Chromophores with hypersensitivity to electric fields for highly sensitive voltag

描述（由申请人提供）：人类大脑中神经回路的功能对于理解脑部疾病、精神疾病、运动障碍和成瘾至关重要。虽然绘制生物结构和神经元之间的连接对于理解电路结构很重要，但直接观察神经元内部和神经元之间的电通信对于完全理解神经回路功能至关重要。为此，二次谐波生成（SHG）显微镜已被开发用于成像神经活动，因为它可以成像生物组织内相对较深的区域，并且因为报告的发色团直接响应膜电位。迄今为止，SHG成像中使用的发色团是不对称的，并且被认为是通过斯塔克位移进行操作的。虽然这些发色团为SHG成像提供了非常有用的信息，但它们的灵敏度似乎受到中度斯塔克位移所需的大视场的限制，这大约是神经元膜电位的10到100倍。这种有限的灵敏度需要更高的光学功率，这可能导致细胞损伤甚至死亡。在此，我们建议采用一种有点违反直觉的方法，即使用对称的发色团，因此在静息电位下没有SHG，但在动作电位下转换为具有大SHG信号和原位灵敏度的不对称发色团。关于这种对称性破缺的实验知识表明，有可能合成地将发色团调到对称破缺的边缘，从而使这一过程可以由外部刺激（如膜电位的变化）诱导。该项目将结合量子化学建模，与生物介质兼容的发色团的计算指导合成，以及生物相关模型膜中发色团的表征，以更全面地描述和理解分子对称性破坏和SHG响应。随着发色团的合成和测试，它们的响应将用于进一步完善量子模型，以更准确地指导后续的合成。哥伦比亚大学Rafael Yuste的实验室将在活的神经元中研究作为SHG成像良好候选者的发色团。尽管电压相关的对称性破缺在很大程度上未经实验测试，但它具有强大的理论基础，因此，尽管存在重大风险，但也有与t相关的潜在转化回报。该计划的最终目标是提供一类灵敏度更高的新发色团，以便广泛的神经科学研究人员可以实际和常规地使用SHG成像来绘制神经回路的功能。

DESCRIPTION (provided by applicant): The functioning of neural circuits in the human brain is fundamentally important for understanding brain disease, psychiatric disorders, movement disorders, and addiction. Although mapping biostructures and the connectivity between neurons is important for understanding circuit structure, directly observing electrical communication within and amongst neurons is critical for complete understanding of neural circuitry function. Towards this end, second harmonic generation (SHG) microscopy has been developed for imaging neural activity because it can image relatively deep within biological tissue and because the reporting chromophores are directly responsive to membrane potentials. To date, the chromophores used in SHG imaging are asymmetric and are believed to operate via a Stark shift. Although these chromophores have provided very useful information about SHG imaging, their sensitivity appears to be limited by the large fields required for moderate Stark shifts, whih are about 10 to 100 times greater than a neuron's membrane potential. This limited sensitivity requires higher optical powers that can cause cell damage and even death. Herein we propose to take a somewhat counterintuitive approach by using chromophores that are symmetrical, and therefore have no SHG at the resting potential, but are converted to asymmetrical chromophores with large SHG signals and sensitivity in situ by the action potential. What is experimentally known about such symmetry breaking suggests that it may be possible to synthetically tune chromophores to the edge of symmetry breaking so that the process can be induced by an external stimulus such as the change in membrane potential. The program will combine quantum chemical modeling, computationally guided synthesis of chromophores that are compatible with biological media, and characterization of the chromophores in biologically relevant model membranes to more fully delineate and understand the molecular symmetry breaking and the SHG response. As chromophores are synthesized and tested, their response will be used to further refine the quantum models to more accurately guide subsequent synthesis. Chromophores identified as good candidates for SHG imaging will be studied in live neurons in the laboratories of Rafael Yuste at Columbia University. Although voltage-dependent symmetry breaking is largely untested experimentally, it has a strong theoretical basis, and so while there is significant risk, there is also potentially transformational reward associated with t. The ultimate goal of the program is to provide a new class of chromophores with much larger sensitivity so that a broad base of neuroscience researchers can practically and routinely use SHG imaging to map the functioning of neural circuits.