Multi‐image based method to correct vignetting effect in light microscopy images

Summary Vignetting is the radial attenuation effect of the image's brightness intensity from the center of the optical axis to the edges. To perform quantitative image analyses it is mandatory to take into account this effect, intrinsic of the acquisition system. Many image processing steps, such as segmentation and object tracking, are strongly affected by vignetting and the effect becomes particularly evident in mosaicing. The most common approach to compensate the attenuation of the image's brightness intensity is to estimate the vignetting function from a homogeneous reference object, typically an empty field, and to use it to normalize the images acquired under the same microscope set‐up conditions. However, several reasons lead to the use of image‐based methods to estimate the vignetting function from the images themselves. In this work, we propose an effective multi‐image based method suitable for real‐time applications. It is designed to correct vignetting in wide field light microscopy images. The vignetting function is computed stemming from a background built incrementally from the proposed background segmentation algorithm, validated on several manually segmented images. The extensive experiments carried out using cell cultures, histological samples and synthetic images prove that our method almost always yields the best results and in worst cases are comparable to those achieved by using homogeneous reference objects.