The purpose of the study is to develop and automate a series of steps for enabling digital 3D tissue volume generation in conventional Brightfield microscopy for histopathology applications. Tissue samples were retrieved from the General Hospital of Athens "Hippocration", Greece. Samples were placed on a microtome that produced consecutive 2 μm sections. Each section was stained using Hematoxylin and Eosin and placed on microscope slides. A histopathologist specified the region of interest (ROI) on each slide. A 2D image was created from each ROI using a LEICA DM2500 microscope with a LEICA DFC 420C camera. Τhe 3D volume was created by stacking consecutive 2D images using a deep learning image interpolation method. The reconstructed 3D tissue volumes were evaluated by an expert histopathologist.Results showed that the 3D volumes might reveal information that is not clearly visible or even undetectable in the conventional 2D Brightfield images. In contrast to other 3D tissue imaging technologies, the proposed method (a) does not depend on the distance of the sample from the objectives producing 3D tissue volumes at any desired magnification, (b) does not require a special instrument, it may be implemented with any conventional Brightfield microscope, and (c) can be used for any given routine application, not only for some specialized clinical studies. The proposed study provides the basis for a feasible, cost-less and time-less upgrade of any standard 2D microscope into a 3D imaging instrument that may enhance the quality of diagnostic assessments in histopathology.