Dyes and other colorants in microtechnique and biomedical research

Coloration, known in biological study as staining, enhances the visibility of living and dead objects under the microscope. This study reviews some of the colorants, chromogenic compounds and reactions exploited in staining, with special attention given to techniques that impart different colours to specific components of cells and tissues. A major mechanism, the first to be recognised, is the uptake of dye cations or anions by oppositely charged macromolecules. Modern investigations have shown that weaker electrical attractions are also important, especially in techniques that use more than one dye and methods for staining uncharged materials. The sizes and shapes of dye molecules or aggregates can also influence where they will lodge in a stained tissue. Reactive textile dyes have few uses in biological study, but there are histochemical methods that can covalently bind chromogens to DNA and to some macromolecular carbohydrates. Metal complexing dyes are universally used in microtechnique. Their mechanisms of action are not all fully understood, especially in the case of the routine haemalum and eosin (H & E) method that is routinely used for pathology. Staining a tiny quantity of a specific substance requires amplification to generate a visible signal. This is achieved with antibodies and catalysts. An immunoglobulin (antibody) molecule bound by a specific tissue component (antigen) can itself serve as antigen to more than one anti-immunoglubulin molecules that are labelled with an enzyme or with biotin. Enzyme labels catalyse the production of insoluble coloured materials. Biotin has a strong and specific affinity for either avidin or streptavidin, proteins with four biotin-binding sites that can be detected with biotinylated enzymes. Antibodies carried on colloidal gold particles are detected directly by electron microscopy or, for light microscopy, by a process known as physical development. The gold particles are catalytic nuclei for deposition of silver in chemical reactions that have also been used in black and white photography. There are some dyes that can be taken up by living cells, and of these some attach to particular organelles or enter specific types of cells. Fluorescent probes can lodge in specific sites such as mitochondria, lysosomes or the cell membrane; other probes can serve as indicators of chemical or electrical changes in living cells. Some lectins (carbohydrate-binding proteins from plants, which can be labelled with histochemically detectable enzymes) are taken up by neurones, and are useful for tracing the trajectories of the axons of these cells. Certain fluorescent dyes are used for the same purpose, as are labelled derivatives of dextrans. Colourless objects, such as aquatic microorganisms, can be observed with a microscope in the living state because their refractive indices are a little higher than that of water. Refraction of light at the surface membrane provides the appearance of a dark line enclosing each organism. Other structures such as nuclei, vacuoles and flagella are visible because they refract or scatter light, but fine structural details are not resolved. The cellulose walls of cells can be seen in unstained, transilluminated sections of plant tissues. Robert Hooke (1635–1703) named cells as structural components of plants but he did not recognise the presence of cells in animals. Lack of added colour seriously limited the microscopic study of animal organs, in which the cells do not have thickened walls. Nevertheless, the major structural features of animal tissues were described before the 1850s, largely from unstained sections and preparations made from dissected, squashed or macerated specimens [1,2]. A note on names: Appendix I (at the end of the paper) gives the trivial and CI names and numbers and some other synonyms for the colorants mentioned in the text.† In this study the traditional or trivial names of colorants (comparable with generic names of drugs) are used. These are the only names used in the literature of biology because most scientists who use colorants do not know the Colour Index (CI). The trivial names and their spellings follow the 10th edition of Conn's Biological Stains,† a book that has been a standard work of reference for biologists since 1925. The trivial names of many dyes can be found in dictionaries. The names are correctly spelled in modern studies on dye chemistry [3] but frequently they are wrongly spelled in biological books, journals and vendors' catalogues. Dyes that are amines (e.g. fuchsine, thionine) have names ending in -ine rather than -in, whereas dyes that are not organic bases (e.g. eosin, phloxin) do not have an -ine ending. Detailed discussion of spellings are available in the literature [4,5]. In the eighteenth century substances such as carmine and indigo were fed to microorganisms, allowed to rise in the