AbstractCommercial exploitation of lignocellulose for biotechnological production of fuels and commodity chemicals requires efficient—usually enzymatic—saccharification of the highly recalcitrant insoluble substrate. A key characteristic of cellulose conversion is that the actual hydrolysis of the polysaccharide chains is intrinsically entangled with physical disruption of substrate morphology and structure. This “substrate deconstruction” by cellulase activity is a slow, yet markedly dynamic process that occurs at different length scales from and above the nanometer range. Little is currently known about the role of progressive substrate deconstruction on hydrolysis efficiency. Application of advanced visualization techniques to the characterization of enzymatic degradation of different celluloses has provided important new insights, at the requisite nano‐scale resolution and down to the level of single enzyme molecules, into cellulase activity on the cellulose surface. Using true in situ imaging, dynamic features of enzyme action and substrate deconstruction were portrayed at different morphological levels of the cellulose, thus providing new suggestions and interpretations of rate‐determining factors. Here, we review the milestones achieved through visualization, the methods which significantly promoted the field, compare suitable (model) substrates, and identify limiting factors, challenges and future tasks. Biotechnol. Bioeng. 2013; 110: 1529–1549. © 2013 Wiley Periodicals, Inc.