We present a new algorithm to analyse information content in images acquired using automated fluorescence microscopy. The algorithm belongs to the group of autofocusing methods, but differs from its predecessors in that it can handle thick specimens and operate also in confocal mode. It measures the information content in images using a 'content function', which is essentially the same concept as a focus function. Unlike previously presented algorithms, this algorithm tries to find all significant axial positions in cases where the content function applied to real data is not unimodal, which is often the case. This requirement precludes using algorithms that rely on unimodality. Moreover, choosing a content function requires careful consideration, because some functions suppress local maxima. First, we test 19 content functions and evaluate their ability to show local maxima clearly. The results show that only six content functions succeed. To save time, the acquisition procedure needs to vary the step size adaptively, because a wide range of possible axial positions has to be passed so as not to miss a local maximum. The algorithm therefore has to assess the steepness of the content function online so that it can decide to use a bigger or smaller step size to acquire the next image. Therefore, the algorithm needs to know about typical behaviour of content functions. We show that for normalized variance, one of the most promising content functions, this knowledge can be obtained after normalizing with respect to the theoretical maximum of this function, and using hierarchical clustering. The resulting algorithm is more reliable and efficient than a simple procedure with constant steps.

Faculty of Informatics Centre for Biomedical Image Analysis Masaryk University Botanicka 68a Brno Czech Republic