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

本项目的目标是开发一种新的仪器，用于同时测量活细胞中细胞骨架蛋白网络的结构和力学特性。一个工作原型振荡光镊细胞热计（OOTC）已经从先前的NSF-SGER资助的工作中开发出来。本项目将通过将现有的OOTC与奥林巴斯旋转盘共聚焦显微镜相结合来实施。近年来，利用光学镊子测量细胞的力学性能已经取得了重大进展。所提出的振荡光镊细胞流速计（OOTC）方法利用锁相放大器对谐波调制粒子运动的相干检测来提高灵敏度、时间分辨率和简单性。研究表明，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力学模量，并具有1 um3的空间清晰度。更重要的是，OOTC能够区分固有的非随机时间变化和布朗运动引起的随机波动；这种能力是传统方法无法实现的，因为生命系统是高度动态的，经常表现出非热的、有节奏的行为，所以这种能力特别有用。然而，尽管OOTC很有能力，除非我们能同时在原位测量细胞骨架结构，否则力学性能数据将与“盲人和大象”的数据一样有信息量。同时和原位测量是至关重要的，因为聚合蛋白网络会在几分之一秒到几小时或几天的时间尺度上改变形状和重组结构。先进的光学荧光成像技术使得通过共聚焦荧光显微镜可以非常详细地产生活细胞的细胞骨架结构和细胞质成分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，这样我们不仅可以感受到而且可以同时看到细胞结构。所开发的仪器将被物理系、生物系和生物工程专业的本科生和研究生用于教育和研究。校园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。本项目的目标是开发一种新的仪器，用于同时测量活细胞中细胞骨架蛋白网络的结构和力学特性。一个工作原型振荡光镊细胞热计（OOTC）已经从先前的NSF-SGER资助的工作中开发出来。本项目将通过将现有的OOTC与奥林巴斯旋转盘共聚焦显微镜相结合来实施。光镊是由聚焦的激光束形成的，用来握住和操纵生物细胞中的微小细胞器，已经发现了测量细胞内机械特性的有效用途。使用一种独特的锁定信号检测方案，一种类似于汽车收音机接收远处无线电台发送的音乐的方法，该方法提供了前所未有的灵敏度、时间分辨率和简单性，可以测量细胞内部的机械特性，同时对活细胞的入侵最小。然而，尽管OOTC很有能力，除非有人能“看到”原位的细胞骨架结构，否则力学性能数据将与“盲人和大象”的数据一样有用。先进的荧光共聚焦成像技术已经可以非常详细地产生活细胞的细胞骨架结构的三维图像。将OOTC与共聚焦显微镜相结合将为OOTC提供眼睛，这样我们不仅可以感受到而且可以同时看到细胞结构。所开发的仪器将被物理系、生物系和生物工程专业的本科生和研究生用于教育和研究。校园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.