Fluorescence fading in quantitative fluorescence microscopy: a cytofluorometry of the automatic recording of fluorescence peaks of very short duration

Rates of photodecomposition were studied in some fluorophores during short (milliseconds) and longer (minutes) illumination periods. A xenon burner served as light source, and care was taken to obtain optimum conditions for activation. The fluorophores studied included (i) the formaldehyde-induced fluorescent product from 5-hydroxytryptamine in mast cells, (ii) Berberine sulphate bound to mast cell polyanions, (iii) Feulgen-Pararosaniline-stained DNA, and (iv) Fluorescein isothiocyanate-conjugated IgG in an antinuclear factor test. All fluorophores showed a significant fading during 3 min illumination. The Fluorescein isothiocyanate-conjugate faded the most rapidly; its fluorescence intensity was reduced to 50% of the initial value after 2 sec continuous illumination. No fluorophore faded significantly during the initial few milliseconds of illumination. On the basis of these findings, an inexpensive measuring device was constructed. It contained a peak-reader and memory circuit triggered by the flash synchronization tap of a camera shutter positioned in the activation beam. The peak-reader has a response time of about 2 msec. Repeated measurements on the various fluorophores indicate that this peak-reading device may be used to measure fluorescence intensity without fading.