Microfluorimetric scanning of sympathetic nerve fibers: quantification of neuronal and extraneuronal fluorescence with the aid of histogram analysis.

A high resolution (0.5 micrometer), two-dimensional microfluorimetric scanning procedure was used to quantitate the formaldehyde-induced fluorescence of noradrenaline in the iris of the rat. Scanning of large areas (50 x 50 micrometer) in the sympathetic innervated dilator muscle revealed an overlap between measurements of nerve fibers and of background (smooth muscle). In order to discriminate between both populations, the scan data were converted into fluorescence histograms (256 fluorescence classes, and subjected to mathematical analysis. The characteristics of the background histogram were obtained from scans of iris preparations devoid of fluorescent nerve fibers (pretreatment of the donor rat with reserpine or sympathetic denervation). A curve-fitting program was applied on those fluorescence classes in the normal iris histogram that represent measurements on pure background and resulted in a full background histogram. After subtraction of this background histogram from the original histogram, a nerve fiber histogram was obtained. The validity of the algorithm was evaluated by scanning iris preparations with varying background intensities. The results showed that quantification of the nerve fiber fluorescence was independent of variation in the background.