Surface micro-topography of the {110} faces of tetragonal lysozyme crystals was observed in situ by laser confocal microscopy (LCM) combined with differential interference contrast microscopy (DIM). We could observe two-dimensional (213) nucleation and subsequent growth of the 2D islands in real time. When steps of neighboring 2D islands coalesced, the contrast of the steps disappeared completely and the 2D islands merged smoothly. This result proved that the height of the steps observed by LCM combined with DIM was always the same and these steps were elementary ones (5.6 nm high). The combination of LCM and DIM was necessary to observe elementary steps with sufficient contrast. The elementary steps on a spiral growth hillock could also be observed using the LCM-DIM system, although their contrast was much smaller than that of the 2D islands because of much smaller inter-step distance. (C) 2003 Elsevier B.V. All rights reserved.