Robust and unbiased estimation of the background distribution for automated quantitative imaging

Abstract

Quantitative analysis of high-throughput microscopy images requires robust automated algorithms. Background estimation is usually the first step and has an impact on all subsequent analysis, in particular for foreground detection and calculation of ratiometric quantities. Most methods recover only a single background value, such as the median. Those that aim to retrieve a background distribution by dividing the intensity histogram yield a biased estimation in images in non-trivial cases. In this work, we present the first method to recover an unbiased estimation of the background distribution directly from an image and without any additional input. Through a robust statistical test, our method leverages the lack of local spatial correlation in background pixels to select a subset of pixels that accurately represent the background distribution. This method is both fast and simple to implement, as it only uses standard mathematical operations and an averaging filter. Additionally, the only parameter, the size of the averaging filter, does not require fine tuning. The obtained background distribution can be used to test for foreground membership of individual pixels, or to estimate confidence intervals in derived quantities. We expect that the concepts described in this work can help to develop a novel family of robust segmentation methods.