Fc gamma receptors (Fc gamma R), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of Fc gamma Rs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of Fc gamma Rs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of Fc gamma Rs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the Fc gamma Rs, revealing the nanoscale distribution of Fc gamma Rs on local areas of macrophages. The experimental results can improve our understanding of Fc gamma Rs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy. (C) 2013 Elsevier Inc. All rights reserved.