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Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging.

Wei-Liang Chen1, Chen-Kuan Chou, Ming-Gu Lin

  • 1National Taiwan University, Department of Physics, No. 1 Section 4 Roosevelt Road, Taipei 10617, Taiwan.

Journal of Biomedical Optics
|November 10, 2009
PubMed
Summary

Combining reflected confocal (RC) and multiphoton microscopy (MPM) with a single laser offers unique tissue imaging. This approach reveals refractive boundaries, cytoplasm, and collagen in skin and cornea, with deeper imaging capabilities for autofluorescence.

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Area of Science:

  • Biomedical Optics
  • Microscopy Techniques
  • Diagnostic Imaging

Background:

  • Reflected confocal (RC) and multiphoton microscopy (MPM) offer distinct clinical diagnostic capabilities.
  • Combining RC and MPM modalities can enhance image contrast, particularly when using a single excitation wavelength.
  • Near-infrared (NIR) excitation enables deeper tissue penetration and visualization.

Purpose of the Study:

  • To investigate the combined use of RC and MPM for enhanced tissue imaging.
  • To evaluate the utility of a single 780-nm femtosecond laser source for simultaneous RC and MPM imaging of skin and cornea.
  • To assess the impact of wavelength and power on image quality and imaging depth.

Main Methods:

  • Utilized a femtosecond titanium-sapphire laser emitting at 780 nm for simultaneous RC and MPM imaging.
  • Applied the technique to ex vivo bovine cornea and porcine skin samples.
  • Investigated RC imaging at multiple wavelengths (488, 543, 633 nm) and varying laser power.

Main Results:

  • NIR RC signal effectively characterized refractive index boundaries in cornea and skin.
  • MPM-derived multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) visualized cytoplasm and collagen, respectively.
  • MAF imaging achieved greater depths than NIR RC imaging.
  • Longer RC wavelengths (up to 633 nm) improved contrast and depth for tissue imaging.
  • Consistent RC image quality was maintained across a 2.7–10.7 mW power range.

Conclusions:

  • Simultaneous RC and MPM imaging with a single NIR laser source provides complementary contrast for tissue characterization.
  • The technique is valuable for imaging refractive boundaries, cytoplasm, and collagen in skin and cornea.
  • Optimizing laser wavelength and power enhances imaging depth and contrast for diagnostic applications.