Liu Kunzan, Cao Honghao, Shashaty Kasey, Yu Li-Yu, Spitz Sarah, Pramotton Francesca Michela, Wan Zhengpeng, Kan Ellen L, Tevonian Erin N, Levy Manuel, Lendaro Eva, Kamm Roger D, Griffith Linda G, Wang Fan, Qiu Tong, You Sixian
Research Laboratory of Electronics, MIT, Cambridge, MA 02139, USA.
Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA.
Sci Adv. 2024 Dec 13;10(50):eadp2438. doi: 10.1126/sciadv.adp2438. Epub 2024 Dec 11.
Label-free imaging through two-photon autofluorescence of NAD(P)H allows for nondestructive, high-resolution visualization of cellular activities in living systems. However, its application to thick tissues has been restricted by its limited penetration depth within 300 μm, largely due to light scattering. Here, we demonstrate that the imaging depth for NAD(P)H can be extended to more than 700 μm in living engineered human multicellular microtissues by adopting multimode fiber-based, low repetition rate, high peak power, three-photon excitation of NAD(P)H at 1100 nm. This is achieved by having more than 0.5 megawatts peak power at the band of 1100 ± 25 nm through 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 multicellular models. 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 cancer research, immune responses, and tissue engineering.
通过NAD(P)H的双光子自发荧光进行无标记成像，能够对活体系统中的细胞活动进行无损、高分辨率可视化。然而，由于光散射，其在厚组织中的应用受到了300μm以内有限穿透深度的限制。在此，我们证明，通过采用基于多模光纤的、低重复率、高峰值功率、在1100nm处对NAD(P)H进行三光子激发，在活体工程化人类多细胞微组织中，NAD(P)H的成像深度可扩展至700μm以上。这是通过使用紧凑型光纤整形器自适应调制多模非线性脉冲传播，在1100±25nm波段获得超过0.5兆瓦的峰值功率来实现的。此外，脉冲能量增加了八倍，能够在活体多细胞模型中更快地成像单核细胞行为。这些结果代表了完整活生物系统深度和动态成像的重大进展。这种模块化设计有望广泛应用于包括癌症研究、免疫反应和组织工程在内的苛刻成像应用。
