Instrument Development: A nanoscale, unbleachable orientation and position sensor for biophysical imaging

在化学部（化学测量和成像）和生物基础设施部（生物研究仪器开发）的支持下，dr。华盛顿大学的Kai-Mei Fu和Paul Wiggins和他们的团队正在寻求建立一代单分子技术，使生物化学过程一次一个分子的可视化。这些方法促进了对细胞中许多最重要的生物过程的基础生物学、化学和物理学的机械理解。具体来说，该团队正在开发一种用于生物物理应用的新型传感器，可以同时测量纳米粒子探针的三维位置和方向。所提出的磁探针成像平台（MagPI）有望广泛应用于一系列生物物理问题，其中方向和位置对生物功能都很重要。研究成果将通过地方、国家和国际会议、出版物和实验室研究网站，以及通过宣传该方法的技术讲习班广泛传播，以促进化学、生物和生物医学研究界采用该方法。这项工作也将支持研究生和本科生在一个丰富的，跨学科的环境，结合物理，化学，工程和生物学的培训。它将为“生物传感和五种感官”的重点单元提供支持，该单元将为小学科学推广项目开发，并通过推广网站向公众提供课程计划。该研究有三个具体目标：(i)有针对性地开发MagPI，以补充商业上可用的预功能化纳米粒子，以便于采用；（ii）将该平台集成到系留粒子运动（TPM）分析中，以证明平台在生物物理背景下的可追溯性；（iii）通过在TPM分析中检测不同转录因子- dna复合物构象之间的预期相互转换，展示该平台的潜力。MagPI探针是不可漂白和不眨眼的，因此非常适合用于探针3d位置和方向的精确跟踪，可以提供重要的新见解，但需要长时间尺度和高时间分辨率成像，这就排除了单分子荧光探针的使用。探头和传感器与现有的对比度产生机制（包括明场、散射和荧光）是互补和兼容的。通过对纳米粒子探针产生的磁偶极子场进行成像，实现了取向和位置传感。磁场成像的物理机制是b场引起金刚石传感器中氮空位中心光致发光强度的位移。

With support from the Divisions of Chemistry (Chemical Measurement and Imaging) and Biological Infrastructure (Instrument Development for Biological Research), Drs. Kai-Mei Fu and Paul Wiggins and their groups at the University of Washington are looking to build on a generation of single-molecule techniques enabling visualization of biochemical processes one molecule at a time. These methods facilitate a mechanistic understanding of the fundamental biology, chemistry and physics underlying many of the most important biological processes in the cell. Specifically, the team is developing a novel sensor for biophysical applications that can simultaneously measure the three-dimensional position and orientation of a nanoparticle probe. The proposed Magnetic-Probe-Imaging platform (MagPI) is expected to be widely-applicable to a range of biophysical problems where orientation and position are both of significance to biological function. Research results will be disseminated broadly through local, national, and international conferences, publications, and laboratory research websites as well as through an advertised technical workshop on the method to facilitate its adoption by the chemical, biological, and biomedical research communities. This work will also support graduate and undergraduate student training in a rich, interdisciplinary environment that combines physics, chemistry, engineering, and biology. It will provide support for a focused unit on "Biosensing and the Five Senses" that will be developed for an elementary-school science outreach program, with lesson plans available to the public through the outreach websiteThe research addresses three specific aims: (i) the targeted development of a MagPI to complement commercially-available pre-functionalized nanoparticles for ease of adoption; (ii) the integration of this platform into a Tethered-Particle-Motion (TPM) assay to demonstrate platform tractability in biophysical contexts; and (iii) a demonstration of the potential for this platform by detecting expected interconversions between distinct transcription-factor-DNA complex conformations in TPM assays. The MagPI probe is unbleachable and unblinking and therefore is ideally suited to applications where precision tracking of probe 3D-position and orientation could provide significant new insights, but long-timescale and high-temporal resolution imaging is required which precludes the use of single-molecule fluorescent probes. The probe and sensor is complementary and compatible with existing contrast generation mechanisms, including brightfield, scattering, and fluorescence. Orientation and position sensing are performed by imaging the magnetic dipole field generated by the nano-particle probe. The physical mechanism for magnetic-field imaging is the B-field-induced shift of the photo-luminescence intensity of nitrogen-vacancy centers in a diamond sensor.