Horton Nicholas G, Wang Ke, Kobat Demirhan, Clark Catharine G, Wise Frank W, Schaffer Chris B, Xu Chris
School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, USA.
Department of Biomedical Engineering, Cornell University, Ithaca, New York, 14853, USA.
Nat Photonics. 2013 Mar 1;7(3):205-9. doi: 10.1038/nphoton.2012.336.
Two-photon fluorescence microscopy (2PM) enables scientists in various fields including neuroscience, embryology, and oncology to visualize and 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 or the insertion of optical probes. Here we demonstrate non-invasive, high resolution, 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.
双光子荧光显微镜（2PM）使神经科学、胚胎学和肿瘤学等各个领域的科学家能够在散射组织深处的细胞水平上可视化组织形态和生理学。然而，组织散射将小鼠脑内2PM的最大成像深度限制在皮层，目前对皮层下结构进行成像需要去除覆盖的脑组织或插入光学探针。在这里，我们展示了在1700nm的光谱激发窗口下，使用三光子荧光显微镜（3PM）对完整小鼠脑内皮层下结构进行非侵入性、高分辨率成像。对小鼠海马体内的血管结构以及红色荧光蛋白（RFP）标记的神经元进行了成像。长激发波长和高阶非线性激发的结合克服了2PM的局限性，使生物学研究能够在组织内更深的深度进行。
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