xylem vessels of plant stems, or mixed with gelatine and injected into arteries, veins or ducts. Microscopic examination revealed the anatomical arrangements of the filled structures, but contrast was not enhanced in the intervening tissues. The first dyes to be applied for improving the visibility of transparent structures were probably saffron for muscle fibres by Anton van Leuwenhoek in 1714 and Carminic Acid (as cochineal) for macerated plant stems by John Hill in 1770. Carmine was used by Marcheso Alfonso Corti in 1851 to make visible the epithelia of the dissected inner ear, but another seven years elapsed before Joseph von Gerlach (1820–1896) found that a more dilute solution of the same dye imparted a strong red colour to the nuclei of animal cells, with the cytoplasm being pink and extracellular materials largely unstained. This discovery in 1858 is generally taken as the starting point of meaningful microscopical staining. Further information about the history of biological microscopy and staining is available in the literature [1,2,6,7]. The second half of the nineteenth century saw rapid expansion of the dye industry, starting in 1856 with the production of mauveine by William Perkin (1838–1907). This, the first commercially successful synthetic dye, soon became obsolete but it was used to stain cell nuclei in living plants [8]. In the following four decades, many synthetic dyes with a variety of chemical structures, were tested as biological stains, especially for thin sections. The invention of the microtome and the development of procedures for fixation, processing and embedding in wax had, by this time, made sections the standard objects for examination in the science of histology. Glass slides and thin glass coverslips were commercially available, as was Canada balsam, a transparent, hydrophobic resinous mounting medium that allowed permanent storage of the preparations. The refractive index of Canada balsam is close to that of glass, as well as most structural macromolecules of tissues that have been dehydrated and equilibrated with a hydrophobic solvent. Modern resinous mountants, made from non-polar synthetic polymers, have the same optical properties. Unless it is naturally pigmented, a thin preparation mounted in a transparent resinous medium is almost invisible when examined with an ordinary microscope. Visibility depends on artificial coloration (staining) of the materials before they are made transparent. A staining procedure must impart conspicuously different intensities of one colour to different structures, as in Gerlach's use of diluted carmine. Alternatively a method may provide more than one colour in a meaningful way, as in techniques that use two or more dyes with different affinities and clearly contrasting colours. The human brain expects included objects, such as cell nuclei or chloroplasts, to be darker than the surrounding cytoplasm. The expectations for extracellular materials vary with the demands of the microscopist. If thin collagen fibres are the objects of interest, these should be more conspicuous than cells and their nuclei. The study of plant anatomy demands colours for different types of cell walls, the nuclei and cytoplasm within the cells being of secondary importance. Before about 1880 dyes were tested as biological stains with little or no regard for the mechanisms that might determine their actions. Of this period Gustav Mann wrote, 'to be an histologist became practically synonymous with being a dyer, with this difference, that the professional dyer knew what he was about, while the histologist, with few exceptions, did not know, nor does he to the present day' [9]. Unfortunately, the latter part of this comment is true even in 2006, especially for those pathologists and academic researchers who look at but do not make microscopical preparations. Long before he discovered the immune system and the first anti-bacterial drug, Ehrlich synthesised several dyes of known chemical constitution and used them to stain animal tissues [10]. He recognised two distinct patterns of staining fixed tissues. Basic dyes (then used industrially to colour wool and silk from alkaline dyebaths) prominently stained cell nuclei. Acid dyes (which were applied to the same proteinaceous textiles from acidic dyebaths) coloured cytoplasm and extracellular fibres. Ehrlich postulated the formation of salt-like compounds between basic dyes and acidic components of cells. The latter were said to be basophilic. Basic components of tissues were termed acidophilic because they combined with acid dyes. In modern terminology, a basic dye has coloured cations and an acid dye has coloured anions. Attraction of dye ions to opposite charges in the structural components of tissues came to be considered the fundamental mechanism of almost all biological staining. The chief basophilic component of cell nuclei is DNA, with its multiple ionised phosphate