A stain that colors basophilic structures, such as chromatin. It can behave as an orthochromatic dye (stains the same color as the dye, i.e., blue) or metachromatic dye (stains violet-red), depending on the pH and chemical nature of the stained substance.
As an orthochromatic stain, it is frequently used to stain nervous tissue, where it stains heterochromatin and Nissl granules (accumulations of rough endoplasmic reticulum cisterns) in neuronal somas, as well as unmyelinated nerve fibers and glial cells. It is also used for staining semithin sections.
Toluidine blue metachromatically stains structures rich in sulfated proteoglycans, such as heparan sulfate, present - for example - in cartilage (chondroblasts and immature matrix) and mast cell granules.
This staining technique uses the reduction of silver nitrate, which is deposited on argyrophilic structures. It can be used alone or in combination with other nuclear or cytoplasmic stains.
The argyrophilic structures, such as the reticulin fibers (or reticular fibers), stain black.
This widely used technique uses two consecutive dyes:
- Hematoxylin: this cationic (basic) dye is used first and stains substances that have acidic groups, such as the phosphate groups of DNA and negatively charged nuclear proteins. Structures stained with cationic dyes like hematoxylin are called basophilic. This is the case with chromatin, so the nuclei are stained with hematoxylin.
- Eosin: it is a weakly anionic (acidic) dye that stains structures rich in basic groups, which are called acidophilic (or eosinophilic when stained with eosin), such as the collagen fibers that stain pink to varying degrees with eosin.
Trichrome stains use two or more dyes to differentially stain various histological structures based on their basophilia/acidophilia or their affinity for a specific dye.
The Goldner's trichrome method (also known as Masson-Goldner) uses Groat's hematoxylin and the dyes acid fuchsin Ponçeau (xylidine Ponçeau or Ponçeau 2R), orange G (in phosphomolybdic acid solution), and light green (acetic acid solution).
Goldner's modification of the original Masson's method stains basophilic structures (such as chromatin) in shades of brown to black, weakly to moderately acidophilic structures in red to orange, and strongly acidophilic structures, such as collagen fibers, in shades of blue to green.
These microscopes utilize changes in the amplitude and phase of the light wave passing through the sample to enhance contrast (differences in transparency) between its various components. This allows the observation of biological structures without the need for staining, making them very useful for live cell and tissue observation, particularly in cell cultures.
Various techniques are used to enhance the contrast of sample elements. The first one, simply called phase contrast, was developed by Zernike, it is based on amplifying the phase delay that occurs in light waves when they pass through objects with different refractive indices.
More recently, other systems have been developed, such as Nomarski-type differential interference contrast microscopy, which uses polarized light, or Hoffman modulation contrast, which laterally illuminates the sample and generates (pseudo)three-dimensional images.
This type of microscopy is based on the detection of fluorescence, which is formed by photons (light) with a specific wavelength emitted by certain molecules called fluorophores. Each fluorophore emits fluorescence characterized by its color, that is, its wavelength. Fluorescence emission occurs when the fluorophore molecules are excited by photons (excitation or incident light) with a characteristic wavelength and higher energy (shorter) than the emitted fluorescence.
In biology, fluorescence microscopy observation is usually performed using epifluorescence microscopes equipped with an illumination system that allows incident light to reach the sample through the objectives. Filters are also required to only allow fluorescence of a specific wavelength (color) to pass through to the eyepieces and the imaging system (photographic or video camera).
Currently, fluorescence confocal microscopy systems are commonly used, which use a coherent incident (excitation) light beam, allowing scanning through planes of the sample and obtaining higher-resolution and fluorescence-sensitive images from relatively thick samples. The obtained plane images (known as optical sections) can be computationally stacked to obtain three-dimensional images.
Biological structures contain many molecules that are inherently fluorescent (autofluorescent), but to detect those that are not, fluorescent molecules called fluorochromes (such as fluorescein and rhodamine) are used, which bind to them. For example, fluorochromes can be used to label antibodies used in immunofluorescence techniques, as well as other biological molecules that have an affinity for certain cellular and tissue components, such as phalloidin, which binds to actin, allowing its visualization. Fluorochromes that have an affinity for DNA, such as DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) or propidium iodide, are also widely used to visualize cell nuclei.
Generally, when the sample emits fluorescence of two or more colors, the acquisition of images is done sequentially, registering one color first, then another, and so on, by interposing the appropriate filters. Images obtained from the same field but with different fluorescence are merged to obtain a composite image.
Histochemical methods use chemical reactions or physical properties of dyes to reveal the presence of organic or inorganic compounds in cells and tissues. Depending on the nature of the compound to be identified, unfixed, frozen, or fixed samples embedded in paraffin or plastic resins or in cell cultures can be used.
