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  Picosecond multi-photon scanning near-field optical microscopy

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

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-FAE9-A Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EBFD-5
Genre: Journal Article

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 Creators:
Jenei, A.1, Author
Kirsch, A. K.2, Author              
Subramaniam, V.2, Author              
Arndt-Jovin, D. J.2, Author              
Jovin, T. M.2, Author              
Affiliations:
1Max Planck Society, ou_persistent13              
2Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              

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Free keywords: Animal; Antibodies; *Chromosomes/ul [Ultrastructure]; Drosophila melanogaster/ge [Genetics]; Fluorescent Dyes/me [Metabolism]; Human; Lasers; *Microscopy, Fluorescence/mt [Methods]; *Mitochondria/ul [Ultrastructure]; Photons; Support, Non-U.S. Gov't; Tumor Cells, Cultured; Ultraviolet Rays; 2-photon fluorescence microscopy; 3-photon induced fluorescence; Ti-sapphire laser; Excitation; Cells; Protein; Spectroscopy
 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.

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Language(s): eng - English
 Dates: 2005-08-161999
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: eDoc: 226752
Other: 11473
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 76 Sequence Number: - Start / End Page: 1092 - 1100 Identifier: -