groups. Other substances that stain with cationic dyes include RNA in cytoplasm and nucleoli, chondroitin sulphates in cartilage, acid glycoproteins in mucus and various substances in plant cell walls. Tissue acidophilia is attributed to proteins that have more basic than acidic amino acid side-chains; those of lysine and arginine are protonated over the whole pH range of staining solutions. It has long been recognised that properties other than charge (especially molecular size and solubility in polar or non-polar liquids) influence penetration and retention of dyes in different parts of cells and tissues (see Ref. 4 for extensive discussion). Electrostatic attraction diminishes with the square of the distance between the centres of charge. This is the strongest type of force that works to bring dye ions into contact with oppositely charged groups in a substrate, and the type with the longest range. Ionic attractions have also been invoked for some staining techniques in which metal salts are used as mordants. Aluminium ions, for example, were envisaged by Baker as positively charged bridges between phosphate anions of DNA and anions of haematein in the haemalum ('alum-haematoxylin') methods for cell nuclei [4,11]. For some other dye–metal combinations, notably chromium(iii) with gallocyanine, it is established that a cationic dye–metal complex is the species that binds to nucleic acids [12]. Similar cationic aluminium–haematein complexes have been suggested [13], and shown to exist and to be capable of binding to DNA [14]. The pH of a typical haemalum staining solution is, however, too low to permit appreciable ionisation of phosphoric acid groups; moreover, nuclear staining occurs after complete extraction of DNA [15]. Mechanisms of staining by haemalum must therefore be more complex than those envisaged by either Baker [11] or Horobin [13]. This subject is discussed in more detail later because sequential application of haemalum and eosin Y has been the standard staining method in diagnostic pathology since 1876 [2]. The necessity of ionic attraction in the mechanism of a staining technique is examined by applying solutions of the dye with a range of pH values. For example, a cationic dye applied to a section of animal tissue at pH 4 will stain nuclei (phosphate ions) and cartilage matrix (sulphate-ester ions). At pH 1 the same dye will stain cartilage matrix but not nuclei, because the weak phosphoric acid groups of DNA are not ionised. If the pH is higher than 5, cationic dyes stain everything because the carboxy groups of proteins, which are present everywhere in animal tissues, are all ionised [16,17]. The ionic attraction of dye cations from a near-neutral solution can be inhibited by adding an inorganic salt that does not significantly change the pH, such as sodium or magnesium chloride [18,19]. The effects of increasing salt concentration are similar to those of lowering the pH. Addition of a salt does not provide a certain method for distinguishing carboxy groups from sulphate-esters [20], but the suppression of dye binding is evidence for the operation of ionic attraction in the staining process. The inorganic ions are assumed to compete with the dye ions for oppositely charged sites in the substrate, but high salt concentrations in a dye solution can have other effects on staining. These effects include enhancement of protein staining by anionic dyes at high pH. Without added salt, an alkaline solution of an anionic dye will stain only those structures that contain proteins rich in arginine and lysine. In vertebrate animals, this amounts to selective staining of eosinophil leucocytes, the Paneth cells of the small intestine and the tails of spermatozoa. With added sodium chloride a dye solution at the same pH stains a wider range of proteinaceous substrates. Inorganic salts also promote the aggregation of dye ions into colloidal particles, which diffuse into the substrate more slowly than the monomeric dye ions. Horobin has provided a detailed discussion of the effects of inorganic salts on staining by anionic and cationic dyes [13]. There are staining methods, including some of those for elastin [18,21] and collagen [22,23] that are not impaired by high salt concentrations, indicating that ionic attraction to a specific substrate is not always essential, even though staining fails when strongly basic amino acid side-chains (arginine and lysine) have been chemically blocked [24,25]. This short descriptive account is based on the work of March [26] and Dapson [27]. For an advanced treatment of hydrogen bonding, see the paper by Prento [28]. Many molecules are dipoles, due to uneven distribution of electrons and partial exposure of atomic nuclei. A water molecule is a dipole. The high density of electrons around the oxygen atom attracts the partly exposed hydrogen nuclei of other water molecules. The attraction between an oxygen (or nitrogen) atom and a hydrogen