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

该项目的目标是开发一种新仪器，用于同时测量活细胞中细胞骨架蛋白网络的结构和机械特性。 振荡光镊细胞流变仪 (OOTC) 的工作原型已经由先前 NSF-SGER 资助支持的工作开发出来。目前的项目将通过将现有的 OOTC 与奥林巴斯转盘共焦显微镜集成来实施。最近，使用光镊测量细胞的机械性能已经取得了重大进展。所提出的方法，振荡光镊细胞流变仪（OOTC），利用锁定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。经证明，OOTC 可以在 0.1-6,000 Hz 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 um3。更重要的是，OOTC 能够区分内在的非随机时间变化和布朗运动引起的随机波动；这种通过传统方法无法实现的能力特别有用，因为生命系统是高度动态的，并且经常表现出非热、有节奏的行为。然而，尽管 OOTC 能力很强，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“盲人摸象”那样信息丰富。同步和原位测量至关重要，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焦荧光显微镜可以详细地生成活细胞的细胞骨架结构和细胞质成分的 3 维图像。将 OOTC 与共焦显微镜集成将为 OOTC 提供眼睛，因此我们不仅可以感觉到，还可以同时看到细胞结构。开发的仪器将供物理、生物系和生物工程项目的本科生和研究生用于教育和研究。校内外的研究人员也可以使用该仪器来研究细胞和组织力学问题。该项目的目标是开发一种新仪器，用于同时测量活细胞中细胞骨架蛋白网络的结构和机械特性。振荡光镊细胞流变仪 (OOTC) 的工作原型已经由先前 NSF-SGER 资助支持的工作开发出来。目前的项目将通过将现有的 OOTC 与奥林巴斯转盘共焦显微镜集成来实施。光镊由聚焦激光束形成，用于固定和操纵生物细胞中的微小细胞器，已发现可有效用于测量细胞内机械特性。该方法采用独特的锁定信号检测方案，类似于汽车收音机接收远处广播电台发送的音乐的方法，为测量细胞内部的机械特性提供了前所未有的灵敏度、时间分辨率和简单性，同时对活细胞的侵入最小。然而，尽管 OOTC 能力很强，除非人们能够在原位“看到”细胞骨架结构，否则机械性能数据将像“盲人摸象”一样提供丰富的信息。先进的荧光共焦成像技术使得生成活细胞细胞骨架结构的详细 3 维图像成为可能。将 OOTC 与共焦显微镜集成将为 OOTC 提供眼睛，因此我们不仅可以感觉到，还可以同时看到细胞结构。开发的仪器将供物理、生物系和生物工程项目的本科生和研究生用于教育和研究。校内外的研究人员也可以使用该仪器来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.