Fading correction for fluorescence quantitation in confocal microscopy

Quantitative analysis in confocal microscopy meets with several problems such as fading of the fluorophore during scanning and attenuation of the fluorescence in thick tissue specimens. The present study reports a quantitative investigation of the enzyme uracil-DNA glycosylase (UDG), which removes uracils from DNA. For this study we developed a fading correction algorithm which takes into account both the number of prior scans in the specimen, and the differences in fading through the specimen from each prior scan, presumably due to differences in 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 distances from the laser focus position. The correction procedure can and should be established for the same specimen, but on a different part of the specimen from that used in the actual biological study. Calibration can thus be done on an unknown or inhomogenous object. For a series of confocal xy-scans through the immunostained cells, a corrected summation image representing total FITC-fluorescence related to UDG was obtained. Both noise removal and fading corrections were performed 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 cellular UDG-content by flow cytometry was also performed in order to make 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.