Quantitation of fluorescence of biogenic monoamines demonstrated with the formaldehyde flourescence method

The present review deals with the possibilities of using the FALCK-HILLARP fluorescence technique for quantitation of biogenic motzoamines present in tissue cells structures, with special reference to neuronally stored monoamines. Since a well standardized technique is of utmost importance for quantitative work, the basic chemistry and the different factors affecting the histochemical reaction and the fluorescence intensity are thoroughly discussed. The main problem when using the present method for monoamine quantitation is that the local intrancuronal concentration of monoamine is generally so high that a concentration dependent quenching of the fluorescence exists. The situation is furthermore complicated by the fact that the fluorescence yield is affected by the subcellular localization of monoamine; the fluorescence yield being higher when the monoamine is extragranularly distributed. It can be concluded that, if changes in the fluorescence intensity are recorded as compared with control, this reflects true changes in amine concentration in most cases. On the other hand, changes in amine concentration may escape detection when the remaining amine concentration is so high that quenching occurs or when a redistribution of amine and/or fluorophor from the storage granules to the extragranular space has taken place. Therefore, in doubtful cases the fluorescence intensity estimations should be correlated with chemical-analytical determinations of monoamine content in comparable pieces of tissue.