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bioRxiv · 10.1101/589317

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

Gao, Y.·Xia, X.·Yu, J.·Chen, T.·Xu, Z.·Xiao, L.·Wang, L.·Yan, F.·Du, Z.·Chu, J.·Zheng, H.·Li, H.·Zheng, W.
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Abstract

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 m, 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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Gao, Y., Xia, X., Yu, J., Chen, T., Xu, Z., Xiao, L., Wang, L., Yan, F., Du, Z., Chu, J., Zheng, H., Li, H., Zheng, W.. 2019-03-27. Volumetric two-photon imaging in live cells and embryos via axially gradient excitation. https://doi.org/10.1101/589317

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