TY - JOUR T1 - Volumetric two-photon imaging in live cells and embryos via axially gradient excitation JF - bioRxiv DO - 10.1101/589317 SP - 589317 AU - Yufeng Gao AU - Xianyuan Xia AU - Jia Yu AU - Tingai Chen AU - Zhili Xu AU - Long Xiao AU - Liang Wang AU - Fei Yan AU - Zhuo Du AU - Jun Chu AU - Hairong Zheng AU - Hui Li AU - Wei Zheng Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/03/27/589317.abstract N2 - 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. ER -