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

卡尔顿大学正在申请设备来支持研究活动，重点是了解机械应变是如何被生物组织维持并通过生物组织传播的。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（~20nm）和时间分辨率（~2ms）。从该实验系统中获得的见解将有助于更好地理解决定组织力学特性的关键结构以及轻度创伤性脑损伤和皮肤病组织损伤的机制。设备的速度和分辨率是通过点扩展函数工程模块（双螺旋相位掩模）和高速相机（Photometrics Kinetix）实现的。该系统允许测量荧光基准标记的3D位移，而无需物理调整显微镜的焦平面，因此测量可以比以前实现的更快（~秒）。使用这种新技术来测量切片组织在机械刺激下的变形是一个新概念，具有快速发展细胞和组织力学领域的潜力，在这些领域，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，作为最近资助的全内反射荧光显微镜的增强，该显微镜将安装在卡尔顿大学。这种点扩展函数工程在超分辨率成像中被迅速采用，但目前在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 hub）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。申请者是组织工程、材料力学和光学显微镜等协同领域的专家，组成一个理想的团队，充分利用所要求的设备。首席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，因此该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（如尖端光学显微镜和样品制备技术）是必不可少的，没有这些工具，HQP将很难在我们的研究界保持受人尊敬的地位。三名在读研究生和两名本科生研究人员立即受益于该设备的使用，随着所要求的设备使新的研究方向得以发展，预计这一数字将会增长。

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.