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

利用荧光显微镜进行快速准确的基准标记跟踪，用于机械生物学实验

• 批准号: RTI-2023-00499
• 负责人: Harris, Andrew
• 金额: $ 10.93万
• 依托单位: Carleton University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=759509
• 关键词: Fast; Accurate; Fiducial; Marker; Tracking

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.

正在请求设备来支持卡尔顿大学的研究活动，重点是了解机械应变如何由生物组织维持和传播。该设备将能够对嵌入软组织样本上的荧光基准标记进行快速三维 (3D) 跟踪，以响应机械刺激，并具有前所未有的空间分辨率 (~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机械特性的关键结构以及轻度创伤性脑损伤和皮肤病中的组织损伤机制。设备的速度和分辨率是通过点扩散函数工程模块（双螺旋相位掩模）和高速相机（Photometrics Kinetix）来实现的。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，因此测量速度比以前快得多（约秒）。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个新概念，并具有细胞和组织力学领域快速发展的潜力，在这些领域中高应变率测量一直具有挑战性。此外，该系统还提供了执行 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。这种点扩散函数工程正在迅速应用于超分辨率成像，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与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 个研究小组和他们的总部。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，组成了一个理想的团队，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拥有丰富的经验，因此该设备将立即有效地使用。为了继续参与国际竞争并为总部提供适当的培训，持续获得跨学科工具（例如尖端光学显微镜和样品制备技术）至关重要，否则将很难在我们的研究界保持受人尊敬的地位。三名目前的研究生和两名本科生研究人员将立即从该设备的使用中受益，随着所需设备能够促进新的研究方向的发展，预计这一数字还会增加。