Optical Amplification Microscopy of Weak Back-Scattered Light

摘要 光学成像提供了足够的空间分辨率、特异性、敏感性和时间分辨率， 在细胞和分子水平上对重要的生物过程提供了大量的见解。虽然 高质量的光学图像可以从体外细胞培养和/或薄组织切片、活细胞 在复杂的三维活体组织环境中进行成像仍然具有相当大的挑战性。因为生物学上的 组织自然不利于光的传播，因为不可避免地存在强烈的环境 环境中的光，在几乎每一种活体生物医学光学成像中都会出现特殊的挑战，即 弱光信号和强光背景(包括组织中的倍散射光和 环境光)。这些挑战大大限制了光学成像在活体中的充分应用 临床前和临床调查。目前，用于光学成像的弱光信号的检测依赖于 大量使用高灵敏度的电子探测器和电子放大器。虽然高端电子产品 光接收器通常是非常敏感的，高灵敏度导致成像系统非常容易 对于随机的光子噪声，这样的环境背景(房间)光，这在实际中是有问题的 应用(例如体内研究和临床实践)。此外，电子探测器不能 将携带图像的弹道光子与多重散射光背景区分开来，这作为一种 生物样品光学成像中的主要噪声源可能是压倒性的和显著的 当成像组织深处的微结构时，分辨率会降低。 在这个拟议的项目中，我们将开发一种使用新型高速光学元件的多模式显微镜 用于光学放大微弱后向散射光信号的参数放大器(OPA)，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显示，OPA将 不仅提供了高水平的信号增益来提高检测灵敏度，而且还提供了固有的非线性 光学门，既能提取成像信号，又能抑制环境光子中的噪声源 和多重散射背景光。我们将系统地探讨这一OPA为以下方面提供的好处 多模式成像，将包括无标签反射、共焦显微镜和光学相干 体层摄影术。在分辨率、对比度、成像深度和减少光损害方面的改进将是 调查过了。该项目的成功完成将展示一种高速、强大、光学的内在活力 将多种模式与增强的性能和新的迷人的成像功能相结合的显微镜 由OPA独家启用。这种活体显微镜不仅将使新的生物学和临床研究成为可能， 而且还推动了基于光放大的光学成像新技术的发展。

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.