Intra-vital microscopy using non-linear optical techniques

这些研究的目的是发展成像技术来监测亚细胞结构和过程，在体内。使用的主要方法是非线性光学显微镜技术。我们一直在系统地开发一种适应生物组织和结构的体内光学显微镜系统，而不是强迫动物在传统的显微镜台上。在过去的一年中，我们取得了以下主要发现：1)与工业界合作，我们已经评估了我们的epi总发射检测系统的商业浓缩版本，以提高多光子激发显微镜的体内检测效率。这个最初的原型表明，在包括肌肉、肾脏和脂肪垫在内的各种组织中，信号噪声比增加了2到5倍。这是将这项技术商业化和将LCE产生的技术传播给一般科学界的第一步。2)为了进一步提高荧光显微镜的信噪比，我们开发了一种提高同时检测多个荧光探针灵敏度的新方法。以前，探针发射光谱的重叠迫使使用限制性带宽滤波器来解析来自不同荧光探针的信号。这种选择性带宽显著降低了荧光成像实验的信噪比。然而，在我们可以使用探针发射光谱密度的先验信息并假设探针的空间重叠最小的情况下，我们证明了通过使用独立分量分析（ICA），我们可以在收集几乎所有发射光的同时确定探针的空间分布。使用这种方法，我们证明了通过简单地使用一个二向镜，造成最小的光损失，用探针发射能量之间的截止频率作为数据的唯一频率编码，我们可以正确地重建样品内探针的分布。这种方法被证明可以显著提高多荧光探针研究的信噪比，根据探针的光谱重叠程度提高2到5倍。3)完成了线粒体在哺乳动物混合纤维型骨骼肌内分布的初步研究。在这些研究中，我们发现氧化纤维中线粒体体积的很大一部分位于纤维中嵌入的毛细血管周围凹槽的外侧区域。通过对几项转基因动物实验的总结和计算分析，线粒体的定位可以得出以下结论：a)氧化纤维中毛细血管的嵌入增加了氧气向纤维扩散的表面积，并在混合纤维系统中选择性地向这些纤维输送氧气。b)线粒体定位于毛细血管周围的外侧空间不是为了改善线粒体的氧气输送。c)线粒体和纤维核在这些侧间隙中的位置很简单，因为这是一个代表机械涡流的位置，收缩元件不能放置，但其他细胞元件可以放置，对收缩装置的影响最小。4)我们已经启动了一个新的项目来评估使用相干反斯托克斯拉曼散射（CARS）在亚微米尺度上对体内代谢物和水进行无创成像。我们已经证明了一种激光激发方案，将允许我们执行皮秒脉冲的CARS和飞秒脉冲的荧光和谐波成像在完整的动物。该系统已经建成，并正在本财政年度的最后一个季度进行测试。

The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. The major approach used was non-linear optical microscopy techniques. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) Working with industry we have evaluated a commercial condensed version of our epi-Total Emission Detection system for improving the detection efficiency of multiphoton excitation microscopy, in vivo. This initial prototype demonstrated a 2 to 5 fold increase in signal to noise in a variety of tissues including muscle, kidney and fat pads. This is the first step in commercialization of this technology and dissemination of technology generated in LCE to the general scientific community. 2) Furthering our interest in improving the signal to noise of fluorescence microscopy we have developed a new approach in improving the sensitivity of detecting multiple fluorescence probes simultaneously. Previously the overlap of the emission spectra of probes forced the use of restrictive bandwidth filters to resolve the signals from different fluorescence probes. This selective bandwidth significantly reduced the signal to noise of the fluorescence imaging experiment. However, in the case where we can use the prior information of the spectral density of the probes emission and make the assumption that the spatial overlap of the probes is minimal, we demonstrated that by using Independent Component Analysis (ICA) we can determine the spatial distribution of the probes while collecting nearly all of the emitted light. Using this approach, we demonstrated that by simply using a dichroic mirror, causing a minimal loss of light, with a cutoff frequency between the emission energies of the probes as the sole frequency encoding of the data we can properly reconstruct the distribution of the probes within the sample. This approach was shown to dramatically improve the signal to noise of multi-fluorescence probe studies by 2 to 5 fold depending on the spectral overlap of the probes. 3) We have completed our initial study on the distribution of mitochondria within mammalian mixed fiber type skeletal muscle. In these studies we discovered that a large fraction of mitochondrial volume in oxidative fibers is located in regions lateral to a groove surrounding capillaries embedded in the fiber. The summary of several transgenic animal studies and computation analysis reveal that the localization of the mitochondria led to the following conclusions: a) The embedding of the capillaries in the oxidative fibers increases the surface area available for oxygen diffusion to the fiber and selectively delivers the oxygen to these fibers in this mixed fiber system. b) The localization of the mitochondria to the lateral space around the capillaries is not to improve oxygen delivery to the mitochondria. This conclusion is primarily based on the geometry of the mitochondria in the lateral spaces c) The location of the mitochondria, and nuclei of the fiber, in these lateral spaces is simple due to the fact that this is a location that represents a mechanical eddy where contractile elements cannot be placed, but other cellular elements can be placed with minimal impact on the contractile apparatus. 4) We have initiated a new program in evaluating the use of Coherent Anti-Stokes Raman Scattering (CARS) for imaging metabolites and water non-invasively on the sub-micron scale in vivo. We have demonstrated a laser excitation scheme that will permit us to perform both pico second pulses for CARS as well as femto second pulses for fluorescence and harmonic imaging in intact animals. This system has been constructed and is undergoing testing the last quarter of this fiscal year.