Extended depth of field for single biomolecule optical imaging-force spectroscopy

Real-time optical imaging combined with single-molecule manipulation broadens the horizons for acquiring information about the spatiotemporal localization and the mechanical details of target molecules. To obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy, we developed an optical method to extend the depth of field in a high numerical aperture objective (≥ 1.2), required to visualize a single fluorophore. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the epidermal growth factor receptor (EGFR) moving along the three-dimensionally elongated filamentous actin bundles connecting cells (intercellular nanotube), while another EGFR on the intercellular nanotube was trapped by optical tweezers in living cells. Our approach is simple, fast and inexpensive, but it is powerful for imaging target molecules axially in single-molecule optical imaging-force spectroscopy.