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Related Experiment Videos

High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy.

Shi-Wei Chu1, Shih-Peng Tai, Chia-Lin Ho

  • 1Graduate Institute of Electro-Optical Engineering and Department of Electrical Engineering, National Taiwan University, Taipei 10617, Taiwan.

Microscopy Research and Technique
|May 13, 2005
PubMed
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This study introduces a novel combined three-photon-fluorescence (3PF) and third-harmonic-generation (THG) microscopy. This advanced technique offers high-resolution 3D imaging with molecular specificity and structural mapping, while minimizing photodamage for biological samples.

Area of Science:

  • Biomedical Optics
  • Microscopy
  • Biophotonics

Background:

  • Nonlinear laser scanning microscopy offers deep optical sectioning.
  • Longer wavelength femtosecond lasers (1,200-1,300 nm) enhance penetration and reduce photodamage.
  • Three-photon-fluorescence (3PF) and third-harmonic-generation (THG) are efficient three-photon processes for imaging.

Purpose of the Study:

  • To demonstrate the first combined 3PF and THG microscopy system.
  • To achieve high-resolution 3D imaging with both functional specificity and structural mapping.
  • To evaluate the noninvasiveness of the technique using a femtosecond laser at longer wavelengths.

Main Methods:

  • Utilized a 1,230-nm Cr:forsterite femtosecond laser.
  • Developed a combined 3PF and THG microscopy setup.

Related Experiment Videos

  • Imaged Hoechst-labeled hepatic cells to showcase simultaneous 3PF and THG signals.

    • Main Results:

    • Achieved simultaneous 3PF and THG imaging with high spatial resolution.
    • Observed strong 3PF from DNA-bound Hoechst-33258 within cell nuclei.
    • THG effectively mapped cellular and organelle membranes without observable photodamage at 100-mW incident power.

    Conclusions:

    • The combined 3PF and THG microscopy provides simultaneous functional and structural imaging.
    • The technique offers superior spatial resolution and deep tissue penetration.
    • This noninvasive imaging modality demonstrates significant potential for biological research.