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Volumetric two-photon imaging in live cells and embryos via axially gradient excitation

By Yufeng Gao, Xianyuan Xia, Jia Yu, Tingai Chen, Zhili Xu, Long Xiao, Liang Wang, Fei Yan, Zhuo Du, Jun Chu, Hairong Zheng, Hui Li, Wei Zheng

Posted 27 Mar 2019
bioRxiv DOI: 10.1101/589317

Two-photon microscopy(TPM) that features subcellular resolution, intrinsic optical sectioning ability, and deep penetration in sample is a powerful tool of bioimaging. However, the process of layer-by-layer scanning to form a 3D image inherently limits the volumetric imaging speed and significantly increases the phototoxicity. Here we develop a gradient TPM technique that enables rapid volumetric imaging by only acquiring two 2D images. By sequentially exciting the specimen with two axially elongated two-photon beams with complementary gradient intensities, the axial positions of fluorophores can be decoded from the intensity ratio of the paired images. We achieve an axial localization accuracy of 0.728 ± 0.657 μm, which is sufficient for rapid 3D subcellular imaging. We demonstrate the flexibility of the gradient TPM on a variety of sparsely labelled samples, including bead phantoms, mouse brain tissues, live macrophages and live nematode embryos. The results show that, compared with conventional TPM, the 3D imaging speed increases 6 folds while the photobleaching and photodamage are extremely reduced.

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