Ultra-deep tissue imaging by super-nonlinear fluorescence microscopy

描述(由申请人提供)：非常希望能够探测活体内部具有亚细胞分辨率的生物活动。为此，光学显微镜是 功能最强大、用途最广的医疗设备。特别是，通过非线性跃迁使用空间受限激发，双光子激发荧光显微镜已经成为成像散射样品(如脑)不可或缺的一部分。然而，由于散射损耗，入射激光功率随成像深度呈指数下降，焦外荧光最终会淹没焦内信号。由此产生的成像对比度损失S/B定义了一个基本的成像深度限制(对于小鼠脑组织约为1 mm)，这不是通过增加激发强度就能克服的。因此，如何成像比基本成像深度限制更深，对包括神经科学、胚胎学和肿瘤学在内的许多生物医学研究提出了巨大的挑战。实现这一目标的新型光学成像技术无疑将开辟新的途径，改变我们监测生命系统的能力。我们建议通过探索一种独特的以探测器为中心的策略来应对这一挑战，而不是流行的以波为中心的方法。我们意识到这些存在一类特殊的成像探测器，它们可以占据亚稳态的开启和关闭状态，这些状态可以被适当波长的外部光操纵。通过利用这类独特的可光开关探针，我们提出了一种新的超非线性(高于其信号对激光强度的二次依赖)荧光显微镜平台，它应该能够提高S/B对比度，并扩展双光子显微镜的基本成像深度限制。具体地说，我们将重点介绍两种看似相反但实际上相关的技术，它们将光开关探针与双光子显微镜相结合，即多光子激活和成像(MPAI)和多光子失活和成像(MPDI)。我们的初步结果证明了MPAI和MPDI在三维组织模体上的有效性。此外，还从实验上验证了信号与激光强度的四阶超非线性关系。因此，我们的目标是进一步发展和完善这项技术，使其能够应用于各种散射样本的成像，特别是脑组织，具有更好的S/B对比度和深度穿透性。具体地说，我们计划(1)系统地评估新一代可光切换探针(包括荧光蛋白和合成染料)；(2)将最有希望的探针应用于脑组织切片成像，(3)最终能够在活体中进行大脑深层MPAI或MPDI，其深度是双光子显微镜所能达到的2.4倍。这项拟议的技术创新有可能改变活体光学显微镜的未来格局，将生物成像带入以前未知的生物医学新领域。

DESCRIPTION (provided by applicant): It is highly desirable to be able to probe biological activities with subcellular resolution deep inside live organisms. To this end, light microscopy is the most powerful and versatile modality. In particular, by employing a spatially confined excitation via a nonlinear transition, two-photon excited fluorescence microscopy has become indispensable for imaging scattering samples such as brain. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast, S/B, defines a fundamental imaging-depth limit (about 1 mm for mouse brain tissues), which cannot be overcome by increasing excitation intensity. Thus, how to image deeper than the fundamental imaging- depth limit poses a grand challenge for many biomedical studies including neuroscience, embryology and oncology. Novel optical imaging techniques that accomplish this goal would undoubtedly open up new avenues, transforming our ability to monitor living systems. We propose to address this challenge by exploring a unique probe-centered strategy as opposed to the popular wave-centered approaches. We realize that these exists a special class of imaging probes that can occupy metastable on- and off- states which can be manipulated by external light at proper wavelengths. By harnessing this unique class of photoswitchable probes, we propose to develop a novel platform of super-nonlinear (higher than quadratic dependence of its signal on the laser intensity) fluorescence microscopy which should be able to promote the S/B contrast and extend the fundamental imaging-depth limit of two-photon microscopy. Specifically, we will focus on two seemingly opposite but actually related techniques which couple photo-switchable probes with two-photon microscopy, namely, multiphoton activation and imaging (MPAI) and multiphoton deactivation and imaging (MPDI). Our preliminary results have demonstrated the validity of both MPAI and MPDI on three-dimensional tissue phantoms. Moreover, the 4th order super-nonlinear dependence of the signal on laser intensity was also verified experimentally. Hence, we aim to further develop and perfect the technique to the stage where it can be applied to imaging various scattering samples, particularly brain tissues, with a much better S/B contrast and depth penetration. Specifically, we plan to (1) systematically evaluate the emerging generation of photoswitchable probes (including fluorescent proteins and synthetic dyes); (2) apply the most promising probes into brain tissue slice imaging and, (3) ultimately be able to perform in vivo deep brain MPAI or MPDI with 2.4 times deeper than what two-photon microscopy can ever achieve. The proposed technical innovation has the potential to change the future landscape of in vivo light microscopy, take bio-imaging into new areas of biomedicine that have been previously uncharted.