabilization. Decoloration is carried out with specific substances depending on our particular sample. Homogenization of the refraction index is the last step, which is done by aromatic solvents, hydrophilic reactives and contrast reactives.
It must be stressed that, whatever the treatment, it may cause modifications in the tissular features, such as retractions or expansions. It is also interesting to remove everything not needed from the sample in order to get the sample as small as possible. The clearing process has to quench auto-fluorescence from the tissue in case we are going to observe the sample under fluorescence microscopes.
Clearing methods can be classified according to the substances they include: organic solvents, hydrogel and hydrophilic. There is a wide variety of protocols for each type of substance. All have advantages and drawbacks. It should be selected the method that best fit our needs.
Organic solvents. This method needs a good fixation, so that aldehydes are commonly selected. An excellent ransparency is accomplished with organic solvents. They can be removed after observation for further processing of the sample. The method has to be adapted to our material and the structures to be studied. However, organic solvents may also have side effects. Thus, they can affect some molecular properties, such as antigenicity, may remove fluorescence, and may change the cellular morphology. They also can retract the tissue, which affect the spatial organization of the sample components. In addition, if the microscope objectives are not special adapted objectives, they may be damaged.
Hydrogels. They are used when the loss of molecules must be prevented. Hydrogels make possible molecules to be attached to hydrogel-tissue mixed structures. Hydrogels normally contain acrylamide monomers disolved at 4 ºC. The solution usually contain glutaraldehyde and formaldehyde. Tissues may be decolored, acrylamide can be polymerized, and lipids are removed.
Hydrophilic. These are methods using water dissolved substances. They can be divided in two groups: those that need just an immersion of the samples, and those that previously need the removing of lipids and a hydration. In the immersion ones, the samples are plunged into solutions with a high light refraction-index, where samples become progressively clear. However, lipids are the main source of light refraction, so that it is interesting to remove them. Thus, methods that include the extraction of lipids render best results.
There are other methods to study large samples of tissue, even complete organisms, such as computational tomography, magnetic resonance, and the positron emission tomography. These are non-invasive techniques that con be done in living organisms. However, they lack enough resolution to perform studies at cellular level.