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.

要求设备支持卡尔顿大学的研究活动，专注于低估机械应变如何通过生物组织维持和传播。该设备将使嵌入在软组织样本上的荧光基准标记的快速三维（3D）跟踪，响应于空间（〜20nm）和时间分辨率（〜2ms）的机械刺激响应于机械刺激。从这个实验系统中获得的见解将使人们更好地了解确定组织机械性能的关键结构以及轻度脑损伤和皮肤病中组织损伤的机制。设备的速度和分辨率是通过点扩展功能工程模块（双螺旋相掩码）和高速摄像头（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，因此可以比以前可实现的速度快得多（〜秒）。使用这项新技术来响应机械刺激的切片组织的变形是一个新概念，并具有快速发展细胞和组织力学领域的潜力，在高应变速率测量方面是具有挑战性的。此外，该系统提供了执行3D超分辨率成像的机会，以增强卡尔顿大学将安装的最近资助的总内反射荧光显微镜。高分辨率成像迅速采用了这种类型的点传播功能工程，但目前在加拿大可用的设备有限。要求的设备将直接支持与卡尔顿大学组织力学有关的广泛研究工作，包括组织工程和应用材料（团队中心）和渥太华卡尔顿生物医学工程研究所（OCIBME），因此将对5个研究小组及其HQP及其HQP有效。申请人是组织工程，材料力学和光学显微镜的协同领域的专家，组成一个理想的团队将充分利用所需的设备。铅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，因此将立即有效地使用设备。为了继续在国际上竞争并为HQP提供适当的培训，必须持续获得跨学科工具，例如尖端的光学显微镜和样品制备技术，必不可少，没有这些工具，将很难在我们的研究社区中保持可观的地位。三名现任研究生和两名本科研究人员将立即从使用本设备中受益，这一数字预计会随着要求的设备的发展而增长，这使新的询问线可以开发。