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Related Experiment Videos

Picosecond multiphoton scanning near-field optical microscopy

A Jenei1, A K Kirsch, V Subramaniam

  • 1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Gottingen, Germany.

Biophysical Journal
|January 23, 1999
PubMed
Summary

We developed simultaneous two- and three-photon excitation for scanning near-field optical microscopy (SNOM). This technique enables high-resolution imaging of biological samples with reduced photodamage, enhancing cellular and chromosomal analysis.

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Area of Science:

  • Biophotonics
  • Microscopy
  • Cell Biology

Background:

  • Scanning near-field optical microscopy (SNOM) offers high resolution but can cause photodamage.
  • Multiphoton excitation in microscopy reduces scattering and photodamage by confining excitation to the focal volume.

Purpose of the Study:

  • To implement simultaneous picosecond pulsed two- and three-photon excitation in SNOM.
  • To image biological specimens with enhanced resolution and minimal photodamage.

Main Methods:

  • Utilized a pulsed Nd:YVO4 laser (1064-nm emission) for simultaneous two-photon excitation (TPE) of visible fluorophores and three-photon excitation (3PE) of UV fluorophores.
  • Employed a shared aperture SNOM with uncoated fiber tips for imaging.
  • Applied TPE and 3PE to human breast adenocarcinoma cells (MCF 7) and Drosophila melanogaster polytene chromosomes.

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Main Results:

  • Successfully imaged cellular organelles and protein bands without observable photodamage.
  • Demonstrated nonlinear dependence of fluorescence intensity on excitation power (5-40 mW).
  • Observed enhanced lateral confinement of excitation in TPE compared to one-photon excitation, with a higher-order dependence on tip-sample displacement.

Conclusions:

  • Simultaneous TPE and 3PE in SNOM is feasible and effective for high-resolution imaging of biological samples.
  • Multiphoton excitation in SNOM confines excitation to a smaller volume, reducing photodamage and improving imaging quality.
  • This technique advances the capabilities of SNOM for sensitive biological applications.