The PAS reaction reveals the presence of all types of carbohydrates (polysaccharides such as glycogen, mucopolysaccharides, glycolipids, glycoproteins, etc.).
It is based on the reaction of Schiff's reagent with the aldehyde groups released from the vic-glycol groups present in carbohydrates after their oxidation with periodic acid. Schiff's reagent contains basic fuchsin (or pararosaniline), which in acidic solution and in the presence of SO2 forms a colorless form (N-sulfonic acid). This reagent reacts with free aldehyde groups, forming an insoluble purple-colored compound. The PAS technique can be used on sections obtained from paraffin-embedded or cryofixed samples.
The Perl's reaction (or Perl's method) is used to detect the presence of iron that forms insoluble deposits of hemosiderin in cells and tissues. However, this method does not detect iron from ferritin, which is water-soluble. It is used on paraffin or frozen sections.
The Perl's reaction is based on the release of ferric ions through the action of hydrochloric acid and their reaction with potassium ferrocyanide, resulting in the formation of a bluish-green precipitate of Prussian blue (ferric ferrocyanide).
The Oil Red O method is used to visualize lipid deposits in cells in the form of lipid droplets (inclusions).
Since lipids are rapidly dissolved by alcohol during the dehydration process, frozen sections are used for their detection.
Oil Red O belongs to the group of lipochromes, substances capable of dissolving in lipids and staining them. Lipochromes are not true dyes but rather colored substances highly soluble in lipids, soluble in non-lipid-soluble substances such as low-concentration alcohols, and do not bind to other tissue structures. For use in histological techniques, these products are dissolved to saturation in 50% or 70% alcohol. Nuclei can be contrasted with hematoxylin, and the preparation should be mounted with glycerin-gelatin.
In most histochemical techniques, frozen tissues and sections obtained from a cryostat are used, as the process of dehydration and embedding would inactivate the enzymes whose presence is to be demonstrated.
Esterases are a group of enzymes that hydrolyze esters of carboxylic acids, primarily located in lysosomes. There are many different types of esterases that act on various substrates, but their activities overlap since many of them are capable of hydrolyzing the same substrate. Therefore, the subdivision of esterases is based on the specific type of ester they efficiently hydrolyze and the effect of various enzyme inhibitors.
The technique used in this case reveals all types of esterase activity as a simple ester, alpha-naphthyl acetate, is used as a substrate. That's why it is called "non-specific esterase." The enzymes release alpha-naphthol, which is revealed with the diazo salt pararosaniline with sodium nitrite, causing the cells with esterase activity to appear reddish-brown in color.
Phosphatases are a group of hydrolytic enzymes that act on esters of phosphoric acid. Some phosphatases specifically act on a particular substrate, such as adenosine triphosphatase, but most of them act on various substrates, and their classification is based on the pH at which they exhibit optimal activity. They can be divided into two groups:
Acid phosphatases, which exhibit optimal activity at low pH (4.7 – 5.5).
Alkaline phosphatases, which exhibit optimal activity at high pH (8.3 – 9.4).
Acid phosphatases are primarily located in lysosomes, while the subcellular localization of alkaline phosphatases is not clear.
For the histochemical demonstration of both groups of phosphatases, naftol-AS-BI-phosphate at the corresponding pH is used as a substrate. The liberated naftol is demonstrated with a diazonium salt: pararosaniline with sodium nitrite for acid phosphatase (cells with this activity are stained reddish-orange) and Fast Red TR for alkaline phosphatase (red color). After the reaction, the nuclei are contrasted with hematoxylin. In the case of acid phosphatase, the sections are dehydrated, cleared, and mounted with Entellan or DPX. However, in the case of alkaline phosphatase, dehydration is not possible, so the preparations are mounted with glycerin-gelatin.
Diaphorases are enzymes belonging to the dehydrogenase group that catalyze the transfer of electrons from NADH or NADPH to other acceptors. The histochemical technique demonstrates the presence of NADP - diaphorase by using NADPH as a substrate and the tetrazolium salt NBT, which is reduced to a blue-colored formazan, as an acceptor to reveal the localization of that enzymatic activity.
Immunodetection techniques (or immunolabeling) are based on the specificity of the antigen-antibody reaction, which allows the identification of a protein (the antigen) whose presence we want to demonstrate by binding it to a specific antibody (primary antibody). Depending on whether they are used to identify cells or tissues, immunocytochemical and immunohistochemical techniques are distinguished, although their principle is the same.
It is common for these techniques to use the method of indirect immunodetection, which involves two stages to increase the sensitivity of detection. In the first step, the primary antibody is used, which binds to its specific antigen wherever it is present. In the second step, another antibody (secondary antibody) against the primary antibody is applied. The secondary antibody is covalently attached to a marker (it is said to be labeled), which allows its visualization. This second step amplifies the result of the reaction because several molecules of the labeled secondary antibody can bind to each molecule of the primary antibody. Consequently, the site where the antigen is located presents more enzyme molecules, resulting in increased sensitivity to detection.
