Epifluorescence, confocal and total internal reflection microscopy for single-molecule experiments: a quantitative comparison.

Epifluorescence, confocal and total internal reflection microscopy are the most widely used techniques for optical single-molecule experiments. Employing these methods, we recorded the emission intensity of the same single molecule as a function of the excitation rate under otherwise identical experimental conditions. Evaluation of these data provides a quantitative comparison of the signal-to-background ratios that can be achieved for the three microscopic techniques.