  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.
