Kunzan Liu, Honghao Cao, Kasey Shashaty, Li-Yu Yu, Sarah Spitz,Francesca Michela Pramotton, Zhengpeng Wan, Ellen L. Kan, Erin N. Tevonian, Manuel Levy,Eva Lendaro, Roger D. Kamm, Linda G. Griffith, Fan Wang, Tong Qiu, Sixian You
Abstract:
Label-free imaging through two- photon autofluorescence of NAD℗H allows for nondestructive, high-resolutionvisualization of cellular activities in living systems. However, its application to thick tissues has been restricted byits limited penetration depth within 300 μm, largely due to light scattering. Here, we demonstrate that the imag-ing depth for NAD℗H can be extended to more than 700 μm in living engineered human multicellular microtis-sues by adopting multimode fiber-based, low repetition rate, high peak power, three-photon excitation of NAD℗Hat 1100 nm. This is achieved by having more than 0.5 megawatts peak power at the band of 1100 ±25 nmthrough adaptively modulating multimodal nonlinear pulse propagation with a compact fiber shaper. Moreover,the eightfold increase in pulse energy enables faster imaging of monocyte behaviors in the living multicellularmodels. These results represent a substantial advance for deep and dynamic imaging of intact living biosystems.The modular design is anticipated to allow wide adoption for demanding imaging applications, including cancerresearch, immune responses, and tissue engineering.