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

Eric W. Hansen

doi:10.1117/12.271273

10 April 1997 Using an adjustable detection time to correct photobleaching effects in fluorescence microscopy

Eric W. Hansen

Proceedings Volume 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV; (1997) https://doi.org/10.1117/12.271273
Event: BiOS '97, Part of Photonics West, 1997, San Jose, CA, United States

Abstract

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.

Citation Download Citation

Eric W. Hansen "Using an adjustable detection time to correct photobleaching effects in fluorescence microscopy", Proc. SPIE 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV, (10 April 1997); https://doi.org/10.1117/12.271273

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