Photonic force microscope based on optical tweezers and two-photon excitation for biological applications

A new scanning probe microscope, the photonic force microscope (PFM), based on optical tweezers and two-photon absorption processes for biological applications is described. Optical tweezers are used to trap a fluorescent latex bead with a diameter of 200 nm in an aqueous solution in all three dimensions. The fluorescent dye is chosen to fulfill the two-photon absorption criterion for the 1064-nm line of a Nd:YVO4 laser. The intensity of the fluorescence emission is utilized as a very sensitive position sensor along the optical axis. Two-dimensional images are formed by laterally scanning the trapped latex bead across biological samples while recording the two-photon-induced fluorescences intensity. A scanning probe image of the outer surface of a small neurite from a cultured rat hippocampal neuron is shown, which is hardly visible under differential interference contrast microscopy. The lateral resolution is given by the bead diameter; the axial resolution is 40 nm. Under the experimental conditions the maximal imaging force applied by the probe is below 5 pN.