Colour vignetting correction for microscopy image mosaics used for quantitative analyses

Image mosaicing permits to achieve one high resolution image, extending the visible area of the sample while keeping the same resolution. However, intensity inhomogeneity of the stitched images can alter measurements and the right perception of the original sample. The problem can be solved by flat-field correcting the images through the vignetting function. Vignetting correction has been widely addressed for grey-level images, but not for colour ones. In this work, a practical solution for the colour vignetting correction in microscopy, also facing the problem of saturated pixels, is described. In order to assess the quality of the proposed approach, five different tonal correction approaches were quantitatively compared using state-of-the-art metrics and seven pairs of partially overlapping images of seven different samples. The results obtained proved that the proposed approach allows obtaining high quality colour flat-field corrected images and seamless mosaics without employing any blending adjustment. In order to give the opportunity to easily obtain seamless mosaics ready for quantitative analysis, the described vignetting correction method has been implemented in an upgraded release of MicroMos (version 3.0, http://sourceforge.net/p/micromos), an open-source software specifically designed to automatically obtain mosaics of partially overlapped images.