that is covalently bonded to oxygen (or nitrogen) is known as a hydrogen bond. It is weaker than the attraction between oppositely charged ions but strong enough to hold water molecules in clusters when in the liquid state. Another type of hydrogen bond can form between the delocalised π-electrons of either a carbon–carbon double bond or an aromatic ring and the hydroxy group of an alcohol or phenol. Many dye ions have oxygen or nitrogen atoms with partial charges (hydroxy, carbonyl, amino, azo and other groups) and all have aromatic rings. In an aqueous solution, water molecules are joined to dye molecules not only by hydrogen bonds but also by ion-dipole forces. For example, sulphonate groups of the dye attract the hydrogen atoms of water. Hydrogen bonds are the strongest nonionic forces that exist between dipoles. Weaker attractions are called van der Waals forces. The constant motion of electrons results in frequent temporary exposure of nuclei and the formation of temporary sites of higher electron density (instantaneous dipoles). When the electron shells of two non-polar molecules are almost in contact, an instantaneous dipole will induce a temporary opposite charge (induced dipole) in the adjacent molecule. The resulting attractions between induced dipoles are known as London forces. Although weak, London forces between closely applied molecules can be multiple, resulting in strong bonding of molecules with mutually conforming shapes. There are two other types of van der Waals force: Debye forces are between permanent dipoles and induced dipoles; and Keesom forces can hold two permanent dipoles together. The bond energy (strength) of a van der Waals attraction is about 1–10% that of a hydrogen bond, which is in turn about one-tenth that of an attraction between ions. For a simple review of the weaker electrical forces in relation to biological staining, see the paper by Dapson [27]. It has been suggested [4,23,29] that hydrogen bonds attach some anionic dyes to uncharged substrates such as cellulose (with many OH groups) and collagen (with many NH and NH2 groups). In aqueous solutions, however, most of the oxygen and nitrogen atoms of dyes are likely to be hydrogen-bonded to water, and van der Waals forces probably account for the affinity of dyes for uncharged substrates [30–32]. Another mechanism involved in biological staining from aqueous solutions is hydrophobic interaction. This is the tendency for hydrophobic parts of dye molecules to enter and adhere to hydrophobic domains of tissue proteins or lipids. Hydrophobicity is due to lack of hydrogen, oxygen or nitrogen atoms capable of forming hydrogen bonds with water. In a solution, hydrophobic molecules occupy spaces between the icosahedral clusters of 280 water molecules that result from intermolecular hydrogen bonding [33]. The hydrophobic molecules may form aggregates, or they may diffuse from the inter-cluster domain of the water (where their concentration is considerably higher than in the solution as a into domains of the substrate that is being the may a high density and be by the action of van der Waals forces It should be that hydrophobic is not a force or a type of bonding, but a of hydrogen bonding water molecules and the of hydrophobic molecules by London forces [27]. Hydrogen bonding may be involved in some biological staining methods in which dyes are applied from such as An is carmine in which a red cationic dye–metal complex binds to which is a in the cytoplasm of and muscle cells. of stained sections to water the dye from attractions der Waals forces and hydrogen bonding to to the binding of some dyes in textile coloration Ref. and in many biological staining methods Ref. Nevertheless, in many on the subject staining by basic and acid dyes is attributed to salt as postulated by Ehrlich [10]. Other also dye binding by forces other than attractions between ions. ionic the weaker forces that hold dye molecules in contact with tissue components from added inorganic but dye binding can be by including a high concentration of in the The molecules are assumed to form hydrogen bonds to the dye and the substrate, the close that is for the operation of the weaker electrical forces It is that a few stains with tissue components to form The known is which with groups. groups can be at specific sites in sections of tissues. include the of or of by acid or and the partial of DNA see In DNA staining, acid the and groups bound to the The of with In the a and a acid group are covalently bound to the Reactive dyes for have few uses in biological staining. included several in the edition of Conn's Biological in the that might with In the 10th edition of dyes were for on the of their in and few textile dyes dyes with amino and hydroxy which are everywhere in sections of tissues. Dyes that colour everything can provide useful between different structural especially in thin but there is no of traditional colorants