Three-dimensional subcellular imaging is essential for biomedical research, but the diffraction limit of optical microscopy compromises axial resolution, hindering accurate three-dimensional structural analysis. This challenge is particularly pronounced in label-free imaging of thick, heterogeneous tissues, where assumptions about data distribution (e.g. sparsity, label-specific distribution, and lateral-axial similarity) and system priors (e.g. independent and identically distributed noise and linear shift-invariant point-spread functions) are often invalid. Here, we introduce SSAI-3D, a weakly physics-informed, domain-shift-resistant framework for robust isotropic three-dimensional imaging. SSAI-3D enables robust axial deblurring by generating a diverse, noise-resilient, sample-informed training dataset and sparsely fine-tuning a large pre-trained blind deblurring network. SSAI-3D is applied to label-free nonlinear imaging of living organoids, freshly excised human endometrium tissue, and mouse whisker pads, and further validated in publicly available ground-truth-paired experimental datasets of three-dimensional heterogeneous biological tissues with unknown blurring and noise across different microscopy systems.
@article{han2024system,title={System- and Sample-agnostic Isotropic Three-dimensional Microscopy by Weakly Physics-informed, Domain-shift-resistant Axial Deblurring},author={Han*, Jiashu and Liu*, Kunzan and Isaacson, Keith B. and Monakhova, Kristina and Griffith, Linda G. and You, Sixian},journal={Nature Communications},volume={16},number={1},pages={745},year={2025},}
Simultaneous Label-free Imaging of Nucleolar Dynamics and Subcellular Metabolic Shifts Across Tissue Contexts
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},}
Simultaneous Label-free Imaging of Nucleolar Dynamics and Subcellular Metabolic Shifts Across Tissue ContextsbioRxiv, 2025
