Picosecond multiphoton scanning near-field optical microscopy

A. Jenei, Achim K. Kirsch, Vinod Subramaniam, Donna J. Arndt-Jovin, Thomas M. Jovin

Research output: Contribution to journalArticle

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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 Mito Tracker 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.

Original languageEnglish
Pages (from-to)1092-1100
Number of pages9
JournalBiophysical Journal
Volume76
Issue number2
Publication statusPublished - 1999

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Photons
Microscopy
Coloring Agents
Fluorescence
Polytene Chromosomes
Photobleaching
MCF-7 Cells
Drosophila melanogaster
Organelles
Adenocarcinoma
Breast
Lasers
Antibodies
DNA
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Jenei, A., Kirsch, A. K., Subramaniam, V., Arndt-Jovin, D. J., & Jovin, T. M. (1999). Picosecond multiphoton scanning near-field optical microscopy. Biophysical Journal, 76(2), 1092-1100.

Picosecond multiphoton scanning near-field optical microscopy. / Jenei, A.; Kirsch, Achim K.; Subramaniam, Vinod; Arndt-Jovin, Donna J.; Jovin, Thomas M.

In: Biophysical Journal, Vol. 76, No. 2, 1999, p. 1092-1100.

Research output: Contribution to journalArticle

Jenei, A, Kirsch, AK, Subramaniam, V, Arndt-Jovin, DJ & Jovin, TM 1999, 'Picosecond multiphoton scanning near-field optical microscopy', Biophysical Journal, vol. 76, no. 2, pp. 1092-1100.
Jenei A, Kirsch AK, Subramaniam V, Arndt-Jovin DJ, Jovin TM. Picosecond multiphoton scanning near-field optical microscopy. Biophysical Journal. 1999;76(2):1092-1100.
Jenei, A. ; Kirsch, Achim K. ; Subramaniam, Vinod ; Arndt-Jovin, Donna J. ; Jovin, Thomas M. / Picosecond multiphoton scanning near-field optical microscopy. In: Biophysical Journal. 1999 ; Vol. 76, No. 2. pp. 1092-1100.
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