Using an adjustable detection time to correct photobleaching effects in fluorescence microscopy

Photobleaching causes progressive fading in successive slices of a through-focus series, so that the last image taken in the series can have a significantly lower signal- to-noise ratio than the first. Bleaching is often successfully minimized by including antifade additives in the specimen preparation and/or by reducing the optical dose to the specimen. However, these measures may not be sufficient in a through-focus series where many slices must be taken. This paper presents a simple approach to ameliorating the effects of bleaching, which is to progressively increase the integration times so as to maintain constant signal level from slice to slice. I refer to the sequence of integration times as an integration schedule. I develop the equations for integration scheduling from the physical assumptions and discuss how the method affects image quality.