Deep and Dynamic Metabolic and Structural Imaging in Living Tissues

TL;DR

This paper introduces a novel multimode fiber-based three-photon excitation method at 1100 nm that significantly extends imaging depth and speed in living tissues, enabling advanced metabolic and structural visualization.

Contribution

It demonstrates a new fiber-shaping technique for deep, fast, and non-destructive imaging of living tissues using three-photon excitation at 1100 nm, surpassing previous depth limitations.

Findings

Imaging depth extended to over 700 μm in living tissues.

Achieved 8-fold increase in pulse energy for faster imaging.

Enables high-resolution, non-destructive visualization of cellular activities.

Abstract

Label-free imaging through two-photon autofluorescence (2PAF) of NAD(P)H allows for non-destructive and high-resolution visualization of cellular activities in living systems. However, its application to thick tissues and organoids has been restricted by its limited penetration depth within 300 $\mu$m, largely due to tissue scattering at the typical excitation wavelength (~750 nm) required for NAD(P)H. Here, we demonstrate that the imaging depth for NAD(P)H can be extended to over 700 $\mu$m in living engineered human multicellular microtissues by adopting multimode fiber (MMF)-based low-repetition-rate high-peak-power three-photon (3P) excitation of NAD(P)H at 1100 nm. This is achieved by having over 0.5 MW peak power at the band of 1100$\pm$25 nm through adaptively modulating multimodal nonlinear pulse propagation with a compact fiber shaper. Moreover, the 8-fold increase in pulse energy at 1100 nm enables faster imaging of monocyte behaviors in the living multicellular models. These results represent a significant advance for deep and dynamic metabolic and structural imaging of intact living biosystems. The modular design (MMF with a slip-on fiber shaper) is anticipated to allow wide adoption of this methodology for demanding in vivo and in vitro imaging applications, including cancer research, autoimmune diseases, and tissue engineering.