Abstract: The last years have seen an increasing use of image analysis techniques in the field of biomedical imaging, in particular in microscopic imaging. The basic step for most of the image analysis techniques relies on a background image free of objects of interest, whether they are cells or histological samples, to perform further analysis, such as segmentation or mosaicing. Commonly, this image consists of an empty field acquired in advance. However, many times achieving an empty field could not be feasible. Or else, this could be different from the background region of the sample really being studied, because of the interaction with the organic matter. At last, it could be expensive, for instance in case of live cell analyses. We propose a non parametric and general purpose approach where the background is built automatically stemming from a sequence of images containing even objects of interest. The amount of area, in each image, free of objects just affects the overall speed to obtain the background. Experiments with different kinds of microscopic images prove the effectiveness of our approach.