Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy

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.