Wei Lu, Chen Zhixing, Min Wei
Department of Chemistry, Columbia University, New York, NY 10027, USA.
Biomed Opt Express. 2012 Jun 1;3(6):1465-75. doi: 10.1364/BOE.3.001465. Epub 2012 May 22.
Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually 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 defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. 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 the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.
双光子荧光显微镜已成为通过检测从空间受限激发体积产生的散射荧光光子来对散射生物样品进行成像的不可或缺的工具。然而，当成像深度更深时，这种光学切片能力最终会失效，因为在散射样品中，离焦荧光会逐渐淹没焦内信号。由此导致的图像对比度损失定义了一个基本的成像深度极限，这是无法通过提高激发效率来克服的。在此，我们提议通过执行受激发射减少荧光（SERF）显微镜来扩展这个深度极限，在SERF显微镜中，焦点处的双光子激发荧光通过一个能够诱导荧光团从激发态产生受激发射的调制聚焦激光束优先开启和关闭。由减少的荧光信号构建的最终图像，由于其对入射激光强度的整体高阶非线性依赖性，显示出显著改善的信背比。我们通过解析理论和数值模拟来证明这一新概念。对于脑组织，SERF预计将把双光子荧光显微镜的成像深度极限扩展超过1.8倍。
