Letters to the editor: New principles for microspectrofluorometric differentiation between DOPA, dopamine and noradrenaline.

Dopa and the primary “atecholamines, dopamine (DA) and noradrenaline (Ntt), are demonstrated with high sensitivity at the cellular level with the formaldehyde (FA) and the glyoxylic acid (GA) fluorescence histochemical methods (5, 6, 9, 10, 12, 13). However, the fluorophores formed from these substances show indistinguishable excitation and emission spectra. Dopa and DA cannot be differentiated in the fluorescence microscope (see references 3, 8 and 12), whereas after acidification of their FA-induced fluorophores dopa and DA can be distinguished from NA by microspectrofluorometry (1, 2, 7). This differentiation procedure can usually be carried out only when the intensity of the intracellular fluorescence is rather high, and if this is not the case the method is usually quite laborious (see reference 2). Thus, there is obviously a great need for new methods for differentiation between dopa, DA and NA in the fluorescence microscope. In a preliminary study (13) we found that in a combined FA and GA reaction, fluorophores with clearly different spectral properties can be formed from dopa and DA. In the present report the usefulness of the two-step reaction with FA and GA for differentiation between dopa, DA and NA has been evaluated. The experiments were performed with the fluorogenic substances enclosed in histochemical protein droplet models. These were prepared by spraying 2% albumin solutions containing 0.5 mg/mI L-dopa, DA or NA on glass cover slips. The histochemical reactions were performed as follows. In the first step, the models were placed in 1-liter closed vessels containing 5 g paraformaldehyde, which either had been equilibrated in air of about 50% relative humidity (11) or excessively dried by heating at + 100#{176}C for 1 hr and subsequent storing over phosphorous pentoxide. The treatment was carried out at different temperatures