Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 49, Issue 3, Pages 305-311Publisher
HISTOCHEMICAL SOC INC
DOI: 10.1177/002215540104900304
Keywords
anti-fading media; anti-fading factor; confocal laser scanning; microscopy
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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.
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