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体内光学显微镜系统，该系统适应生物组织和结构，而不是将动物置于传统的显微镜载物台上。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。这些研究将通过定量3D电子显微镜研究进行随访。4)今年的一项重大技术努力是汽车显微镜的发展，以研究水在完整组织中的运动。我们已经证明，飞秒脉冲在生物组织中产生水汽车信号的效率要高得多，而不会影响质子和氘之间的选择性，这是示踪剂研究所需的。在我们的应用中，信噪比的这种改善接近200倍。我们已经证明，水的运动可以跟踪在亚微米尺度上与汽车显微镜在完整的骨骼肌使用氘作为示踪剂。我们目前正在表征复杂生物组织中产生的光学伪影，以开发适当的光学或成像处理技术来提供补偿。

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. These studies will be followed up with quantitative 3D electron microscopy studies. 4) A major technological effort this year was the development of CARS microscopy to study the motion of water in intact tissues. We have demonstrated that femto second pulses are much more efficient in generating water CARS signals in biological tissues without compromising the selectivity between proton and deuterium, required for tracer studies. This improvement in signal to noise approached 200 fold in our application. We have demonstrated that water motion can be tracked on the submicron scale with CARS microscopy within intact skeletal muscle using deuterium as a tracer. We are currently characterizing the optical artifacts generated in complex biological tissues to develop appropriate optical or imaging processing techniques to provide compensation.