Fluorescence imaging of bulk stained tissue is a popular technique to monitor the activities of neuronal population. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. While in single cell staining experiments, background can be measured from a neighboring non-stained region, such a region often does not exist in bulk stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of the method and give example applications in ratiometric calcium imaging of brain slices. Using these results, we show that our method significantly improves the quantitative measurements of [Ca2+] dynamics in bulk stained neuronal population.