Deep and dynamic metabolic and structural imaging in living tissues
Overview
Metabolic and structural imaging of the entire 500-μm-deep living blood-brain barrier microfluidic model, comprising vascular endothelial cells, pericytes, and astrocytes.
To achieve deep-tissue imaging in living organisms, two-photon autofluorescence (2PAF) microscopy of NAD(P)H is a powerful technique, offering non-invasive, high-resolution visualization of cellular metabolic processes. However, light scattering traditionally limits the penetration depth of this method to within 200 μm. We have overcome this limitation by developing a high-power, multimode fiber-based light source that modulates multimodal nonlinear pulse propagation with a compact fiber shaper. This innovative approach extends the imaging depth of 2PAF microscopy to over twice its previous limit. The modular design provides flexibility and facilitates the widespread adoption of this technology for demanding in vivo and in vitro imaging applications, including in areas such as cancer research, immune responses, and tissue engineering.
Source: A multimode fiber source generating 0.5 MW peak power at 1100±25 nm was achieved by adaptively modulating multimodal nonlinear pulse propagation with a compact fiber shaper.
Imaging: Three-photon excitation at 1100 nm enabled structural and metabolic imaging at depths exceeding 700 μm in living engineered human multicellular microtissues.
Biology: Revealed depth-associated redox heterogeneity in the blood-brain barrier microfluidic model and speed-associated redox heterogeneity in monocyte behaviors.
Concept of the imaging platform using multimode fiber source.
Results
Beam optimization for high-quality imaging
Comparison of images and beam properties acquired before (initial) and after (optimized) optimizing the fiber shaper.
Three-photon NAD(P)H imaging improves depth
Deep NAD(P)H imaging with 1100 nm MMF source through the entire 720 μm of depth of the 3D microvascular network. Three-photon imaging uses longer excitation wavelength that reduces the scattering in tissue. The higher-order confinement improves the imaging signal-to-background ratio (SBR) deep in tissue.
Deep imaging in blood-brain barrier microfluidic model
Deep metabolic and structural imaging of the entire 500-μm-deep living blood-brain barrier microfluidic model, comprising vascular endothelial cells, pericytes, and astrocytes. Based on the structural and metabolic features, same type of cells can cluster. Additionally, the ability to image deep into tissue reveals depth-associated redox heterogeneity that can help to understand biology.
Dynamic imaging of monocyte behaviors
Dynamic metabolic and structural imaging of the monocyte behaviors in the vasculature network, showing relation between metabolic and motility behaviors in individual cells as well as statistics in a large population.
References
2024
Spectral-temporal-spatial Customization via Modulating Multimodal Nonlinear Pulse Propagation
Tong Qiu, Honghao Cao, Kunzan Liu, Li-Yu Yu, Manuel Levy, Eva Lendaro, Fan Wang, and Sixian You
Multimode fibers (MMFs) are gaining renewed interest for nonlinear effects due to their high-dimensional spatiotemporal nonlinear dynamics and scalability for high power. High-brightness MMF sources with effective control of the nonlinear processes would offer possibilities in many areas from high-power fiber lasers, to bioimaging and chemical sensing, and to intriguing physics phenomena. Here we present a simple yet effective way of controlling nonlinear effects at high peak power levels. This is achieved by leveraging not only the spatial but also the temporal degrees of freedom during multimodal nonlinear pulse propagation in step-index MMFs, using a programmable fiber shaper that introduces time-dependent disorders. We achieve high tunability in MMF output fields, resulting in a broadband high-peak-power source. Its potential as a nonlinear imaging source is further demonstrated through widely tunable two-photon and three-photon microscopy. These demonstrations provide possibilities for technology advances in nonlinear optics, bioimaging, spectroscopy, optical computing, and material processing.
@article{qiu2024spectral,title={Spectral-temporal-spatial Customization via Modulating Multimodal Nonlinear Pulse Propagation},author={Qiu, Tong and Cao, Honghao and Liu, Kunzan and Yu, Li-Yu and Levy, Manuel and Lendaro, Eva and Wang, Fan and You, Sixian},journal={Nature Communications},volume={15},number={1},pages={2031},year={2024},publisher={Nature Publishing Group UK London}}
Deep and Dynamic Metabolic and Structural Imaging in Living Tissues
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, and Sixian You
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.
@article{liu2024deep,title={Deep and Dynamic Metabolic and Structural Imaging in Living Tissues},author={Liu, Kunzan and Cao, Honghao and Shashaty, Kasey and Yu, Li-Yu and Spitz, Sarah and Pramotton, Francesca Michela and Wan, Zhengpeng and Kan, Ellen L. and Tevonian, Erin N. and Levy, Manuel and Lendaro, Eva and Kamm, Roger D. and Griffith, Linda G. and Wang, Fan and Qiu, Tong and You, Sixian},journal={Science Advances},volume={10},number={50},pages={eadp2438},year={2024},}
