Two-photon focal modulation microscopy for high-resolution imaging in deep tissue
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Updated: Feb 4, 2026

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain
Published on: January 13, 2022
Yao Zheng1,2, Jiajia Chen2, Xin Shi2
1Center for Neuroscience, Department of Neurobiology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China.
Abstract:
Two-photon microscopy (2PM) is one of the most widely used tools for in vivo deep tissue imaging. However, the spatial resolution and penetration depth are still limited due to the strong scattering background. Here we demonstrate a two-photon focal modulation microscopy. By utilizing the modulation and demodulation techniques, background rejection capability is enhanced, thus spatial resolution and imaging penetration depth are improved. Compared with 2PM, the transverse resolution is increased by 70%, while the axial resolution is increased to 2-fold. Furthermore, when applied in conventional 2PM mode, it can achieve inertial-free scanning in either transverse or axial direction with in principle unlimited scanning speed. Finally, we applied 2PFMM in thick scattering samples to further examine the imaging performance. The results show that the signal-to-background ratio of 2PFMM can be improved up to five times of 2PM at the depth of 500 μm. Fluorescent imaging in the mouse brain tissue. 3D Thy1-GFP hippocampal neurons imaged by (A) 2PM compared with (B) 2PFMM; (C-H) xy maximum-intensity projection imaged by 2PM compared with 2PFMM. Scale bar 50 μm.
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