ELSEVIER Computer Methods and Programs in Biomedicine 44 (1994) 61-67 computer methods and programs in biomedicine Shading correction and calibration in bacterial fluorescence measurement by image processing system M.H.F. Wilkinson Laboratory for Medical Microbiology, University of Groningen, Oostersingel 59, 9713 EZ Groningen, The Netherlands Received 26 October 1993; revision received 17 January 1994; accepted 21 March 1994 Abstract An image processing system with applications in bacterial (immuno-)fluorescence measurement has been developed. To reach quantitative results, correction for non-uniformities in system sensitivity, both as a function of time (calibra- tion for drifts) and as a function of image coordinates (shading correction), is essential. Both problems can be handled simultaneously by acquiring images of a uniformly fluorescent, solid standard as a reference image. To measure bacter- ial fluorescence, the average fluorescence intensity of isolated areas of interest (the bacteria) is computed, and corrected using the reference images. Two shading correction methods are theoretically and experimentally compared: direct averaging in the corrected image, and (weighted and unweighted) averaging using the raw image and a separate shading image to determine the weights and correct for shading during the averaging. The latter method proved computational- ly 3.5-6.5 times faster on average and reduced propagation of truncation errors during computation, resulting in 40% less noise, for 8-bits/pixel images. Keywords: Bacterial fluorescence; Quantitative microscopy; Image processing 1. Introduction In recent years, there has been a rapidly growing interest in quantitative microscopy. Within this trend, an important focus is on fluorescence micro- scopy [1]. The ability to measure ion concentrations [2], membrane potentials [31 or antibody titres [4], to name but a few possibilities, is a powerful tool in the research of many biological processes at the microscopic level. In our laboratory, a microbio- logical digital image processing system has been * Corresponding author. developed, with applications in both morphology and quantitative indirect immuno-fluorescence [5,6]. Designed as a low cost image processing system, it is based on personal computers equipped with image processor boards. Titres of serum antibodies against faecal flora or cultured bacteria can be determined with a high degree of accuracy using this method [4,7]. The system measures fluorescence by deter- mining the average surface brightness of specified areas of interest (AOIs) thought to coincide with bacteria. To date, our method focuses solely on bac- teria, but the method could equally apply to eukaryote cells, organelles or any other features in 0169-2607/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0169-2607(94)01480-4