Lumicks C-Trap Optical Tweezers with Confocal Fluorescence Microscope

Lumicks C-Trap 光镊与共焦荧光显微镜

• 批准号: 10629714
• 负责人: Bennett Van Houten
• 金额: $ 55.23万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2024-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10629714
• 关键词: Agreement; Cancer Center; Cancer Etiology; Cells; Color; Computer software; Confocal Microscopy; DNA; DNA Damage; DNA Repair; DNA-Protein Interaction; Data; Data Analyses; Doctor of Philosophy; Ensure; Experimental Designs; Faculty; Fluorescence; Funding; Genome Stability; Image; Interest Group; Kinetics; Lasers; Malignant Neoplasms; Measurement; Measures; Microfluidics; Microscope; Minor; Molecular Motors; Neurodegenerative Disorders; Pennsylvania; Proteins; Pythons; Reproducibility; Request for Proposals; Research Personnel; Role; Scanning; Science; Site; Speed; System; Technology; Temperature; Time; Universities; Virus; Work; biophysical techniques; fluorescence microscope; instrument; laser tweezer; mechanical force; member; millisecond; molecular assembly/self assembly; new technology; optic trap; optic tweezer; optical traps; programs; single molecule; virology

Title: Lumicks C-Trap optical tweezers with confocal fluorescence microscope. PI: Bennett Van Houten, PhD Abstract This proposal requests the purchase of a Lumicks C-trap correlative single-molecule and force microscope, which consists of four optical traps, and a 3-color confocal fluorescence platform for Major and Minor user groups at the University of Pittsburgh and Carnegie Mellon University. The C-trap is the only commercially available instrument that fully integrates laser traps with confocal microscopy. Single molecule analysis has helped revolutionize the field of protein-DNA interactions and each of the users have a focused funded project that would be greatly facilitated by access to this instrument. Through a lease agreement we have been using the instrument since Feb of 2021 and several user groups have successfully generated preliminary data on the instrument with up to three different fluorescent proteins. This instrument represents the first placement of this novel technology within the entire Common Wealth of Pennsylvania, and the new four laser trap module is planned to be added June 2022 to keep up with this cutting-edge technology. The C-trap allows investigators to use biophysical approaches to interrogate protein-DNA interactions and follow molecular motors and assembly of protein machines at specific sites on DNA. This instrument combines several unique features that makes single molecule and force measurements accessible to wide group of users that might be relatively new to the single molecule field. The instrument has an automated microfluidic system with a five chamber imaging flow cell, which provides a wide variety of experimental designs. The instrument is fully temperature controlled so kinetics of protein-DNA interactions can be followed at different temperatures. The laser traps have high temporal and spatial control and can measure down to sub pN forces accurately. The force measurements are real-time correlated with the confocal scanning which can scan at speeds up to 5 msec per scan allowing to capture rapid single molecule kinetics. The software that controls the instrument and allows data analysis is easy to use through a Python user interface. Scripting of experimental work-flow as well as data analysis allows highly reproducible conditions to be established. The two user groups consists of members of the Genome Stability and Cancer Virology Programs at the UPMC-Hillman Cancer Center, where the instrument is housed, as well as other minor users throughout the campus. These groups are interested in the role of DNA damage and repair in cancer and neurodegenerative diseases, as well as how specific viruses can cause cancer. Five Major users have been identified for the purpose of this application and they in turn have requested a majority of the time. The five Minor users will work on the instrument in the remaining time. The Lumicks C-trap is being managed as a user facility at the Hillman Cancer Center and will afford the capacity for advanced single molecule and mechanical force measurements and will ensure sustained and efficient use by faculty working in the biomedical sciences in Pittsburgh

标题：具有共聚焦荧光显微镜的Lumicks C-Trap光学镊子。 PI：Bennett Van Houten，博士 抽象的 该提案要求购买Lumicks C-Trap相关单分子和强制显微镜， 由四个光学陷阱组成，以及针对主要用户和次要用户的3色共聚焦荧光平台 匹兹堡大学和卡内基·梅隆大学的小组。 C陷阱是唯一的商业上 可用的仪器将激光陷阱与共聚焦显微镜完全整合在一起。单分子分析具有 帮助彻底改变了蛋白质-DNA相互作用的领域，每个用户都有一个重点资助的项目 通过使用该乐器，这将极大地促进。通过租赁协议，我们一直在使用 自2021年2月以来，该工具和几个用户组成功地生成了有关 具有多达三种不同荧光蛋白的仪器。该乐器代表了此仪器的第一个位置 宾夕法尼亚州全部共同财富以及新的四个激光陷阱模块的新技术是 计划添加2022年6月，以跟上这项尖端技术。 C陷阱允许调查人员 使用生物物理方法来询问蛋白-DNA相互作用并跟随分子电机和 在DNA上特定位点的蛋白质机器组装。该乐器结合了几个独特功能 使一组可能相对较新的用户可以访问单分子和力测量 到单分子场。该仪器具有带有五个腔室成像的自动微流体系统 流动池，提供多种实验设计。该仪器是完全控制的 因此，可以在不同温度下遵循蛋白-DNA相互作用的动力学。激光陷阱高 时间和空间控制，可以准确地测量到子PN力。力测量是 实时与共聚焦扫描相关，该扫描可以以每次扫描最高5毫秒的速度扫描，可以 捕获快速的单分子动力学。控制仪器并允许数据分析的软件是 易于通过Python用户界面使用。实验工作流以及数据分析的脚本 允许建立高度可重复的条件。两个用户组由 仪器所在的UPMC-Hillman癌症中心的基因组稳定和癌症病毒学计划 在整个校园内以及其他次要用户。这些小组对DNA的作用感兴趣 癌症和神经退行性疾病的损害和修复，以及特定病毒如何引起 癌症。出于此应用程序的目的，已经确定了五个主要用户 大部分时间都要求。五个未成年人将在剩余时间内从事该乐器。这 Lumicks C-Trap正在Hillman Cancer Center作为用户设施进行管理，并将负担能力 高级单分子和机械力测量，将确保持续有效地利用 在匹兹堡的生物医学科学工作