Immunodetection techniques can be used on isolated cells and tissues where the antigen's structure has been preserved, for example, using unfixed cell suspensions or sections obtained from a cryostat or gentle fixations with formalin and embedding in low-melting-point paraffin to prevent antigen denaturation.
Generally, the nuclei are stained with a dye that does not interfere with immunodetection, either with DNA-affine fluorochromes (e.g., DAPI) or dyes for bright-field microscopy, such as hematoxylin.
Depending on the type of marker used, the following immunohistochemical methods are distinguished:
- Immunofluorescence. Fluorochromes such as fluorescein, rhodamine, Texas Red, and others are used as markers. The visualization of the results is done using fluorescence microscopy, and the structures that bind the primary antibody show the fluorescence emitted by the fluorochrome.
- Immunoenzymatic. Enzymes such as peroxidase are used as markers, and their activity is revealed using the corresponding histochemical method, usually visible with bright-field microscopy. Structures positive for the primary antibody appear stained with the color of the developer used to detect the histochemical reaction product.
In the microscopic observation of histological sections, the image seen depends on the plane in which the original structure was sectioned. It is important to note that when cutting a three-dimensional structure, the orientation of the sectioning plane determines the shape of the two-dimensional section obtained on a slide.
When the structure is heterogeneous in its content (for example, it contains various layers or components), the orientation and level at which the section is made also determine which of them can be seen in the section.
Therefore, it is necessary to know and indicate the orientation of the sectioning plane when performing the microscopic description of the sections to correctly interpret the morphology and components of the structure.
The embedding of samples in plastic resins (such as Epon, Araldite, or similar) allows obtaining thinner sections compared to paraffin embedding.
Although initially this embedding technique was used as a method for selecting regions for obtaining ultra-thin sections for transmission electron microscopy, nowadays it is frequently used for light microscopy as well.
The thickness of semi-thin sections varies between 0.5 and 5 μm and they are stained using various techniques such as toluidine blue or hematoxylin-eosin. By using low-melting-point resins, histochemical and immunolabeling techniques can also be performed.
Electron microscopy utilizes a radiation source that emits an electron beam with a much shorter wavelength than visible light to "illuminate" the structures being observed. This enables achieving a much higher resolution compared to light microscopy.
On the other hand, electrons have a much lower penetration power in materials, including gases, than photons. This requires working under high vacuum and specific sample preparation. Additionally, the resulting electron microscopy images provide information in shades of gray (although in some cases, the images can be post-colorized using false color scales).
Electron microscopy images can be formed based on the information carried by two classes of electrons interacting with the sample, resulting in two types of electron microscopy:
This type utilizes electrons that pass through the sample to form the images, which requires using extremely thin samples. In the case of biological samples, the cuts are ultra-thin, with a thickness ranging from 70 to 90 Å. To obtain them, the samples need to be embedded in plastic resins (Araldite, Epon) that are hard enough to be sectioned that thinly.
For biological samples, since electrons do not provide colors, heavy metals are used to enhance the contrast of cellular and tissue components to electrons. Osmium (osmium tetroxide) is primarily used to "fix and stain" lipophilic structures such as cellular membranes, and uranium (uranium acetate) is used for proteins and nucleic acids.
The major advantage of TEM is its high resolution power, approaching 0.2 nm (2 Å). This has allowed the observation and description of the structure of cellular components and organelles as well as microorganisms. Under certain conditions, even the structure of proteins and nucleic acids can be observed.
A particular method in TEM is the technique of cryofracture, in which metal replicas of the samples are observed. For this, the samples are frozen, fractured by hitting them with a blade, and the exposed surfaces are coated (shadowed) with a thin layer of metal (usually platinum). The organic parts are removed, and the metal replica is observed under the transmission electron microscope.
This method provides relief images and is particularly suitable for the observation of cellular membranes.
In this case, the images are formed by utilizing the information carried by electrons emitted by the sample (known as secondary electrons) when the sample is exposed to the electron beam from the "illumination" source. Structurally, this allows obtaining three-dimensional images of the sample's surface with a large depth of field but not its interior. Furthermore, to minimize the interactions between electrons scattered from different parts of the sample (which result in blurred images), the sample is illuminated by sequentially scanning the surface.
Since the focus is on the electrons of the beam interacting with the sample's surface, in the case of biological samples, no sectioning is required. However, the sample's surface is usually coated with a thin (less than 10 nm) metallic layer, typically gold, which facilitates the emission of secondary electrons.
The resolution power of SEM is much lower than that of TEM, approximately 1 nm, but sufficient magnification (around x30,000) can be achieved for the study of biological samples, including cytohistological levels.