[Metachromasia, double refraction and dichroism caused by Toluidine Blue reaction (author's transl)]

Abstract

The present paper is concerned with those optical phenomena which may be caused in histological structures by the toluidine blue reaction. They relate to absorption as well as refraction properties. The paper gives a detailed description of how the toluidine blue reaction is carried out on histological preparations. The analysis of absorption results leads, in connection with physicochemical considerations, to a new explanation of the cause of toluidine blue metachromasia. In this, the assumption is made that the conditions of attachment of protons to the hydrogen-bearing nitrogen of dye molecules are largely responsible for the occurrence of metachromasia, the way in which the attachment of the proton to this quaternary nitrogen is influenced being of no consequence in this particular connection. It may result from both a combination of homogeneous toluidine blue molecules into associates and a shift to the alkaline region of the pH value of the dye solution. However, the term metachromasia is used to describe only that shift of the absorption spectrum which is produced by associates. The thus produced metachromasia ("concentration metachromasia") involves the development of a so-call "gamma-band" (with an absorption peak of gamma = 546 nm), there being a causal rather than a fundamental difference between the latter band and the normal metachromatic mu-band (with an absorption peak of gamma = 540 nm). Whereas in the case of concentration metachromasia and for an aqueous solution there may be observed an association of dye molecules having a maximum intermolecular distances of less than 5 angström units, which is due to the large number of toluidine blue molecules per unit volume, mu-metachromasia is usually observed in connection with chromotropic structures. This association results in the lone-pair electrons of the hydrogen-bearing nitrogen atom being included in the intermolecular resonance system, which in turn results in the attachment of a proton to this nitrogen being weakened, which leads to the assumption of a red color. The difference to gamma-metachromasia lies in the fact that even by treatment with dilute solutions, in which the dye is present in a monomeric form, such a metachromasia may be produced on account of the sufficiently dense charge pattern of the chromotropic structure. This particular pattern tends to induce an association of dye molecules which in turn are associated, through hydrogen bridges, with the anionic groups. The orthochromatic toluidine blue reaction was found to be a possible method of detecting bridge-bond-active hydrogen in histological structures. The cause of the assumption of a blue color, which is observed in this connection, is a monomeric attachment of the dye. Additional factors leading to ortho-chromatism are also discussed...