Solutions to common issues in widefield microscopy: vignetting, mosaicing and depth of focus

In this thesis we have developed solutions to common issues regarding widefield microscopes, facing the problem of the intensity inhomogeneity of an image and dealing with two strong limitations: the impossibility of acquiring either high detailed images representative of whole samples or deep 3D objects. First, we cope with the problem of the non-uniform distribution of the light signal inside a single image, named vignetting. In particular we proposed, for both light and fluorescent microscopy, non-parametric multi-image based methods, where the vignetting function is estimated directly from the sample without requiring any prior information. After getting flat-field corrected images, we studied how to fix the problem related to the limitation of the field of view of the camera, so to be able to acquire large areas at high magnification. To this purpose, we developed mosaicing techniques capable to work on-line. Starting from a set of overlapping images manually acquired, we validated a fast registration approach to accurately stitch together the images. Finally, we worked to virtually extend the field of view of the camera in the third dimension, with the purpose of reconstructing a single image completely in focus, stemming from objects having a relevant depth or being displaced in different focus planes. After studying the existing approaches for extending the depth of focus of the microscope, we proposed a general method that does not require any prior information. In order to compare the outcome of existing methods, different standard metrics are commonly used in literature. However, no metric is available to compare different methods in real cases. First, we validated a metric able to rank the methods as the Universal Quality Index does, but without needing any reference ground truth. Second, we proved that the approach we developed performs better in both synthetic and real cases.