Lateral features of size 1-10 nm are created on graphite, using an RF magnetron gas-condensation cluster beam deposition source. Specifically, size-selected gold clusters, Au-55(+) and Au-70(+), are pinned to the graphite surface, in order to explore the immobilization of protein molecules. Refined sample preparation protocols enable the utilization of the atomic force microscope (AFM) to visualize two proteins, histidine affinity tagged green fluorescent protein (pHAT-GFP) and Human Oncostatin M, both in air, and in physiological buffer solution, which mimics their natural environment. Both protein islands (complexes) and individual (or a few) protein molecules are identified. The impetus for single molecule science studies lies in the possible observation of the structural conformation changes of proteins as they perform their individual functions in their native environments. The manner in which specific proteins organize themselves spatially is a key consideration in understanding how they function, e.g., in disease control. The cluster approach creates sufficiently dilute arrays of truly nanoscale features that single molecule optical experiments may also be feasible in the future. Experiments on the re-usability of the nanocluster films provide further proof of the resilience and versatility of this type of nanostructured surface for protein immobilization work. (C) 2003 Elsevier B.V. All rights reserved.