MRI: Development of an Optical Tweezers-based Time-Lapse 3D Imaging Cytorheometer

本计画的目标是发展一种新的仪器，以同时量测活细胞中细胞骨架蛋白质网络的结构与力学性质。 振荡光镊细胞流变仪（OOTC）的工作原型已经从先前的NSF-SGER赠款支持的工作中开发出来。目前的项目将通过整合现有的OOTC与奥林巴斯旋转圆盘共聚焦显微镜。利用光镊技术测量细胞的力学性质，近年来取得了重大进展。所提出的方法，振荡光镊细胞流变仪（OOTC），利用相干检测谐波调制粒子运动的锁定放大器，以增加灵敏度，时间分辨率和简单性。已经证明，OOTC可以在0.1- 6,000 Hz的频率范围内以每秒100个数据的速度测量动态机械模量，空间分辨率为1 um 3。更重要的是，OOTC能够区分固有的非随机时间变化与随机波动，由于布朗运动;这种能力，不能通过传统的方法实现，是特别有用的，因为生活系统是高度动态的，往往表现出非热的，有节奏的行为。然而，尽管OOTC很有能力，但除非我们能够同时原位测量细胞骨架结构，否则力学性能数据将像“盲人与大象”一样提供信息。同时和原位测量是至关重要的，因为聚合蛋白质网络在几分之一秒到几小时或几天的时间尺度内改变形状并重组其结构。先进的光学荧光成像技术使得通过共聚焦荧光显微镜能够产生活细胞的细胞骨架结构和细胞质组分的非常详细的三维图像。将OOTC与共聚焦显微镜相结合将为OOTC提供眼睛，因此我们不仅可以感觉到而且可以同时看到细胞结构。所开发的仪器将用于物理，生物系和生物工程课程的教育和研究的本科生和研究生。校内外的研究人员也可以使用该仪器研究细胞和组织力学问题。本计画的目标是发展一种新的仪器，以同时量测活细胞中细胞骨架蛋白质网络的结构与力学性质。振荡光镊细胞流变仪（OOTC）的工作原型已经从先前的NSF-SGER赠款支持的工作中开发出来。目前的项目将通过整合现有的OOTC与奥林巴斯旋转圆盘共聚焦显微镜。利用聚焦激光束形成的光镊可以固定和操纵生物细胞中的微小细胞器，是测量细胞内力学性质的有效工具。使用一种独特的锁定信号检测方案，这种方法类似于汽车收音机如何拾取远处电台发送的音乐，所提出的方法提供了前所未有的灵敏度，时间分辨率和简单性，用于测量细胞内部的机械特性，对活细胞的侵入最小。然而，尽管OOTC很有能力，但除非人们能够“看到”原位的细胞骨架结构，否则机械性能数据将像“盲人与大象”一样提供信息。先进的荧光共聚焦成像技术已经使得产生活细胞的细胞骨架结构的非常详细的三维图像成为可能。将OOTC与共聚焦显微镜相结合将为OOTC提供眼睛，因此我们不仅可以感觉到而且可以同时看到细胞结构。所开发的仪器将用于物理，生物系和生物工程课程的教育和研究的本科生和研究生。校内外的研究人员也可以使用该仪器研究细胞和组织力学问题。

The goal of this project is to develop a new instrument for simultaneous measurements of the structural and the mechanical properties of cytoskeletal protein network in living cells. A working prototype oscillating optical tweezer cytorheometer (OOTC) has already been developed from a work supported by a prior NSF-SGER grant. The present project will be implemented by integrating the existing OOTC with an Olympus spinning disk confocal microscope. The use of optical tweezers to measure mechanical properties of cells has been making significant processes recently. The proposed approach, Oscillating Optical Tweezer Cytorheometer (OOTC), takes advantage of the coherent detection of harmonically modulated particle motions by a lock-in amplifier to increase sensitivity, temporal resolution and simplicity. It has been demonstrated that OOTC can measure the dynamic mechanical modulus in the frequency range of 0.1- 6,000 Hz at a rate as fast as 100 data per second with 1 um3 spatial definition. More importantly, OOTC is capable of distinguishing the intrinsic non-random temporal variations from random fluctuations due to Brownian motion; this capability, not achievable by a conventional approach, is particular useful because living systems are highly dynamic and often exhibit non-thermal, rhythmic behavior. However, as capable as is OOTC, unless we can simultaneously measure the cytoskeletal structures in situ, the mechanical properties data would be as informative as that of "Blind men and the Elephant". Simultaneous and in situ measurements are critical because the polymeric protein network changes shape and reorganizes its structure in time scales from a fraction of a second to hours or days. Advanced optical fluorescent imaging techniques have made it possible to produce 3 dimensional images of the cytoskeletal structure and cytoplasmic components of living cells in great detail by confocal fluorescent microscopy. Integrating OOTC with a confocal microscope will provide eyes to OOTC so we not only feel but also see the cellular structures at the same time. The developed instrument will be used by both undergraduate and graduate students from the physics, biology departments and bioengineering program for education and research. Researchers on and off campus can also use the instrument for studying cell and tissue mechanics problems. The goal of this project is to develop a new instrument for simultaneous measurements of the structural and the mechanical properties of cytoskeletal protein network in living cells. A working prototype oscillating optical tweezer cytorheometer (OOTC) has already been developed from a work supported by a prior NSF-SGER grant. The present project will be implemented by integrating the existing OOTC with an Olympus spinning disk confocal microscope. Optical tweezers, formed by focused laser beam to hold and manipulate minute organelles in biological cells, have found effective use for measuring intracellular mechanical properties. Using an unique lock-in signal detection scheme, an approach similar to how a car radio pick up music sent by a distant radio station, the proposed approach provides unprecedented sensitivity, temporal resolution and simplicity for measuring the mechanical properties of the cell interior with minimal invasion to living cells. However, as capable as is OOTC, unless one can "see" the cytoskeletal structures in situ, the mechanical properties data would be as informative as that of "Blind men and the Elephant". Advanced fluorescent confocal imaging techniques have made it possible to produce 3 dimensional images of the cytoskeletal structure of living cells in great detail. Integrating OOTC with a confocal microscope will provide eyes to OOTC so we not only feel but also see the cellular structures at the same time. The developed instrument will be used by both undergraduate and graduate students from the physics, biology departments and bioengineering program for education and research. Researchers on and off campus can also use the instrument for studying cell and tissue mechanics problems.