Optical Amplification Microscopy of Weak Back-Scattered Light

总结 光学成像提供了足够的空间分辨率、特异性、灵敏度和时间分辨率， 在细胞和分子水平上对重要的生物学过程提供了实质性的见解。虽然 可以从体外细胞培养物和/或薄组织切片、活体细胞 在复杂的三维活体组织环境中成像仍然是相当具有挑战性的。因为生物 组织自然地不有利于光的传播，并且由于不可避免地存在强环境光， 光在环境中，特殊的挑战出现在几乎每一个体内生物医学光学成像，即 弱光信号和强光背景（包括组织中的多次散射光和 环境光）。这些挑战极大地限制了光学成像在体内的全面应用 临床前和临床研究。目前，用于光学成像的弱光信号的检测依赖于 主要依靠使用高灵敏度的电子探测器和电子放大器。虽然高端电子 光接收器通常非常灵敏，高灵敏度导致成像系统非常容易 随机光子噪声，例如环境背景（房间）光，这对于实际应用是有问题的。 应用（例如体内研究和临床实践）。此外，电子探测器无法 将承载图像的弹道光子与多次散射光背景区分开， 生物样品的光学成像中的主要噪声源，可以是压倒性的和显著的 当对组织深处的微结构进行成像时，会降低分辨率。 在这个拟议的项目中，我们将开发一种多模态显微镜，利用一种新型的高速光学 参量放大器（OPA）光学放大微弱的反向散射光信号，并展示其能力 通过研究小鼠皮肤中的体内细胞凋亡事件。根据我们的初步数据，OPA将 不仅提供高水平的信号增益以提高检测灵敏度，而且提供固有的非线性 光学门，用于提取承载成像的信号并拒绝来自环境光子的噪声源 和多重散射的背景光。我们会有系统地探讨这项外地加工措施所带来的好处， 多模式成像，包括无标记反射共焦显微镜和光学相干 断层扫描分辨率、对比度、成像深度的改进以及减少的光损伤将是 研究了该项目的成功完成将展示一种高速，稳健，光学活体 显微镜结合了多种模式，具有增强的性能和新的迷人的成像功能 由外行星联盟独家授权这种活体显微镜不仅可以进行新的生物学和临床研究， 也促进了基于光放大器的新型光学成像技术的发展。

SUMMARY Optical imaging, which offers sufficient spatial resolution, specificity, sensitivity, and temporal resolution, has provided substantial insights into important biological processes at the cellular and molecular levels. Although high quality optical images can be obtained from in vitro cell cultures and/or thin tissue sections, intravital cell imaging in a complex, three-dimensional living tissue environment remains quite challenging. Because biological tissue naturally does not favor the propagation of light, and because of the inevitable presence of strong ambient light in the environment, special challenges arise in virtually every in vivo biomedical optical imaging, namely weak light signals and strong light backgrounds (including the multiply-scattered light in the tissue and the environment light). These challenges have significantly limited the full application of optical imaging in in vivo pre-clinical and clinical investigations. Currently, the detection of weak light signals for optical imaging relies heavily on the use of highly sensitive electronic detectors and electronic amplifiers. Although high-end electronic photo receivers are often very sensitive, the high sensitivity leads to the imaging systems being extremely prone to random photon noise, such environmental background (room) light, which is problematic for practical applications (e.g. in vivo studies and clinical practice). Furthermore, electronic detectors are incapable of distinguishing image-bearing ballistic photons from the multiply-scattered light background, which as a predominant source of noise in optical imaging of biological samples, can be overwhelming and significantly degrade resolution when imaging microstructure deep in tissue. In this proposed project, we will develop a multimodal microscope that utilizes a novel high speed optical parametric amplifier (OPA) to optically amplify weak back-scattered light signals, and demonstrate its capabilities by investigating in vivo cellular apoptosis events in murine skin. As shown by our preliminary data, the OPA will not only provide a high level of signal gain to improve detection sensitivity, but also provide an inherent nonlinear optical gate to both extract imaging-bearing signals and reject the noise sources from environmental photons and multiply-scattered background light. We will systematically explore the benefits afforded by this OPA for multimodal imaging that will include label-free reflectance confocal microscopy and optical coherence tomography. Improvement in resolution, contrast, imaging depth, and reduced photo-damage, will be investigated. The successful completion of this project will demonstrate a high-speed, robust, optical intravital microscope that combines multiple modalities with enhanced performance and new fascinating imaging functions uniquely enabled by the OPA. This intravital microscope will not only enable new biological and clinical studies, but also promote the development of new optical imaging technologies based on optical amplifiers.