Fluorescence imaging below the diffraction limit using Near-field Scanning Optical Microscope (NSOM) with a metallic probe

Abstract

Recently, fluorescence measurement systems using NSOM have been developed. Such NSOM systems opened the possibility of spectroscopic characterization of nanoscopic field, thereby allowing for a single molecule detection including single molecule orientation, excited state lifetime, energy transfer, and so on. Such information couldn't be obtained by the use of macroscopic detection mechanics. But the single molecule fluorescence have been done with aperture probe NSOMs, thus limiting the lateral resolution to the aperture size (100 nm). Apertureless probes, such as metallic probes and cantilevers of atomic force microscope systems, are expected to bring the resolution near the size of the tip (< 50 nm). But the fluorescence background needs to be rejected because the illumination spot is diffraction limited.