Two-photon fluorescence microscopy (2PM)1 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 2PM within the mouse brain to the cortical layer, 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 (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.

High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 50 neurons in the hippocampal stratum pyramidale at ~1-mm depth within an intact mouse brain. our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues.

Deep tissue in vivo two-photon fluorescence imaging of cortical vasculature in a mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.6 mm in mouse cortex is achieved in vivo, approximately reaching the fundamental depth limit in scattering tissue.

Two-photon fluorescence microscopy (2PM) 1 enables scientists in various fields including neuroscience 2,3 , embryology 4 , and oncology 5 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 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6,7 . Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.Optical imaging plays a major role in both basic biological research and clinical diagnostics, providing a non-invasive or minimally-invasive microscopic imaging capability to investigate biological tissue. Optical image acquisition through significant depths of biological tissue, however, presents a major scientific challenge since tissue is extremely heterogeneous and the strong scattering of the various tissue components has historically restricted high-resolution optical imaging to thin sections or to superficial layers. The development of 2PM has significantly extended the penetration depth of high-resolution optical imaging, particularly for in vivo applications [8][9][10][11][12] . In the last 20 years, 2PM has enabled, in many fields for the first time, direct visualization of the normal behaviour of Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence and requests for materials should be addressed to C. Xu (chris.xu@cornell.edu). † These authors contributed equally to this work * cx10@cornell.edu Author Information Reprints and permissions information is available at www.nature.com/reprints.The authors declare no competing financial interests.Supplementary Information is linked to the online version of the paper at www.nature.com/naturephotonics. Author Contributions 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. 3,13 . Two-photon excitation of fluorescent molecules in tissue depends on the ability of sufficient excitation light to reach the focus of the objective unscattered (i.e., ballistic excitatio...

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-μm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.