An efficient autofocus algorithm for indirect immunofluorescence applications

Photobleaching effect is a major issue in developing autofocus algorithms for indirect immunofluorescence (IIF) assay since it quickly flattens the fluorescence intensity of the samples. To overcome this limitation we propose an autofocus algorithm, suited for IIF, aiming at minimizing the number of images required to focus the sample. We test our algorithm with an heterogeneous set of IIF images containing the substrates most widely used in clinical practice, comparing popular focus functions in order to find out the most appropriate for our procedure. Experimental results prove that our algorithm focus the sample using less images than other popular algorithms proposed in the literature.