Laser and Microscope Development for Multicolor Nonlinear Imaging Deep in Tissue

 描述(申请人提供)：非线性显微镜是一种强大的技术，用于研究组织中正常和疾病状态下的细胞过程。现有的显微镜设计和激励激光器目前限制了成像通道的数量和成像深度。增加这些数量将使在更多种类的解剖结构中研究更复杂的细胞相互作用成为可能。最近的工作已经确定了使用多光子荧光和谐波产生显微镜进行组织深层成像的最佳条件(光脉冲能量、持续时间、波长和重复率)。然而，提供所需光脉冲并与生物成像实验室兼容的光源不可用。康奈尔大学的科学家提议开发激光和新的显微镜设计，这些设计将极大地增强深层组织和多通道成像的能力。在飞秒脉冲光纤激光器的设计中，将采用非线性脉冲传输的新概念。这将利用光纤的主要实用优势，扩展目前用于非线性显微镜的激光器的性能。具体地说，这些源将提供峰值功率为1兆瓦、波长在800至1350 nm之间可调、重复频率在1至10 MHz之间的100飞秒脉冲。激光器最终将是紧凑和坚固的，这将促进在研究实验室内外使用非线性显微镜技术。新的激光将通过对测试样品的一系列成像实验以及小鼠大脑和脊髓的活体成像进行评估。这些实验还将测试对每种成像情况的最佳条件的理解。一种利用这些新激光能力的高光谱多光子显微镜将被开发出来。这将使用多个激发和检测波长来极大地增加获取的信息量，使每个像素总共获得80个激发/发射荧光信息。当与密集的荧光标记策略相结合时，这种高光谱显微镜有可能将活体成像从主要用于检验假说的工具转变为生物发现的技术。

 DESCRIPTION (provided by applicant): Nonlinear microscopy is a powerful technique for investigating cellular processes that underlie normal and disease states in tissue. Existing microscope designs and excitation lasers currently limit the number of imaging channels and the imaging depth. Increasing these will enable studies of more-complex cell interactions in a greater variety of anatomical structures. Recent work has identified the optimal conditions (light pulse energy, duration, wavelength, and repetition rate) for imaging deep in tissue using multiphoton fluorescence and harmonic-generation microscopies. However, sources that supply the needed light pulses and are compatible with bioimaging laboratories are not available. Scientists from Cornell University propose to develop lasers and new microscope designs that will greatly enhance the capabilities for deep-tissue and multi-channel imaging. New concepts in nonlinear pulse propagation will be employed in the design of femtosecond-pulse fiber lasers. These will extend the performance of lasers currently employed in nonlinear microscopy, with the major practical advantages of fiber. Specifically, the sources will supply 100-femtosecond pulses with peak power of 1 megawatt, wavelength-tunable between 800 and 1350 nm and at repetition rates between 1 and 10 MHz. The lasers will ultimately be compact and robust, which will facilitate the use of nonlinear microscopy techniques within and beyond research laboratories. The new lasers will be evaluated through a series of imaging experiments on test samples and through in vivo imaging in the brain and spinal cord of mice. These experiments will also test understanding of the optimum conditions for each imaging situation. A hyperspectral multiphoton microscope that capitalizes on the capabilities of these new lasers will be developed. This will employ multiple excitation and detection wavelengths to vastly increase the amount of information acquired to a total of 80 channels of excitation/emission fluorescence information per pixel. When combined with dense fluorescent labeling strategies, this hyperspectral microscope has the potential to transform in vivo imaging from a tool used primarily for testing hypotheses into a technique for biological discovery.