Pulse-shaping for Multiphoton FRET Microscopy in Vivo

描述（由申请人提供）：多光子FRET显微镜的脉冲整形在体内多光子显微镜已被证明是一个宝贵的工具，在散射组织和活体动物成像。然而，由于多光子显微镜所使用的典型激光光源的光谱范围有限和费用昂贵，阻碍了蛋白质-蛋白质相互作用的多光子定量研究。宽带钛蓝宝石激光源在单个激光源中提供了惊人的激发波长范围，允许同时激发多个荧光团。然而，宽频带激励缺乏选择性。脉冲整形提供了一个理想的解决方案，提供高效和选择性的多光子激发，与减少光漂白的额外好处。本提案旨在将脉冲整形与体内定量多光子FRET显微镜相结合，为研究界引入一种令人兴奋的新工具。这项新技术将允许快速和定量成像许多常用的两个光子荧光团和FRET对，包括许多荧光蛋白。我们将首先演示多重荧光激发和检测溶液中常用的荧光蛋白和FRET对，使用光谱分解和FRET化学计量算法来获得相对自由和相互作用蛋白浓度的定量测量。然后，我们将在活细胞成像应用中使用优化的脉冲形状，以提供细胞膜受体信号的定量测量。我们将比较基于脉冲形状的技术与单光子FRET化学计量学和定量多光子FRET成像采用荧光寿命方法（FRET- flim）。与FRET- flim和当前基于激光调谐的多路多光子成像相比，我们预计图像采集速度将提高几个数量级，从而使体内多光子FRET显微镜成为可能。在优化多光子FRET的脉冲成形方法中，我们还将深入了解光漂白和光损伤减少机制，这些机制将广泛适用于其他多光子成像技术。

DESCRIPTION (provided by applicant): Pulse-shaping for Multiphoton FRET Microscopy In Vivo Multiphoton microscopy has proven to be an invaluable tool for imaging in scattering tissue and live animals. However, quantitative multiphoton studies of protein-protein interactions have been hindered by the expense and limited spectral range of the typical laser sources available for multiphoton microscopy. Broadband titanium-sapphire laser sources offer a stunning range of excitation wavelengths in a single laser source, allowing simultaneous excitation of multiple fluorophores. However, with broadband excitation comes a lack of excitation selectivity. Pulse-shaping offers an ideal solution for providing efficient and selective multiphoton excitation, with the added benefit of reduced photobleaching. This proposal aims to combine pulse-shaping with quantitative multiphoton FRET microscopy in vivo, introducing an exciting new tool to the research community. This new technology will permit rapid and quantitative imaging of many commonly-used two photon fluorophores and FRET pairs, including many fluorescent proteins. We will initially demonstrate multiplex fluorescence excitation and detection of commonly-used fluorescent proteins and FRET pairs in solution, employing spectral-unmixing and FRET stoichiometry algorithms to obtain quantitative measures of relative free and interacting protein concentrations. We will then employ the optimized pulse-shapes in live-cell imaging applications to provide quantitative measures of cell membrane receptor signaling. We will compare the pulse-shaping-based technology with one-photon FRET stoichiometry and quantitative multiphoton FRET imaging employing fluorescence lifetime methodology (FRET-FLIM). Compared to FRET-FLIM and current multiplex multiphoton imaging based on laser-tuning we anticipate orders of magnitude enhancement in image acquisition speed, enabling multiphoton FRET microscopy in vivo. In optimizing the pulse-shaping methodology for multiphoton FRET we will also gain insight into photobleaching and photodamage reduction mechanisms that will be widely applicable to other multiphoton imaging techniques.