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

该奖项支持开发一种新型多光子显微镜，该显微镜将能够在15个数量级的时间内测量生物分子动力学。该显微镜将使用微流控技术提供微秒级时间尺度的混合，以及低(毫升/小时)的样品消耗率。通过使用显微镜探测沿出口通道增加的距离(对应于增加的时间)的样品流，将实现微秒到秒的时间尺度延迟。该光学系统将实施三脉冲回波峰移光谱(3PEPS)，这是一种四波混频方法，已被证明对测量含有辅因子的蛋白质的波动光谱很有用。该显微镜最初将用于两个方面的工作：1)研究蛋白质折叠过程中柔韧性和异质性的演变；2)表征光活性黄色蛋白质光循环中中间物种的动力学。直接测量生物分子在广泛时间范围内的分子运动对于加深对生物学的微观理解至关重要。为了充分理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)到几秒甚至几分钟。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，以开发将促进生物分子动力学知识的仪器。这一新的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更广泛应用，并将进一步开发在生物学研究中前景广阔的技术：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，从而有利于本科生和研究生水平的跨学科生物物理学教育。

This award 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.