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，取得了以下主要发现：1）微创双光子激发荧光显微镜（TPEFM）正在用于研究细胞内的亚细胞代谢过程，在完整的动物中，在正常的体内条件下，使用各种外源性和内源性荧光探针。我们已经完成了下一代运动校正系统，该系统使用共振扫描显微镜和图形处理单元（GPU）来快速处理数据，以接近真实的时间执行完整的三维校正方案。这允许对体内生物组织内的运动进行三维校正。 这种新颖的接口的一个真正的真实的时间3D成像技术与GPU提供了第一个真实的时间运动校正方案的活体显微镜。 2)我们已经适应了这种技术，以产生大视场（厘米）的图像与微米的平面分辨率。这提供了一个新的观点的整体微血管和细胞结构的组织以前没有。这种方法已被应用于大脑，心脏，肝脏和骨骼肌。3)在这些研究中，我们已经完成了应用自适应光学校正激发光畸变的初步研究。使用这种方法，我们已经能够大大提高这些测量的穿透力和信噪比。目前正在将该系统全面纳入我们的标准范围。4)利用TPEFM的固有性质，我们已经完成了一个成像计划，收集几乎所有的发射光从探头在成像实验的发展。这种方法被称为总排放检测（TED）。我们目前已经修改了这个最初的概念，包括一个表面收集计划与体内测量兼容（所谓的epi-TED）。该系统已经显示出将来自体内荧光成像实验的信号收集提高2-4倍。显然，这种方法是目前在体外或体内成像任何荧光探针的最有效的方法。

The purpose of these studies was 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) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have completed a next generation system for motion correction that performs a complete 3 dimensional correction scheme in near real time using a resonant scanning microscope and a graphical processing unit (GPU) to rapidly process the data. This permits the 3 dimensional correction for motion within the biological tissues in vivo. This novel interface of a true real time 3D imaging technique with a GPU provides the first real time motion correction scheme for intra-vital microscopy. 2) We have adapated this technology to generate large field of view (centimeters)images with micron in plane resolution. This provides a novel view of the overall microvascular and cellular structure of tissues previously not available. This approach has been applied to the brain, heart, liver and skeletal muscle. 3) We have completed our preliminary studies on the application of adaptive optics to correct for the distortion of the excitation light in these studies. Using this approach we have been able to greatly improve the penetration and signal to noise of these measurements. Full implementation of this system into our standard scope is now underway. 4) Using the inherent nature of TPEFM we have completed the development of an imaging scheme that collects nearly all of the emitted light from a probe during the imaging experiment. This approach termed Total Emission Detection (TED). We have currently modified this initial concept to include a surface collecting scheme compatible with in vivo measurements (so called epi-TED). This system has shown to improve the signal collection from fluorescence imaging experiments in vivo by a factor of 2-4 fold. Clearly, this approach is currently the most efficient method of imaging any fluorescent probe in vitro or in vivo.