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.

We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 mum are performed.

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...

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...