for this The dye used in staining is in with a to of ions. This known as is frequently called even though that is not a which can form coloured complexes with a range of is made by of In the preparation of modern haemalum solutions half the is to haematein by adding a of sodium of the haematein that is by use of the solution and by The of of haematein is a known as is because it a strong colour to the nuclear of cells. haemalum does not stain many structures other than nuclei. The of nuclear staining by haemalum is not to the DNA and nuclear staining can be after complete extraction of DNA from the sections. The staining is however, by chemical of amino and groups These that the strongly basic proteins with DNA in the substrate of nuclear staining by This with the results of and who and studies of aluminium–haematein These that a haemalum solution at pH known to contain cationic dye–metal stained DNA and RNA in fixed cell Their staining solution, however, was the dilute one haematein with that was used in the and it to stain the cells. The haemalum solutions used in for staining nuclei contain from to haematein and to at a pH close to and they stain nuclei in a few dilute and haemalum solutions Aluminium ions can by or ionic to all parts of a section of tissue binding of to DNA occurs even at low pH and can in reactions applied to tissue stained with haemalum In a technique for amplification and of DNA is a method for and In the presence of at concentrations with those in haemalum solutions, cationic dyes do not stain nucleic acids and selective for macromolecular and in the the is frequently applied to objects seen in sections stained with haemalum and eosin even though it has been known for many years that haemalum does not as a cationic The mechanism of nuclear staining by haemalum is understood, and investigations are and its are closely to and and can be used to make a staining solution with haemalum that colours cell nuclei red rather than is a useful red nuclear to follow coloration of acidic with Other dyes used in with salts as cationic colorants, staining structures other than nuclei. are and Acid which are dyes. an dye, is used in with chromium(iii) rather than ions The of the complex was by and Horobin [12]. Carmine is a of Carminic Acid from cochineal) with and it is as a cationic complex In several staining techniques two dyes with or charges are applied to sections or and they bind to different of staining with anionic dyes include method with eosin Y and method with Acid and and the method with Acid and Dyes frequently used with Acid include and also known as and water is a of closely which contain up to major components and several the on the labelled have a major component the of does not influence its staining which have been exploited since Ehrlich it into in [2]. With these the dye anions bind to cytoplasm and the anions to collagen fibres. The for the coloration were based on assumed in of the tissue. The dye ions the section more and the from the of The more of collagen fibres ions of but the ions were more in The coloured anions can also be to have higher affinity for protein than the on account of their sizes and numbers of anionic sites [13]. with and with different dye to the of selective staining due to different of and different affinities of the dyes for collagen and cytoplasm. Some are to with the of For example, with different of dyes cell nuclei may the same colour as either cytoplasm with or collagen with found that of a tissue with acid all staining by anionic dyes with small molecules but not the staining of collagen by anionic dyes. found that with acid the uptake of into collagen by this not the of staining. achieved suppression of all including that of by sections with a of acid and was by of the groups with a acid postulated that the dyes such as Acid and eosin stained cytoplasm by ionic attraction to protonated amino groups whereas the dye anions were to collagen by groups of arginine (which are by and in by hydrogen bonds to the hydroxy groups of and The of ionic attraction from that staining of cytoplasm and collagen is by of inorganic anions to the staining The hydrogen bonding mechanism was also suggested by and who that many dyes were as collagen stains when applied as solutions in aqueous In a later van der Waals forces and hydrophobic to hydrogen Prento hydrogen bonding because collagen staining was by of but not of sodium to the staining Although it works by hydrogen bonding, may not the hydrogen bonding of dyes to substrates. discussed hydrogen-bonded molecules may the dye ions and substrate molecules too for the operation of van der Waals forces. The of to collagen staining that the dye was not being in a hydrophobic in the did the staining of cytoplasm by Other methods use of multiple anionic dyes are the stains, in which two or anionic dyes are used in with an

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