Chromophores with hypersensitivity to electric fields for highly sensitive voltag

描述（由申请人提供）：人脑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。尽管绘制生物结构和神经元之间的连接对于理解回路结构很重要，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，二次谐波发生（SHG）显微镜被开发用于神经活动成像，因为它可以在生物组织内相对较深的位置成像，并且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。尽管这些发色团提供了有关 SHG 成像的非常有用的信息，但它们的灵敏度似乎受到中等斯塔克位移所需的大场的限制，该场大约比神经元膜电位大 10 到 100 倍。这种有限的灵敏度需要更高的光功率，这可能会导致细胞损伤甚至死亡。在此，我们建议采取一种有点违反直觉的方法，即使用对称的生色团，因此在静息电位下没有 SHG，但通过动作电位将其转化为具有大 SHG 信号和原位敏感性的不对称生色团。实验上对这种对称性破缺的了解表明，有可能将发色团综合调整到对称性破缺的边缘，以便可以通过外部刺激（例如膜电位的变化）来诱导该过程。该项目将结合量子化学建模、计算引导的与生物介质兼容的发色团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缺和二次谐波响应。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合成。哥伦比亚大学 Rafael Yuste 实验室将在活神经元中对被确定为二次谐波成像良好候选者的发色团进行研究。尽管电压相关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，因此虽然存在重大风险，但也存在与 t 相关的潜在转化奖励。该项目的最终目标是提供一类具有更高灵敏度的新型发色团，以便广大神经科学研究人员能够实际且常规地使用 SHG 成像来绘制神经回路的功能图。

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.