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Dual-wavelength multimodal multiphoton microscope with SMA-based depth scanning.

Wentao Wu1,2, Qihao Liu1, Christoph Brandt1

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We developed a novel multimodal multiphoton microscopy system for deep tissue imaging. This advanced microscopy technique utilizes dual-wavelength lasers and depth scanning to visualize biological structures over 200 µm deep.

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Area of Science:

  • Biomedical Engineering
  • Optical Imaging
  • Microscopy

Background:

  • Multiphoton microscopy (MPM) offers deep tissue imaging capabilities.
  • Existing MPM systems often lack multimodal signal acquisition and efficient depth scanning.
  • Advanced imaging techniques are crucial for in-situ analysis of biological tissues.

Purpose of the Study:

  • To develop and demonstrate a multimodal multiphoton microscopy (MPM) system with integrated depth scanning.
  • To enable simultaneous acquisition of various optical signals for comprehensive tissue analysis.
  • To achieve deep penetration imaging in diverse biological samples.

Main Methods:

  • Utilized an Er-doped femtosecond fiber laser providing dual output wavelengths (1580 nm and 790 nm) for three-photon and two-photon excitation.
  • Incorporated a shape-memory-alloy (SMA) actuated miniaturized objective for precise depth scanning.
  • Acquired image stacks combining two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and third harmonic generation (THG) signals.

Main Results:

  • Successfully demonstrated depth scanning capability up to 200 µm in biological tissues.
  • Acquired multimodal image stacks from animal, fungus, and plant tissue samples.
  • Validated the system's performance in capturing TPEF, SHG, and THG signals simultaneously.

Conclusions:

  • The developed multimodal MPM system provides enhanced deep tissue imaging capabilities.
  • The integration of dual-wavelength excitation and depth scanning significantly improves imaging versatility.
  • This technology holds potential for advanced research in various biological fields requiring high-resolution, deep imaging.