Flat field correction for high‐throughput imaging of fluorescent samples

Summary 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. Lay description Microscopy images tend to be shaded, being usually brighter in the centre and darker at corners of the image field. This effect is also called vignetting, see http://en.wikipedia.org/wiki/Vignetting. Here we describe a software approach which corrects for shading yielding so‐called flat field images. These flat field images do not only show uniform background intensity, but the intensity of each single object (e.g. cells) in the image is corrected for the location dependent shading effect. Thus, any subsequent quantification of the object intensity is no longer impaired by the shading effect. The software uses regular sample images for estimating a pair of intensity profiles needed for the correction, no specific additional reference images are required. The profiles are automatically estimated, that is, without user interference, but with several internal steps of quality control. The approach is best suited for high‐throughput applications.