Towards In Vivo Force Spectroscopy Using Optomagnetic Tweezers

描述（由申请人提供）：单分子力谱（SMFS）用于研究驱动细胞和蛋白质生物活性的基本力。几乎所有这些生物物理测量都是使用纯化的生物分子在体外进行的。当研究细胞的功能和机制时，这种方法由于未能考虑细胞内环境的影响而产生固有的不完整结果。因此，开发能够在体内传感的力谱系统将提高生物物理测量能力，并有助于澄清驱动细胞功能的相互作用的起源。更广泛地说，对细胞过程的更完整的理解将有助于研究人员发现和开发包括癌症在内的各种疾病的治疗方法。 该提案概述了一种新型仪器的开发，该仪器结合了光学和磁性镊子的操纵能力，以促进高分辨率定位以及在活细胞内应用相对较大范围的力。为了实现这种类型的仪器，光学镊子系统将被改造为能够产生可控旋转磁场的磁性装置。磁各向异性的Janus球，直径小于200纳米，将被制造作为探针，可以被光学捕获和磁驱动。它们的大小将通过内吞作用促进细胞摄取。一旦进入细胞内部，红外激光束或超快激光脉冲与外部磁致动相结合，将使探针运动性和力的应用不会损害细胞的健康。先进的伺服控制方案将允许精确施加力和测量细胞内力学。最初将探针插入活粒细胞中以评估仪器性能。一旦参数得到优化，体内力谱仪将提供给一般的研究团体，用于表征无数的细胞系。 公共卫生相关性（由申请人提供）：细胞是人类生命的基本功能单位，但其许多内部和外部相互作用仍然是神秘的。 通过插入细胞的显微探针施加力和扭矩，我们的目标是量化和操纵这些相互作用。准确的定量将为细胞过程提供新的见解，最终可用于帮助理解驱动包括癌症在内的各种疾病的机制。

DESCRIPTION (provided by applicant): Single molecule force spectroscopy (SMFS) is used to study the fundamental forces that drive the biological activity of cells and proteins. Nearly al of these biophysical measurements are performed in vitro using purified biomolecules. When studying the function and mechanics of cells, this approach yields inherently incomplete results by failing to account for the influence of the intracellular environment. Therefore, the development of a force spectroscopy system capable of in vivo sensing will advance biophysical measurement capability and help clarify the origins of the interactions driving cellular function. Broadly, a more complete understanding of cellular processes will aid researchers in the discovery and development of treatments for various disorders including cancer. This proposal outlines the development of a novel instrument that combines the manipulation capabilities of optical and magnetic tweezers to facilitate high resolution positioning as well as the application of a relatively large range of forces inside a living cell. To realize an instrumen of this type, an optical tweezers system will be retrofitted with a magnetic apparatus capable of generating controllable rotating magnetic fields. Magnetically anisotropic Janus spheres, less than 200 nm in diameter, will be fabricated to serve as probes that can be optically trapped and magnetically actuated. Their size will facilitate cellular uptake via endocytosis. Once inside the cell infrared laser beams or ultrafast laser pulses combined with external magnetic actuation will enable probe motility and force application without compromising the health of the cell. Advanced servo control schemes will allow the accurate application of forces and measurement of intracellular mechanics. Initially probes will be inserted into live granulocytes to access instrument performance. Once parameters are optimized the in vivo force spectroscopy instrument will be made available to the general research community for the characterization of a myriad of cell lines. PUBLIC HEALTH RELEVANCE (provided by applicant): The cell is the fundamental functional unit of human life yet many of its internal and external interactions remain mysterious. Through the application of forces and torques via microscopic probes inserted into the cell, we aim to quantify and manipulate these interactions. Accurate quantification will provide new insight into cellular processes, which could eventually be used to help understand the mechanisms driving various diseases including cancer.