Quantitative Comparison of Anti-Fading Mounting Media for Confocal Laser Scanning Microscopy

Abstract

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 ( EM1); and background fluorescence ( B). The fluorescent intensity at a certain point following nth scan is given as EMn = EM1 • 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.