Application of electromagnetic centroids to colocalization of fluorescing objects in tissue sections

Light microscopy as well as image acquisition and processing suffer from physical and technical prejudices which preclude a correct interpretation of biological observations which can be reflected in, e.g., medical and pharmacological praxis. Using the examples of a diffracting microbead and fluorescently labelled tissue, this article clarifies some ignored aspects of image build-up in the light microscope and introduce algorithms for maximal extraction of information from the 3D microscopic experiments. We provided a correct set-up of the microscope and we sought a voxel (3D pixel) called an electromagnetic centroid which localizes the information about the object. In diffraction imaging and light emission, this voxel shows a minimal intensity change in two consecutive optical cuts. This approach further enabled us to identify z-stack of a DAPI-stained tissue section where at least one object of a relevant fluorescent marker was in focus. The spatial corrections (overlaps) of the DAPI-labelled region with in-focus autofluorescent regions then enabled us to co-localize these three regions in the optimal way when considering physical laws and information theory. We demonstrate that superresolution down to the Nobelish level can be obtained from commonplace widefield bright-field and fluorescence microscopy and bring new perspectives on co-localization in fluorescent microscopy.