Intra-vital microscopy using non-linear optical techniques

这些研究的目的是开发成像技术来监测体内的亚细胞结构和过程。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活体光学显微镜系统，该系统适用于生物组织和结构，而不是迫使动物登上传统的显微镜舞台。在过去的一年里，我们取得了以下主要发现：1)与工业界合作，我们评估了我们的表观总发射检测系统的商业精简版本，以提高多光子激发显微镜的体内检测效率。这个最初的原型显示，包括肌肉、肾脏和脂肪垫在内的各种组织的信噪比增加了2到5倍。这是将这项技术商业化并将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，我们开发了一种新的方法来提高同时检测多个荧光探针的灵敏度。以前，探测器发射光谱的重叠迫使使用限制带宽的滤光片来分辨来自不同荧光探测器的信号。这种选择性带宽显著降低了荧光成像实验的信噪比。然而，在我们可以利用探测器发射的光谱密度的先验信息并假设探测器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)，我们可以确定探测器的空间分布，同时收集几乎所有发射的光。使用这种方法，我们证明了通过简单地使用二向色镜，造成最小的光损失，以探头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探头在样品中的分布。这种方法被证明显著提高了多荧光探针研究的信噪比，这取决于探针的光谱重叠。3)初步完成了线粒体在哺乳动物混合纤维型骨骼肌中的分布研究。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嵌入纤维的毛细血管周围凹槽的侧面。几项转基因动物研究和计算分析的总结表明，线粒体的定位导致了以下结论：a)在氧化纤维中嵌入毛细血管增加了氧气向纤维扩散的表面积，并选择性地将氧气输送到这种混合纤维系统中。B)将线粒体定位于毛细血管周围的侧隙并不是为了改善向线粒体的氧气输送。这一结论主要是基于线粒体在侧隙中的几何形状c)线粒体和纤维的核在这些侧隙中的位置很简单，这是因为这是一个代表机械涡流的位置，其中不能放置收缩元件，但可以放置其他细胞元件，而对收缩装置的影响最小。4)我们启动了一项新的计划，以评估相干反斯托克斯拉曼散射(CARS)在体内对代谢产物和水进行非侵入性成像的应用。我们已经演示了一种激光激发方案，它将允许我们在完整的动物身上同时执行微微秒脉冲和毫微秒脉冲，用于荧光和谐波成像。这一系统已经建成，并正在接受本财政年度最后一个季度的测试。

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.