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Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy.

Ramón Carriles1, Dawn N Schafer, Kraig E Sheetz

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This review covers high-speed multiphoton microscopy, detailing scanning technologies, nonlinear optical contrast mechanisms, and optimization techniques. It explores new applications in microfluidics and medical imaging.

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Area of Science:

  • Optics and Photonics
  • Biomedical Imaging
  • Microscopy

Background:

  • Multiphoton microscopy offers advanced imaging capabilities.
  • High-speed imaging presents unique technical challenges and limitations.

Purpose of the Study:

  • To review the current state of multiphoton microscopy, focusing on high-speed applications.
  • To discuss various scanning technologies, nonlinear contrast mechanisms, and optimization strategies.
  • To highlight emerging applications in microfluidics and medical sciences.

Main Methods:

  • Review of existing literature on multiphoton microscopy techniques.
  • Analysis of scanning methods: line scan, multifoci, multidepth, and novel detection.
  • Examination of nonlinear optical contrast mechanisms: multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and third harmonic generation (THG).

Main Results:

  • Detailed description of scanning technologies and their impact on imaging speed and quality.
  • Comprehensive review of MPEF, SHG, and THG contrast mechanisms.
  • Discussion on optimizing nonlinear optical signals through focal volume control and photobleaching mitigation.

Conclusions:

  • Multiphoton microscopy is advancing rapidly, with high-speed capabilities enabling new research avenues.
  • Novel applications in microfluidic chemical analysis and medical imaging using two-photon absorption and self-phase modulation are promising.
  • Optimization of imaging parameters is crucial for maximizing signal and minimizing photodamage.