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

受激发射减少荧光 (SERF) 用于打破和扩展双光子显微镜的基本成像深度

• 批准号: 9025791
• 负责人: Wei Min
• 金额: $ 20万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9025791
• 关键词: Alzheimer' s Disease; Animals; Area; Biological; Biomedical Research; Brain; Dependence; Electronics; Embryology; Exhibits; Fluorescence; Fluorescence Microscopy; Health; Huntington Disease; Image; Imaging Device; Imaging Techniques; In Vitro; Lasers; Life; Medical; Microscopy; Molecular; Monitor; Mus; Names; Neurosciences; Optics; Organism; Performance; Photons; Resolution; Sampling; Science; Signal Transduction; Staging; Techniques; Testing; Time; Tissue imaging; Work; brain tissue; contrast imaging; fluorescence imaging; fluorescence microscope; fluorophore; improved; in vivo; innovation; nervous system disorder; novel; oncology; optical imaging; relating to nervous system; simulation; tissue phantom; two-photon

 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.

 描述（由适用提供）：两光子荧光显微镜已成为不可或缺的工具，可以通过检测从空间上限制的刺激体积中检测荧光照片来对散射生物学样品进行成像。然而，这种光学切片能力最终在成像更深时会崩溃，因为异常荧光逐渐淹没了散射样品中的聚焦点信号。产生的图像对比度丢失（S/B〜1）定义了基本成像深度（小鼠脑组织约1 mM），这不能通过简单地增加兴奋能力来克服。本文中，我们建议通过执行刺激的发射降低荧光（SELF）显微镜来扩展此深度极限，在这种情况下，焦点处的两光子激发荧光更优选地通过调制和聚焦的激光束打开和关闭，该激光束能够诱导荧光团的刺激发射。来自他们激动的状态。由于其对入射激光强度的总体三阶非线性依赖性，发现由还原荧光信号构成的所得农奴图像被发现可显着改善信噪比对比度。我们计划（1）在实验室中构建第一个农奴显微镜； （2）在体外构造3D发出幻象，并使用它们来测试和验证新的农奴显微镜的成像性能； （3）在动物脑上应用农奴显微镜，并在体内表现出高对比度超深组织成像。在技​​术上很简单且易于构建，农具显微镜仅需将红色CW激光束（以及相关的调制电子设备）添加到标准的两光子荧光显微镜中。在数值模拟的支持下，预期该提出的技术将将两光子显微镜的成像深度限制延长1.8倍。