Flat field correction for high‐throughput imaging of fluorescent samples

Vignetting of microscopic images impacts both the visual impression of the images and any image analysis applied to it. Especially in high‐throughput screening high demands are made on an automated image analysis. In our work we focused on fluorescent samples and found that two profiles (background and foreground) for each imaging channel need to be estimated to achieve a sufficiently flat image after correction. We have developed a method which runs completely unsupervised on a wide range of assays. By adding a reliable internal quality control we mitigate the risk of introducing artefacts into sample images through correction. The method requires hundreds of images for the foreground profile, thus limiting its application to high‐throughput screening where this requirement is fulfilled in routine operation.