Developmental Resource for Biophysical Imaging and Opto- Electronics

“生物物理成像和光电开发资源”的全球使命包括创建，开发，促进和应用新的仪器技术，用于动态细胞和分子过程的可视化和测量，最终目标是解决生物研究中的基本问题。激光物理、非线性光学、微电子和计算机技术的进步推动了光学显微镜的复兴，产生了强大的新型光学和微探针技术。近年来，仪器研究和发展已经产生了基于双光子激发（TPE）的非线性激光显微镜，它是通过同时吸收两个低能光子的分子来产生产生荧光或光化学的高能激发。这项资源发明的动机是需要瞬时空间分辨光化学以及三维分辨，无背景荧光显微镜。这项研究计划的大部分目的是实现这项发明的潜力，并通过探索性的生物物理应用建立其方法和效用。我们特别提出了针对活体神经系统制剂的动态测量的发展，如通过笼化分子点光解的神经递质微药理学，快速（视频速率）比例钙成像，以及厚的，高度散射的生物标本的方法优化。资源开发包括纳米空间位移测量，单个分子的纳米尺度跟踪，以及最近的一种皮牛顿灵敏度光学力探针，其在扫描模式下运行，通过光学力显微镜（OFM）提供表面映射。这些仪器为细胞表面受体动力学、感觉转导、细胞运动机制和第二信使信号机制的生物物理学研究提供了新的资源。多路仪器的资源开发，以适应这些探针的组合，原子力显微镜（较大的力）和荧光成像和分子跟踪活细胞。利用这些新技术，研究目标是在体内测量和绘制皮牛顿相互作用力、配体-受体对的动力学、细胞表面顺应性和个体分子间力。具体研究目标：(1)双光子激光扫描显微镜（TPLSM）技术——设计开发具有荧光衰减时间成像、视频速率比例成像、可编程笼状神经递质释放和单粒子跟踪功能的TPLSM研究工作站。(2)激光显微镜和双光子激发的探索性应用——开发新的仪器技术来利用TPLSM的优势，特别是(a)厚、强散射、活制剂的技术，(b)微药理学的局部快速笼激活技术，(c)神经系统制剂的特定应用，以及(d)植物组织的特定应用。(3)光化学和光物理——细胞环境中的激光激发——确定光化学过程的机制和控制，这些过程最终限制了活细胞中单光子和双光子荧光显微镜的有效性。了解单光子和双光子光漂白、细胞光损伤、光消融和笼活化动力学的作用机制。应用光消融和笼活化生物系统。(4)非线性激光显微镜测量分子迁移率——开发使用双光子激发荧光光漂白恢复（FPR）、荧光活化再分配（FAR）和荧光相关光谱（FCS）进行三维分辨分子迁移率测量的仪器和技术。适用于第二信使反应、受体-分泌偶联和神经递质释放的光调节。(5)利用荧光相关光谱（FCS）测量蛋白质动力学和折叠——利用FCS在空间定域双光子激发的辅助下作为蛋白质折叠和动力学的一种新的快速探针。(6)纳米显微镜和皮牛顿力——开发用于扫描细胞表面受体相互作用的多模纳米仪器和技术，以及用于低至皮牛顿水平的力-速度关系的实际测量。将光学力显微镜（OFM）与光学镊子操作、纳米分子跟踪、荧光成像、原子力显微镜（AFM）和可能的近场荧光激发相结合。

The global mission of the "Developmental Resource for Biophysical Imaging and Opto-electronics" consists of the creation, development, facilitation and application of new instrumentation technologies for the visualization and measurement of dynamic cellular and molecular processes with the ultimate objective of solving fundamental problems in biological research. A renaissance in light microscopy, driven by advances in laser physics, nonlinear optics, microelectronics and computer technology has engendered powerful new optical and microprobe technologies. Instrumentation research and development has recently produced nonlinear laser microscopy based on two photon excitation (TPE) which occurs by simultaneous molecular absorption of two low energy photons to create a high energy excitation that generates fluorescence or photochemistry. This Resource invention was motivated by a need for instantaneous spatially resolved photochemistry as well as three dimensionally resolved, background-free fluorescence microscopy. Much of this research program aims to realize the potential of this invention and to establish its methodology and utility through exploratory biophysical applications. We propose developments aimed particularly at dynamical measurements in living nervous system preparations such as neurotransmitter micropharmacology by point photolysis of caged molecules, fast (video rate) ratiometric calcium imaging, and optimization of methods for thick, highly scattering biological specimens. Resource developments include nanometer spatial displacement measurements, nanometer scale tracking of individual molecules, and most recently a picoNewton-sensitivity optical force probe whose operation in a scanning mode provides surface mapping by Optical Force Microscopy (OFM). These instruments are enabling new Resource biophysics research on cell surface receptor dynamics, sensory transduction, cell motility mechanisms, and second messen ger signalling mechanisms. Resource development of multiplexed instrumentation that accommodates cmmbinations of these probes, atomic force microscopy (for larger forces) and fluorescence imaging and molecular tracking on living cells is proposed. Using these novel technologies, research objectives are the in vivo measurement and mapping of picoNewton interaction forces, dynamics of ligand-receptor pairs, cell surface compliances and individual intermolecular forces. Specific Research Objectives: (1) Technology of two photon laser scanning microscopy (TPLSM) -- Design and develop TPLSM research workstations capable of fluorescence decay time imaging, video rate ratiometric imaging, programmable caged neurotransmitter release and single particle tracking. (2) Exploratory applications of laser microscopy and two photon excitation -- Develop new instrumentation techniques to exploit the advantages of TPLSM, in particular (a) techniques for thick, strongly scattering, living preparations, (b) localized fast cage activation for micropharmacology, (c) specific applications to nervous system preparations, and (d) specific applications to plant tissue. (3) Photochemistry and photophysics -- laser excitation in the cellular environment -- Determine mechanisms and controls of photochemical processes that ultimately limit the effectiveness of both one and two photon fluorescence microscopy in living cells. Understand operative mechanisms of one and two photon photobleaching, cellular photodamage, photoablation and the kinetics of cage activation. Apply photoablation and cage activation to biological systems. (4) Nonlinear laser microscopy measurements of molecular mobility -- Develop instrumentation and techniques for 3-d resolved molecular mobility measurements using two photon excited fluorescence photobleaching recovery (FPR), Fluorescence Activation Redistribution (FAR), and Fluorescence Correlation Spectroscopy (FCS). Apply to photoregulation of second messenger responses, receptor-secretion coupling, and neurotransmitter release. (5) Measurement of Protein Dynamics and Folding by Fluorescence Correlation Spectroscopy (FCS) -- Apply FCS aided by spatially localized two photon excitation as a new fast probe of protein folding and dynamics. (6) Nanometer microscopy and picoNewton forces -- Develop multimode nanometer instrumentation and techniques for scanning cell surface receptor interactions and for practical measurements of force-velocity relations down to the picoNewton level. Combine Optical Force Microscopy (OFM) with optical tweezers manipulation, nanometer molecular tracking, fluorescence imaging, atomic force microscopy AFM), and possible near field fluorescence excitation.