Fast and Accurate Fiducial Marker Tracking with Fluorescence Microscopy for Mechanobiology Experiments

正在请求提供设备，以支持卡尔顿大学的研究活动，重点是了解生物组织如何承受和传播机械应变。该设备将能够快速三维（3D）跟踪嵌入在软组织标本上的荧光基准标记，以响应机械刺激，具有前所未有的空间（~20 nm）和时间分辨率（~2 ms）水平。从这个实验系统中获得的见解将导致更好地理解的关键结构，确定组织的机械性能和组织损伤的机制，在轻度创伤性脑损伤和皮肤病。该设备的速度和分辨率通过点扩散函数工程模块（双阻相位掩模）和高速相机（Photometrics Kinetix）实现。该系统允许荧光基准标记物的3D位移被测量，而无需物理地调整显微镜的焦平面，因此测量可以比以前实现的（~秒）快得多。使用这种新技术来测量切片组织响应机械刺激的变形是一种新概念，并具有快速推进细胞和组织力学领域的潜力，其中高应变率测量一直具有挑战性。此外，该系统提供了执行3D超分辨率成像的机会，作为对最近资助的将安装在卡尔顿大学的全内反射荧光显微镜的增强。这种类型的点扩散函数工程正迅速被用于超分辨率成像，但目前加拿大可用的设备有限。所要求的设备将直接支持卡尔顿大学组织力学相关的广泛研究工作，包括组织工程和应用材料（TEAM中心）和渥太华卡尔顿生物医学工程研究所（OCIBME），因此将立即使5个研究小组及其HQP受益。申请人是组织工程，材料力学和光学显微镜协同领域的专家，形成一个理想的团队将充分利用所需的设备。主要申请人在单分子成像、细胞和组织力学方面具有丰富的经验，因此设备将立即有效地使用。为了继续在国际上竞争并为HQP提供适当的培训，持续获得跨学科工具，如尖端的光学显微镜和样品制备技术是必不可少的，没有这些工具，我们很难在研究界保持令人尊敬的地位。目前有三名研究生和两名本科生研究人员将立即从使用这一设备中受益，随着所要求的设备能够开发新的调查路线，这一数字预计将增加。

Equipment is being requested to support research activities at Carleton University focused on understating how mechanical strain is sustained by and transmitted through biological tissues. The equipment will enable rapid three-dimensional (3D) tracking of fluorescent fiducial markers embedded on soft tissue specimens in response to mechanical stimulation with unprecedented levels of both spatial (~20nm) and temporal resolution (~2ms). Insights gained from this experimental system will lead to a better understanding of the key structures that determine tissue mechanical properties and the mechanisms of tissue injury in mild traumatic brain injury and dermopathies. The speed and resolution of the equipment is achieved through a point spread function engineering module (Double Helix phase mask) and a high-speed camera (Photometrics Kinetix). This system allows 3D displacements of fluorescent fiducial markers to be measured without physically adjusting the focal plane of the microscope, and therefore measurements can be made much faster than previously attainable (~seconds). Using this new technology to measure the deformation of sliced tissue in response to mechanical stimulation is a new concept and holds the potential for rapid advancement of the cell and tissue mechanics fields, where high strain rate measurements have been challenging. In addition, this system offers the opportunity to perform 3D super-resolution imaging as an enhancement to a recently funded Total Internal Reflection Fluorescence microscope that will be installed at Carleton University. Point spread function engineering of this kind is rapidly being adopted for super-resolution imaging, but there is currently limited equipment available in Canada. The requested equipment will directly support broad research efforts pertaining to tissue mechanics at Carleton University including the Tissue Engineering and Applied Materials (TEAM hub) and the Ottawa Carleton Institute for Biomedical Engineering (OCIBME) and therefore will be of immediate benefit to 5 research groups and their HQP. The applicants are experts in the synergistic fields of tissue engineering, material mechanics and optical microscopy, forming an ideal team will make full use of the requested equipment. The lead applicant has extensive experience in single molecule imaging, cell, and tissue mechanics and therefore the equipment will be used immediately and effectively. To continue to compete internationally and provide proper training to HQP, sustained access to interdisciplinary tools such as cutting-edge optical microscopy and sample preparation techniques are essential, without which it will be hard to maintain a respectable standing within our research community. Three current graduate students and two undergraduate researchers stand to benefit immediately from the use of this equipment, a number that is anticipated to grow as the requested equipment enables new lines of inquiry to develop.