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

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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.