Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM(1)); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM(n) = EM(1) * A ((n-1)). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.