Stimulated emission reduced fluorescence (SERF) for breaking and extending the fundamental imaging-depth of two photon microscopy

 描述(申请人提供)：双光子荧光显微镜通过检测来自空间受限激发体积的荧光光子，已成为对散射生物样品成像不可缺少的工具。然而，当成像更深时，这种光学切片能力最终会被破坏，因为散焦的荧光逐渐淹没了散射样品中的焦内信号。由此产生的图像对比度损失(S/B~1)定义了一个基本的成像深度限制(对于小鼠脑组织约为1 mm)，这不是简单地增加激发功率就能克服的。在这里，我们建议通过执行受激发射减少荧光(SERF)显微镜来扩展这一深度限制，在受激发射折减荧光显微镜中，焦点处的双光子激发荧光优先被调制和聚焦的激光束开启和关闭，该光束能够诱导荧光团从激发态发出受激发射。由此得到的由还原的荧光信号构建的SERF图像由于其总体三阶非线性依赖于入射激光强度，被发现表现出显著改善的信号与背景的对比度。我们计划(1)建立实验室第一台农奴显微镜；(2)构建体外3D问题模型，并用它们来测试和验证新型农奴显微镜的成像性能；(3)将农奴显微镜应用于动物大脑，并展示体内高对比度的超深部组织成像。SERF显微镜技术简单，易于制造，因为只需要在标准双光子荧光显微镜上添加一束红色连续激光(连同相关的调制电子设备)。在数值模拟的支持下，所提出的技术有望将双光子显微镜的成像深度极限延长1.8倍。

 DESCRIPTION (provided by applicant): Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting fluorescence photons originated from a spatially confined excitation volume. However, such optical sectioning capability eventually breaks down when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast (S/B~1) defines a fundamental imaging-depth limit (around 1 mm for mouse brain tissues), which cannot be overcome by simply increasing excitation power. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from their excited states. The resulting SERF image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall third-order nonlinear dependence on the incident laser intensity. We plan to (1) build the first SERF microscope in the lab; (2) construct in vitro 3D issue phantoms, and use them to test and validate the imaging performance of the new SERF microscope; and (3) apply SERF microscopy on animal brain, and demonstrate high-contrast ultra-deep tissue imaging in vivo. SERF microscope is technically straightforward and easy to build, as only a red CW laser beam (together with the associated modulation electronics) is needed to be added onto a standard two-photon fluorescence microscope. As supported by numerical simulations, the proposed technique is expected to extend the imaging depth limit of two-photon microscopy by a factor of 1.8.