Collaborative Research: Development of a Novel Multiphoton Microscopefor Measuring Biomolecular Dynamics Over 15 Orders of Magnitude in Time

该奖项是针对与DBI-0454763的合作。它支持一种新型多光子显微镜的开发，这种显微镜将能够在时间上测量超过15个数量级的生物分子动力学。该显微镜将使用微流体技术来提供微秒级时间尺度的混合，以及低（ml/小时）的样品消耗率。微秒到秒的时间尺度延迟将通过使用显微镜沿着出口通道在增加的距离（对应于增加的时间）上探测样品流来实现。光学系统将实现三脉冲回波峰移光谱（3PEPS），这是一种四波混合方法，已被证明可用于测量含协因子蛋白质波动的光谱。该显微镜最初将用于两方面的工作：1)研究蛋白质折叠过程中的柔韧性和异质性的演变；2)表征光活性黄色蛋白在光循环中的中间物种动力学。在广泛的时间范围内直接测量生物分子的分子运动对于进一步从微观上理解生物学至关重要。为了充分了解分子运动和生化事件之间的关系，必须记录分子动力学的时间尺度是巨大的——从飞秒（10-15秒）到秒甚至分钟。这项合作工作汇集了光学物理学家、化学工程师和生物物理学家的专业知识，以开发将推进生物分子动力学知识的仪器。这种新型仪器将促进强大的飞秒非线性光学技术在生物化学研究中的广泛应用，并将进一步发展在生物研究中有很大前景的技术：多光子显微镜和微流体。这项研究将为学生提供在包括分子生物学家、化学工程师和光学物理学家在内的多学科团队中工作的机会，从而有利于本科和研究生层次的跨学科生物物理学教育。

This award is for a collaboration with DBI-0454763. It supports the development of a novel multiphoton microscope that will be capable of measuring biomolecular dynamics over 15 orders of magnitude in time. The microscope will use microfluidics technology to provide microsecond time-scale mixing, and low (ml/hour) sample consumption rates. Microsecond to second time scale delays will be achieved by using a microscope to probe the sample flow at increasing distances (corresponding to increasing time) along the outlet channel. The optical system will implement three pulse echo peak shift spectroscopy (3PEPS), a four-wave mixing method that has proven useful for measuring the spectrum of fluctuations of cofactor-containing proteins. The microscope will initially be employed in two lines of work: 1) studying the evolution of flexibility and heterogeneity during protein folding and 2) characterizing the dynamics of intermediate species in the photocycle of photoactive yellow protein. Direct measurements of the molecular motions of biomolecules over a broad time range is crucial to furthering a microscopic understanding of biology. To fully appreciate the relation between molecular motions and biochemical events, the time scale over which molecular dynamics must be recorded is enormous - from femtoseconds (10-15 seconds) to seconds or even minutes. This collaborative work brings together the expertise of an optical physicist, chemical engineer, and biophysicist, to develop instrumentation that will advance knowledge in biomolecular dynamics. This new instrumentation will stimulate broader application of powerful femtosecond nonlinear optical techniques to biochemical studies, and will further develop technologies with great promise in biological studies: multiphoton microscopy and microfluidics. This research will benefit interdisciplinary biophysics education at the undergraduate and graduate levels by providing opportunities for students to work in multidisciplinary teams including molecular biologists, chemical engineers, and optical physicists.