Deep and dynamic metabolic and structural imaging in living tissues

Area of Science:

• Biophotonics
• Cellular Imaging
• Tissue Engineering

Background:

• Label-free imaging of NAD(P)H autofluorescence offers nondestructive, high-resolution visualization of cellular activities.
• Limited penetration depth (approx. 300 μm) in thick tissues restricts current NAD(P)H imaging applications due to light scattering.

Purpose of the Study:

• To extend the imaging depth for NAD(P)H autofluorescence in living multicellular microtissues.
• To enable deeper and faster dynamic imaging of cellular behaviors in complex biological models.

Main Methods:

• Utilized multimode fiber-based, low repetition rate, high peak power three-photon excitation of NAD(P)H at 1100 nm.
• Employed adaptive modulation of nonlinear pulse propagation with a compact fiber shaper to achieve >0.5 MW peak power.
• Applied the technique to living engineered human multicellular microtissues.

Main Results:

• Successfully extended NAD(P)H imaging depth to over 700 μm in living multicellular microtissues.
• Achieved an eightfold increase in pulse energy, enabling faster imaging of cellular dynamics.
• Demonstrated high-resolution, label-free visualization of cellular activities at unprecedented depths.

Conclusions:

• Three-photon excitation of NAD(P)H significantly overcomes the penetration depth limitations of two-photon imaging in thick biological samples.
• The developed modular system facilitates deep and dynamic imaging of intact living biosystems.
• This advancement holds potential for applications in cancer research, immunology, and tissue engineering.