One-photon and two-photon excited fluorescence microscopies based on polarization-control: Applications to tip-enhanced microscopy

Abstract

One-photon and two-photon excited fluorescence microscopies using either radial or azimuthal polarization have been developed and applied to the imaging of quantum dots. In both cases (one-photon and two-photon excitations), the fluorescence image profile of each quantum dot is in good agreement with the electric field intensity distribution of a tightly focused spot using a high numerical aperture objective lens. While this polarization dependence of the absorption/emission of quantum dots (or other dye molecules) is useful for characterizing the orientation of the quantum dots, most of the biological applications that employ quantum dots or dye molecules as labels require the information describing not only the orientation but also the precise position of each dot. In order to improve the sensing accuracy of the dot’s position, we employ a modified near-field fluorescence microscopy system that utilizes a tip-enhancement technique and radially polarized two-photon excitations. For the tip enhancement, a commercially available silicon cantilever tip has been successfully utilized instead of metallic tips, as the latter tip can drastically quench the near-field fluorescence. Our tip-enhanced two-photon excited fluorescence microscopy technique enables visualization of the quantum dots distributed on a cover slip beyond the diffraction limit of light. We demonstrate that our approach is advantageous not only due to its high spatial resolution but also due to its high sensitivity by showing that the fluorescence signal is not detectable without the aid of the tip enhancement in some cases.