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Scattering correction through Fourier-domain intensity coupling in two-photon microscopy (2P-FOCUS).

Daniel Zepeda1, Yucheng Li1, Yi Xue1

  • 1Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Dr., Davis, California 95616, USA.

Photonics Research
|July 4, 2025
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Summary

We developed a new two-photon microscopy system (2P-FOCUS) that corrects light scattering in biological tissues. This system enhances deep tissue imaging by modulating light intensity in the Fourier domain, improving signal by tens of folds.

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Area of Science:

  • Biomedical optics
  • Microscopy
  • Photonics

Background:

  • Light scattering in biological tissues limits imaging depth.
  • Current scattering correction methods require transmission access or iterative optimization.
  • Limited photon availability exacerbates scattering challenges.

Purpose of the Study:

  • Introduce a novel two-photon microscopy system for scattering correction.
  • Overcome limitations of existing scattering correction techniques.
  • Enable deep tissue imaging in challenging conditions.

Main Methods:

  • Developed a two-photon microscopy system with Fourier-domain intensity coupling for scattering correction (2P-FOCUS).
  • Utilized intensity modulation in the Fourier domain, leveraging nonlinear interference and two-photon excitation.
  • Employed random patterns for scattering probing and a single-shot algorithm for rapid correction mask generation.
  • Enabled subregion-specific mask customization to overcome memory effect limitations.

Main Results:

  • Demonstrated focusing and imaging through bone and ex vivo mouse brain samples.
  • Significantly enhanced two-photon fluorescence signals (tens of folds) compared to uncorrected imaging.
  • Achieved scattering correction in a volume of 230 μm × 230 μm × 510 μm, exceeding the memory effect range.
  • Completed scattering measurement, calculation, and correction within seconds.

Conclusions:

  • 2P-FOCUS effectively corrects light scattering in biological tissues.
  • The system enables rapid, cost-effective deep tissue imaging.
  • 2P-FOCUS offers broad adoption potential for advanced biomedical imaging applications.