Picosecond multiphoton scanning near-field optical microscopy

Biophys J. 1999 Feb;76(2):1092-100. doi: 10.1016/S0006-3495(99)77274-7.

Abstract

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies
  • Chromosomes / ultrastructure*
  • Drosophila melanogaster / genetics
  • Fluorescent Dyes / metabolism
  • Humans
  • Lasers
  • Microscopy, Fluorescence / methods*
  • Mitochondria / ultrastructure*
  • Photons
  • Tumor Cells, Cultured
  • Ultraviolet Rays

Substances

  • Antibodies
  • Fluorescent Dyes