Laser and Microscope Development for Multicolor Nonlinear Imaging Deep in Tissue

 描述（由申请人提供）：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发激光器目前限制了成像通道的数量和成像深度。增加这些将使研究更复杂的细胞相互作用在更广泛的解剖结构。最近的工作已经确定了最佳条件（光脉冲能量，持续时间，波长和重复率）的成像组织深处使用多光子荧光和谐波产生显微镜。然而，提供所需的光脉冲，并与生物成像实验室兼容的来源是不可用的。 康奈尔大学的科学家们提出开发激光器和新的显微镜设计，这将大大提高深层组织和多通道成像的能力。 非线性脉冲传输的新概念将用于飞秒脉冲光纤激光器的设计。这些将扩展目前在非线性显微镜中使用的激光器的性能，并具有光纤的主要实用优势。具体来说，这些光源将提供峰值功率为1兆瓦的100飞秒脉冲，波长可在800至1350 nm之间调谐，重复频率在1至10 MHz之间。激光器最终将是紧凑和坚固的，这将有利于在研究实验室内外使用非线性显微镜技术。 新的激光器将通过一系列测试样本的成像实验和通过小鼠大脑和脊髓的体内成像进行评估。这些实验还将测试对每种成像情况的最佳条件的理解。 将开发利用这些新激光器能力的高光谱多光子显微镜。这将采用多个激发和检测波长，以极大地增加所获取的信息量，达到每个像素总共80个激发/发射荧光信息通道。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体内成像从主要用于测试假设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.