Fading correction for fluorescence quantitation in confocal microscopy

Quantitative analysis in confocal microscopy meets with several probl ems such as fading of the fluorophore during scanning and attenuation of the fluorescence in thick tissue specimens. The present study rep orts a quantitative investigation of the enzyme uracil-DNA glycosylas e (UDG), which removes uracils from DNA. For this study we developed a fading correction algorithm which takes into account both the numbe r of prior scans in the specimen, and the differences in fading throu gh the specimen from each prior scan, presumably due to differences i n laser intensity at various axial distances from the focus position. On this point, our findings are in contrast with results reported in other well known papers, and indicate different fading at various di stances from the laser focus position. The correction procedure can a nd should be established for the same specimen, but on a different pa rt of the specimen from that used in the actual biological study. Cal ibration can thus be done on an unknown or inhomogenous object. For a series of confocal xy-scans through the immunostained cells, a corre cted summation image representing total FITC-fluorescence related to UDG was obtained. Both noise removal and fading corrections were perf ormed on each image in the series before the summation image was made . Estimates of total amounts of UDG localized in the cells and nuclei , respectively, could then be obtained. Measurement of the total cell ular UDG-content by flow cytometry was also performed in order to mak e a comparison of the two methods for quantitative analysis. For both methods a range of approximately 4.5 was obtained between total UDG- content of cells at the 5 and 95 percentage points.