Nanoscope for Individual Molecules in Biophysics and Biotechnology

许多基本的生物信号，从DNA转录到对感染的免疫反应，都可以被单个或几个分子或细胞内激活和控制，因此，测量单个生物分子在体内和体外的动态功能对于有效的生物物理研究是必要的。该奖项的目的是创建有效的仪器来观察和理解这些敏感的生命过程，并作为副产品来提高分析微芯片生物技术的分子灵敏度。需要对单个生物分子进行纳米精度的非侵入性光学检测和定位，并测量体内或体外单个分子功能的动力学。荧光的光学显微成像是一种很有前途的方法，但单分子过程是微不足道的，尽管多年来在特殊情况下已经可以检测到单个分子。该奖项的目的是开发一种用户友好的、良性的光学纳米显微镜，用于在活细胞上进行纳米级的单分子检测和定位，用于生物物理研究。最近对单个分子荧光的研究揭示了在单分子水平上混淆了许多生物物理应用的光物理病理学，但幸运的是，已经发现了有希望的研究方法，可能会产生令人满意的光学探针。光学纳米技术的发展依赖于多光子激光显微镜(MPM)和荧光相关光谱(FCS)。通过精确的纳米结构制备、新的优化光学技术和新的软件开发，将这些方法结合起来，为设计和构建专门设计的用于优化单分子动力学测量的光学纳米显微镜复合体提供了基础，该复合体使用FCS和MPM成像进行细胞和组织结构的纳米分子定位。将购买的设备包括开发、建造和优化光学纳米显微镜所需的部件，时间约为三年。单分子光学探测器的发展利用光物理仪器测量从数百皮秒到数百秒的光学动力学，时间相关的单光子计数、荧光相关光谱、光子爆发分析和光谱记录，所有这些都是在单分子水平上的。人力资源开发是这一计划的固有副产品，通过对参与的博士后和博士后学生以及几个学科的访问科学家进行跨学科培训。根据我们过去的经验，他们可以成为有效的生物物理研究人员，作为学术人员、技术管理人员和研发领导者。

Many essential biological signals, ranging from DNA transcription to immunological responses to infection, can be activated and controlled by a single or a few molecules or in the cell; therefore, the capability for measurement of dynamical functions of individual biomolecules in vivo and in vitro is necessary for effective biophysical research. The objective of this award is to create effective instrumentation for observing and understanding these sensitive life processes, and as a byproduct to enhance the molecular sensitivity of analytical microchip biotechnology. Non-invasive optical detection of individual biomolecules and location with nanometer precision and measurement of the dynamics of individual molecular functions in vivo or in vitro is needed. Optical microscopic imaging of fluorescence is a promising approach, but access to single molecule processes is tenuous, although individual molecules have been detectable in special cases for many years. The aim of this award is to develop a user-friendly, benign optical nanoscope for single molecule detection and localization at nanometer scales on living cells for biophysical research. Recent research on fluorescence of individual molecules has revealed photophysical pathology that confounds many biophysical applications at the single molecule level, but fortunately promising research approaches have been discovered that may yield satisfactory optical probes. The optical nanoscope development relies on multiphoton laser microscopy (MPM) and fluorescence correlation spectroscopy (FCS). Combining these methods enhanced by precision nanostructure fabrications, new optimized optics and new software developments provides the basis to design and construct a purpose-designed optical Nanoscope complex for optimized single molecule dynamical measurements using FCS with MPM imaging for nanometer molecular localization in cellular and tissue structures. The equipment to be purchased consists of the components required to develop, construct and optimize the optical nanoscope on a timeline of about three years. The development of single molecule optical probes utilizes photophysical instrumentation to measure optical dynamics from hundreds of picoseconds to hundreds of seconds with time correlated single photon counting, fluorescence correlation spectroscopy, photon burst analysis, and spectral recording, all at the single molecule level. Human resource development comprises an inherent byproduct of this program through interdisciplinary training of participating predoctoral and postdoctoral students and visiting scientists in several disciplines. They can be expected on the basis of our past experience to become effective biophysical researchers as academic faculty, technology managers, and research and development leaders.