A microscopical assay using a densitometric application of image analysis to quantify neurotransmitter dynamics.

We have attempted to demonstrate the technical requirements and performance of a microscopical assay using a densitometric application of image analysis to measure immunohistochemical stain intensity. Not surprisingly, the techniques required were more demanding than those used for the quantification of field and object parameters in the nervous system. The following areas of methodology have been shown to be important: (1) use of buffers free of metallic ions for tissue processing, (2) selection and titration of first and second layer antibodies, (3) reduction and control of fading of fluorescence, (4) selection of microscopical and imaging equipment to give accurate, sensitive and uniform representations of low-light biological images, and (5) use of appropriate image analysis algorithms in order to generate binary images that match the spatial and intensity distributions of immunostaining. Incorporation of these techniques into our assay system gave sensitive measurements of the time-scale of uptake of 5-hydroxy-tryptamine (5-HT) into sympathetic nerve terminals. The microscopical assay appears to have advantages over alternative approaches used for studies of neurotransmitter dynamics, particularly in small, heterogeneous tissue samples.