Pulse-shaping for Multiphoton FRET Microscopy in Vivo

描述（由申请人提供）：脉冲成形的多光子FRET显微镜在体内多光子显微镜已被证明是一个宝贵的工具，在散射组织和活动物成像。然而，蛋白质-蛋白质相互作用的定量多光子研究受到多光子显微镜可用的典型激光源的费用和有限的光谱范围的阻碍。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，随着宽带激发而来的是激发选择性的缺乏。脉冲整形提供了一个理想的解决方案，提供有效的和选择性的多光子激发，减少光漂白的额外好处。该提案旨在将联合收割机脉冲整形与体内定量多光子FRET显微术相结合，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的许多常用的双光子荧光团和FRET对，包括许多荧光蛋白。我们将首先展示多重荧光激发和检测常用的荧光蛋白和FRET对溶液中，采用光谱解混和FRET化学计量算法，以获得相对自由和相互作用的蛋白质浓度的定量测量。然后，我们将在活细胞成像应用中采用优化的脉冲形状，以提供细胞膜受体信号传导的定量测量。我们将比较基于脉冲整形的技术与单光子FRET化学计量和定量多光子FRET成像采用荧光寿命方法（FRET FLIM）。相比FRET-FLIM和目前的多路复用多光子成像的激光调谐的基础上，我们预计在图像采集速度的数量级增强，使多光子FRET显微镜在体内。在优化多光子FRET的脉冲整形方法，我们也将深入了解光漂白和光损伤减少机制，将广泛适用于其他多光子成像技术。 公共卫生相关性：脉冲整形的多光子FRET显微镜在体内拟议的工作将开发多功能技术，允许快速和定量成像和分析的多蛋白质相互作用，通过多光子荧光共振能量转移（FRET），彻底改变我们的能力，调查细胞信号在体内。开发的基本光学方法和算法将很容易转移到其他研究人员，使这项技术在正常发育和人类疾病的生物医学研究中得到广泛应用。在优化多光子FRET应用的脉冲整形方法中，我们还将深入了解减少光漂白和光损伤的方法，这些方法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. PUBLIC HEALTH RELEVANCE: Pulse-shaping for Multiphoton FRET Microscopy In Vivo The proposed work will develop versatile technology to permit rapid and quantitative imaging and analysis of multiple protein interactions via multiphoton fluorescence resonance energy transfer (FRET), revolutionizing our ability to investigate cell signaling in vivo. The fundamental optical methods and algorithms developed will be transferred readily to other investigators, enabling widespread use of this technology in biomedical studies of normal development and human disease. In optimizing the pulse-shaping methodology for multiphoton FRET applications we will also gain insight into methods to reduce photobleaching and photodamage that will be widely applicable to other multiphoton imaging techniques.