Serial block face imaging is a microscopy technique in which the top of a specimen is cut or ground away and a mosaic of images is collected of the newly revealed cross-section. Images collected from each slice are then digitally stacked to achieve 3D images. The development of fully automated image acquisition devices has made serial block face imaging more attractive by greatly reducing labour requirements. The technique is particularly attractive for studies of biological activity within cancellous bone as it has the capability of achieving direct, automated measures of biological and morphological traits and their associations with one another. When used with fluorescence microscopy, serial block face imaging has the potential to achieve3Dimagesof tissue aswell as fluorescent markers of biological activity. Epifluorescencebased serial block face imaging presents a number of unique challenges for visualizing bone specimens due to noise generated by sub-surface signal and local variations in tissue autofluorescence. Here we present techniques for processing serial block faceimagesof trabecularboneusingacombination of non-uniform illumination correction, precise tiling of the mosaic in each cross-section, cross-section alignment for vertical stacking, removal of sub-surface signal and segmentation. The resulting techniques allow examination of bonesurface texture thatwill enable3Dquantitativemeasures of biological processes in cancellous bone biopsies.