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Related Experiment Videos

Continuous wave two-photon scanning near-field optical microscopy

A K Kirsch1, V Subramaniam, G Striker

  • 1Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Biophysical Journal
|September 3, 1998
PubMed
Summary
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We demonstrate continuous-wave two-photon excitation for near-UV fluorophores in scanning near-field optical microscopy (SNOM). This technique successfully imaged stained chromosomes and cell nuclei, showing faster fluorescence decay in two-photon excitation mode.

Area of Science:

  • Microscopy
  • Spectroscopy
  • Biophysics

Background:

  • Scanning near-field optical microscopy (SNOM) offers high resolution imaging.
  • Two-photon excitation (TPE) can improve imaging depth and reduce photobleaching.
  • Near-UV absorbing fluorophores are crucial for biological sample labeling.

Purpose of the Study:

  • To implement and evaluate continuous-wave two-photon excitation (TPE) for near-UV absorbing fluorophores in SNOM.
  • To compare TPE with single-photon excitation (SPE) in SNOM for biological imaging.
  • To investigate the fluorescence intensity dependence on excitation power and tip-sample displacement.

Main Methods:

  • Utilized a 647-nm Ar-Kr mixed gas laser for TPE of DNA dyes (DAPI, Hoechst 33342) and ethidium bromide.

Related Experiment Videos

  • Employed a shared aperture SNOM with uncoated fiber tips.
  • Imaged polytene chromosomes of Drosophila melanogaster and 3T3 Balb/c cell nuclei.

    • Main Results:

    • Successfully imaged labeled chromosomes and cell nuclei using TPE-SNOM.
    • Observed a nonlinear, second-order dependence of fluorescence intensity on excitation power (8-180 mW).
    • Measured faster fluorescence decay in TPE mode compared to SPE mode as a function of tip-sample displacement.

    Conclusions:

    • Continuous-wave TPE is effective for near-UV fluorophores in SNOM.
    • TPE-SNOM provides viable imaging of biological structures.
    • TPE exhibits distinct fluorescence decay characteristics compared to SPE in SNOM.