Super-multiplex optical imaging: development of novel spectroscopy and probes to illuminate complex biomedicine

生物系统在空间上天生是异质的，在时间上是动态的，在空间上是复杂的 大自然。一个巨大的挑战是如何研究在每个相关领域的广大互动参与者 长度尺度，从蛋白质复合体中的蛋白质网络到内部相互作用的细胞器 细胞，组织内的各种细胞类型，以及功能器官内的协同组织。因此， 同时监测生物体内大量相互作用的物种的能力 具有足够的时空分辨率的系统对于表征和 对潜在复杂性的理解。 然而，目前还没有合适的技术来应对这一重大挑战。这个 流行的“组学”技术不具备所需的时空分辨率，尤其是 用于三维样本或活体样本。光学显微镜只能成像少数(2~5)个 一次不同的目标，受到基本的荧光“颜色障碍”的限制。 打破光学显微镜的颜色障碍，弥合“组学”与 在这里，我们提出了一个全新的技术平台。新型振动光谱学 包括电子预共振受激拉曼散射(EPR-SRS)和受激拉曼散射 将利用拉曼激发荧光(SREF)来实现最灵敏的拉曼 到目前为止的图像。我们的初步数据已经证明了单分子的敏感性。我们将进一步 通过探索二维激发光谱技术发展到~100 颜色，设计和合成成像探针库，开启超分辨率超 多路成像。成像技术随后将在几个广泛的影响中实施 应用包括超多路组织病理学、全脑结构成像 复杂性，以及活细胞的超多参数深度表型。 光学显微镜的创新改变了许多生物学问题的解决方式 地址。就像共焦显微镜是生物医学实验室和双光子技术的主力一样 荧光显微镜已经改变了活体脑成像，我们设想我们新提出的 超多路光谱和显微技术将打破目前的技术瓶颈， 革命性的多色光学成像，并成为全系统研究的新标准 一般情况下，复杂的系统。

Biological systems are inherently heterogeneous in space, dynamic in time, and complex in nature. A grand challenge is how to study the vast number of interacting players at every relevant length scale, ranging from protein network in protein complexes, to interacting organelles within cells, to various cell types within tissues, and to synergistic tissues within functional organs. Hence, the ability to simultaneously monitor a large number of interacting species inside biological systems with sufficient spatial-temporal resolution is indispensable for characterization and understanding of the underlying complexity. However, there is currently no suitable technology that can meet this grand challenge. The prevalent “omics” technologies do not have the required spatial-temporal resolution, especially for three-dimensional samples or living specimen. Optical microscopy can only image a few (2~5) different targets at once, limited by the fundamental “color barrier” of fluorescence. To break the color barrier of light microscopy and to bridge the gap between “omics” and imaging, here we propose a radically new technology platform. Novel vibrational spectroscopy including electronic-pre-resonance stimulated Raman scattering (epr-SRS) and stimulated Raman excited fluorescence (SREF) will be exploited, to achieve the most sensitive Raman imaging to date. Our preliminary data have proved single-molecule sensitivity. We will further develop the technique by exploring the two-dimensional excitation spectroscopy to reach ~100 colors, designing and synthesizing a library of imaging probes, opening up super-resolution super- multiplex imaging. The imaging technology will then be implemented in several broad-impact applications including super-multiplex tissue pathology, mapping brain-wide architecture complexity, and super-multiparameter deep phenotyping of living cells. Innovations in optical microscopy have changed the way many biological problems are addressed. Just like confocal microscopy is the work-horse in biomedical labs and two-photon fluorescence microscopy has transformed in vivo brain imaging, we envision our newly proposed super-multiplex spectroscopy and microscopy will break the current technical bottleneck, revolutionize multicolor optical imaging, and become a new standard for system-wide study of complex systems in general.