IDBR - Development of an Ultrafast Phase-shaping Contrast Microscope

项目摘要：本项目将开发一种超快速相位成形对比显微镜（UPSCM），为生物研究提供增强的成像能力和新的成像对比模式。UPSCM将在多功能仪器中利用宽带激光器的幅度和相位整形，该仪器将具有三种工作模式，以满足广泛的成像需求。第一种成像方式将允许快速和定量的多光子荧光共振能量转移（MFRET）成像。拟议的MFRET成像模式比现有技术快几个数量级，为研究动物和组织模型中的蛋白质-蛋白质相互作用开辟了新的机会，并将很容易扩展到视频速率成像速度。UPSCM的第二种成像方式将是双色泵探针对比显微镜。这种模式将利用泵-探针对比机制，包括受激发射、激发态吸收和基态漂白，以实现非荧光和弱荧光内源性物种的敏感成像。最后，第三种成像方式将采用单一形状的激光脉冲，旨在提供分子对比以及洞察分子的能量景观。多光子显微镜彻底改变了我们观察生物世界的能力。多光子激发显著提高成像深度到组织，并提供了新的对比模式。多光子荧光显微镜已被广泛使用，并已显示出增加荧光的信号对背景，同时减少光损伤和光漂白，允许在较长时间内更敏感地检测荧光信号。然而，在典型的多光子荧光显微镜中，使用可调谐飞秒激光对单个荧光探针成像，使得同时观察多种化学物质变得困难。提出的UPSCM将采用极宽带飞秒激光器，允许对常用荧光探针进行选择性和同步多光子荧光成像。这种方法将促进转基因动物中蛋白质-蛋白质相互作用的研究，使健康和患病动物的细胞信号传导的新研究成为可能。除了增强的多光子荧光成像，UPSCM将为非荧光内源性物种提供新颖、敏感和选择性的对比，这将广泛适用于旨在提高当前对基本细胞过程和疾病诊断方法的理解的成像研究。

Project AbstractThe project will develop an Ultrafast Phase-Shaping Contrast Microscope (UPSCM) to provide enhanced imaging capabilities and new modes of imaging contrast for biological research. The UPSCM will exploit the amplitude and phase-shaping of a broadband laser in a versatile instrument that will have three modes of operation to meet a wide range of imaging needs. The first imaging modality will permit rapid and quantitative multiphoton fluorescence resonance energy transfer (MFRET) imaging. The proposed MFRET imaging modality is orders of magnitude faster than current technology, opening up new opportunities for studying protein-protein interactions in animal and tissue models and will be readily extended to video-rate imaging speeds. The second imaging modality of the UPSCM will be two-color pump-probe contrast microscopy. This modality will exploit pump-probe contrast mechanisms including stimulated emission, excited state absorption and ground-state bleach to enable sensitive imaging of nonfluorescent and weakly fluorescent endogenous species. Finally, the third imaging modality will employ a single shaped laser pulse designed to provide molecular contrast as well as insight into the energy landscape of the molecule.Multiphoton microscopy has revolutionized our ability to visualize the biological world. Multiphoton excitation markedly enhances the depth of imaging into tissues, and provides novel modes of contrast. Multiphoton fluorescence microscopy has been widely used and has been shown to increase signal-to-background for fluorescence while reducing photodamage and photobleaching, permitting more sensitive detection of fluorescence signals over extended periods of time. However, in a typical multiphoton fluorescence microscope, a tunable femtosecond laser is used to image a single fluorescent probe, making the simultaneous observation of multiple chemical species difficult. The proposed UPSCM will employ an extremely broadband femtosecond laser to allow selective and simultaneous multiphoton fluorescence imaging of commonly used fluorescent probes. This methodology will facilitate studies of protein-protein interactions in transgenic animals, enabling new studies of cellular signaling in healthy and diseased animals. In addition to enhanced multiphoton fluorescence imaging, the UPSCM will provide novel, sensitive and selective contrast for nonfluorescent endogenous species that will be broadly applicable to imaging studies aimed at improving current understanding of basic cellular processes and methods for disease diagnosis.