Axial scanning method for single biomolecule optical imaging combined with tweezers (Conference Presentation)

Optical imaging combined with single-molecule force spectroscopy broadens the horizons for the spatiotemporal localization and the mechanical details of target molecules. We developed a simple optical method to extend the depth of field in a high numerical aperture objective (NA = 1.2 and 1.4) that requires to visualize a single fluorophore. This method enables us to obtain an optical signal outside the focal plane without unintended interruption of the force signal in single-molecule optical imaging-force spectroscopy. By axial scanning, using an electrically tunable lens with a fixed sample, we were successfully able to visualize the diffusion of proteins along DNA that is three-dimensionally stretched by optical tweezers. We also demonstrated the performance of the ETL system in the single particle tracking analysis of epidermal growth factor receptor (EGFR) on filamentous actin bundles connecting cells, in the presence of the mechanical force applied to the intercellular nanotube, using an optical trap combined with a line scanning confocal microscope. In fact, ETL has been exploited in various microscopes. These microscopes are intended to have a wide axial scan range of hundreds of micrometers in an objective lens with low NA (< 1.0) for deep-tissue imaging, except for one study, which used a high NA objective lens (NA = 1.4) in wide-field microscopy for the static image of protein-complexes in HeLa cells. Our study is a first demonstration of an ETL being used for a single-molecule force-optical imaging system in extended depth of field.