Ultrasonication is an energy intensive process used to enhance organics' solubilisation and used in promoting methane yield from wastewater sludge. Applying limited ultrasound without full disruption of floc structure or cell walls can significantly enhance methane yield from sludge at reduced energy cost. This work probed the spatial distribution of extracellular polymeric substances, cells in the original wastewater floes, and the yielded fragments following weak ultrasonication using a process of multiple staining and a confocal laser scanning microscope technique. The stereological information of the three-dimensional architecture of the original wastewater floc and ultrasonicated fragments was extracted and compared. The local structural changes following weak ultrasonication were presented for the first time, which should also correlate with the enhanced methanogenesis thereby produced. (C) 2007 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.