Intra-vital microscopy using non-linear optical techniques

使用非线性光学技术的活体显微镜检查

• 批准号: 8746581
• 负责人: Robert Balaban
• 金额: $ 105.1万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746581
• 关键词: Animals; Area; Biological; Blood capillaries; Cell Nucleus; Cell physiology; Cellular Structures; Cellular biology; Communities; Data; Detection; Diffusion; Elements; Evaluation; Fatty acid glycerol esters; Fiber; Fluorescence; Fluorescence Microscopy; Frequencies; Goals; Image; Imaging Techniques; Industry; Kidney; Lasers; Lateral; Light; Location; Mechanics; Medicine; Microscope; Microscopy; Mitochondria; Monitor; Motion; Muscle; Noise; Optics; Oxygen; Physiologic pulse; Physiological; Physiology; Resolution; Sampling; Scheme; Signal Transduction; Skeletal Muscle; Spatial Distribution; Staging; Structure; Surface; System; Techniques; Technology; Testing; Tissues; Transgenic Animals; Water; Work; base; capillary; commercialization; computerized data processing; density; fluorescence imaging; improved; in vivo; independent component analysis; interest; meter; new technology; novel strategies; programs; prototype; research study; submicron

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.

这些研究的目的是开发成像技术来监测体内亚细胞结构和过程。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适用于生物组织和结构的体内光学显微镜系统，而不是将动物置于传统的显微镜平台上。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epi-总发射检测系统的商业浓缩版本，以提高体内多光子激发显微镜的检测效率。该初始原型在包括肌肉、肾脏和脂肪垫在内的各种组织中显示出2至5倍的信噪比增加。这是将这一技术商业化和向一般科学界传播LCE产生的技术的第一步。 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，探针的发射光谱的重叠迫使使用限制性带宽滤波器来分辨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（伊卡），我们可以在收集几乎所有发射光的同时确定探测器的空间分布。使用这种方法，我们证明，通过简单地使用分色镜，造成最小的光损失，与探针的发射能量之间的截止频率作为唯一的频率编码的数据，我们可以正确地重建样品内的探针的分布。这种方法被证明是显着提高信噪比的多荧光探针研究的2至5倍，这取决于探针的光谱重叠。3)我们已经完成了对哺乳动物混合纤维型骨骼肌线粒体分布的初步研究。在这些研究中，我们发现氧化纤维中的大部分线粒体体积位于嵌入纤维中的毛细血管周围的凹槽的侧面区域。几个转基因动物研究和计算分析的总结表明，线粒体的定位导致以下结论：a）氧化纤维中毛细血管的嵌入增加了可用于氧扩散到纤维的表面积，并选择性地将氧递送到该混合纤维系统中的这些纤维。B）线粒体定位于毛细血管周围的侧向空间不是为了改善向线粒体的氧递送。该结论主要基于线粒体在侧向空间中的几何形状。c）线粒体和纤维核在这些侧向空间中的位置是简单的，因为这是代表机械涡流的位置，其中不能放置可收缩元件，但可以放置其他细胞元件，对可收缩装置的影响最小。4)我们已经启动了一个新的计划，在评估使用相干反斯托克斯拉曼散射（汽车）成像代谢物和水的非侵入性的亚微米尺度在体内。我们已经证明了一种激光激发方案，这将使我们能够在完整的动物中进行皮科脉冲汽车以及飞秒脉冲荧光和谐波成像。该系统已经建立，并正在本财政年度的最后一个季度进行测试。