BACKGROUND REJECTION AND SIGNAL-TO-NOISE OPTIMIZATION IN CONFOCAL ANDALTERNATIVE FLUORESCENCE MICROSCOPES

In the confocal microscope, tightly focused illumination and spatially filtered detection are combined to reduce out-of-focus background and to produce high-quality images that display thin optical sections wit hin thick fluorescent specimens. We define background as the detected light that originates outside a resolution volume and signal as the de tected light that originates within the same volume. Background reject ion is measured by the signal-to-background ratio (S/B) and is calcula ted for confocal, spinning-disk, line-illumination, slit-detection, an d conventional fluorescence microscopes as a function of both the spat ial filter size and the specimen thickness. Spatial filter sizes that reject background and optimize the signal-to-noise ratio (S/N) are cal culated for each microscope. These calculations are normalized so that the time-averaged illumination at each point in the specimen is the s ame for each microscope. For thick specimens, we show that the S/B obt ained with a confocal microscope can be more than 100 times greater th an the S/B available with a conventional microscope, and we find that the optimal confocal S/N can be a factor of 10 greater than the S/N in the conventional microscope.