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Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy.

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  • 1Université de Limoges, XLIM, UMR CNRS 7252, 123 Avenue A. Thomas, 87060 Limoges, France.

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This study introduces nonlinear endogenous fluorescence lifetime imaging microscopy (FLIM) for malignant tissue diagnosis. This novel endoscopic technique uses nicotinamide adenine dinucleotide (NADH) and a fibered microscope for enhanced imaging depth and optical sectioning.

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(060.2310) Fiber optics(170.2150) Endoscopic imaging(170.2520) Fluorescence microscopy(170.3880) Medical and biological imaging(170.6920) Time-resolved imaging(180.4315) Nonlinear microscopy

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Area of Science:

  • Biomedical Optics
  • Endoscopic Imaging
  • Fluorescence Microscopy

Background:

  • In situ fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH) shows promise for malignant tissue diagnosis.
  • Two-photon nonlinear excitation offers intrinsic optical sectioning and improved imaging depth, crucial for endoscopic applications.

Purpose of the Study:

  • To demonstrate nonlinear endogenous FLIM in a fibered microscope with proximal detection for the first time.
  • To establish a foundational step towards a nonlinear endomicroscope for improved diagnostic capabilities.

Main Methods:

  • Utilized a double-clad microstructured fiber with lengths exceeding 3 meters and excitation pulse durations of approximately 50 fs.
  • Employed two-photon nonlinear excitation for intrinsic optical sectioning and enhanced imaging depth.
  • Collected fluorescence photons via the fiber's inner cladding and analyzed the impulse response function (IRF) contributions.

Main Results:

  • Demonstrated nonlinear endogenous FLIM applied to NADH in cultured cells.
  • Showcased that the inner cladding's contribution to the IRF, due to modal and chromatic dispersions, is minimal (< 600 ps) and stable up to 8m fiber length.
  • Confirmed the suitability for short lifetime measurements, essential for rapid diagnostic applications.

Conclusions:

  • Nonlinear endogenous FLIM in a fibered microscope is feasible and provides stable measurements for NADH lifetime.
  • The phasor representation is an effective visualization tool suitable for the high-speed requirements of endoscopy.
  • This work represents a significant advancement towards developing a practical nonlinear endomicroscope for clinical diagnostics.