Digital Deconvolution Microscopy: Development, Evaluation and Utilization in 3D quantitative studies of E-cadherin expression in skin of Bufo arenarun tadpoles

Deconvolution of three-dimensional (3D) fluorescence microscopy images using computational restoration algorithms may be seen as a cheaper alternative to modern 3D microscopy techniques. We have recently developed a digital deconvolution microscope based upon a standard upright fluorescence microscopy setup, which allows us to perform precise optical-sectioning procedures by positioning the sample at different focal depths. The acquired images are then treated using computational methods which use information regarding the image-formation process (deconvolution using the Point Spread Function, PSF). Processed images are used to perform a more accurate 3D quantification and analyses. The main objective of the present work is to evaluate the technique’s steps and to analyze the usefulness of this method with the aid of a biological model. For that reason, we selected a 3D cell-adhesion skin model, based upon a specimen commonly used by our research group. In the present work we show what must be considered in order to obtain an adequate and precise quantification.