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In vivo three-photon microscopy of subcortical structures within an intact mouse brain


Two-photon fluorescence microscopy1 enables scientists in various fields including neuroscience2,3, embryology4 and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

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Figure 1: Wavelength-dependent attenuation length in brain tissue and measured laser characteristics.
Figure 2: In vivo 3PM images of Texas-Red–dextran labelled mouse brain vasculature.
Figure 3: In vivo 3PM images of RFP-labelled neurons in mouse brain.
Figure 4: Fluorescence signal attenuation curves of in vivo experiments.
Figure 5: Resolution characterization of 3PM.


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This work is partially funded by grants from the National Institutes of Health (NIH; R01CA133148, R01EB014873 and R21RR032392). N.G.H. is supported by the National Science Foundation Graduate Research Fellowship Program (DGE-0707428). The authors acknowledge discussions with D. Dombeck, as well as N. Nishimura and J. Rubin, regarding preparation of the ex vivo brain slices.

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Authors and Affiliations



C.X. initiated and supervised the study. N.G.H., K.W., D.K. and C.G.C. performed the experiments and data analysis. N.G.H., K.W., D.K. and C.X. contributed to the writing and editing of the manuscript. C.B.S. and C.X. contributed to the design of the experiments. F.W. and C.X. contributed to the laser source design.

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Correspondence to Chris Xu.

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The authors declare no competing financial interests.

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Horton, N., Wang, K., Kobat, D. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photon 7, 205–209 (2013).

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