In Vivo Three-Photon Microscopy of Subcortical Structures within an Intact Mouse Brain

Abstract

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...

Temporal focusing microscopy using three-photon excitation fluorescence with a 92-fs Yb-fiber chirped pulse amplifier

“…Here, we demonstrate a simple way to reduce the excitation volume along the axial direction by utilizing three-photon excitation fluorescence (3PEF) with TF microscopy (3PEF-TF). The cubic dependence of the 3PEF signal on the excitation intensity allows the excitation volume in TF microscopy to be further confined in a similar manner to point-scanning 3PEF microscopy [12]. Third harmonic generation, the intensity of which is proportional to the cubic of the excitation intensity, has been applied to TF microscopy with line illumination [13].…”

Wide-field multiphoton imaging with TRAFIX

“…It has also been shown that by relying on such higher order nonlinear process, fluorescence is confined to a smaller volume, reducing out-of-focus light. 17,22 Novel laser sources have recently been developed to generate high energy pulses to optimize the absorption of three photons. Such ultrashort Compressive sensing is demonstrated a posteriori, by using 5% and 15% of the recorded Hadamard patterns and minimizing to the l1 norm.…”

“…pulsed lasers typically have pulse duration and repetition rate below 70 fs and 1.25 MHz, [17][18][19][20][21]23 respectively. The Ti:Sapphire laser source in our system is typically used to generate 2P excitation, therefore, it is not optimal for 3P microscopy.…”

High power NIR fiber-optic femtosecond Cherenkov radiation and its application on nonlinear light microscopy

“…Since its first demonstration, nonlinear light microscopy (NLM), including multi-photon fluorescence microscopy [1][2][3][4][5][6] and multi-harmonic generation microscopy [7][8][9], has attracted much biological and medical interest due to its capability to provide molecular and structural information with high 3D spatial resolutions. Recently, enabled by femtosecond near-infrared (NIR) solid-state light sources working in the 0.7-1.3 μm bio-penetration window, NLM with a solid-state NIR light source offers the advantages of deeper penetration and much reduced photodamage and photobleaching effects [2,4,8].…”