A5078546850- Advanced Fluorescence Microscopy Techniques
- Cardiomyopathy and Myosin Studies
- Enzyme Structure and Function
- Cellular Mechanics and Interactions
- Force Microscopy Techniques and Applications
- Lipid Membrane Structure and Behavior
- Protein Structure and Dynamics
- Advanced Biosensing Techniques and Applications
- Advanced Electron Microscopy Techniques and Applications
- Near-Field Optical Microscopy
- Mass Spectrometry Techniques and Applications
- Genomics and Chromatin Dynamics
- Biosensors and Analytical Detection
- RNA and protein synthesis mechanisms
- Plant Molecular Biology Research
- Gold and Silver Nanoparticles Synthesis and Applications
- Advanced Proteomics Techniques and Applications
- Advanced biosensing and bioanalysis techniques
- Optical Coherence Tomography Applications
- Muscle Physiology and Disorders
- Integrated Circuits and Semiconductor Failure Analysis
- Photonic and Optical Devices
- Protein Tyrosine Phosphatases
- Plasmonic and Surface Plasmon Research
- Galectins and Cancer Biology
University of Oxford
2015-2024
Arizona Oncology
2014
Bristol-Myers Squibb (Germany)
2005
Watching proteins' weight Careful measurements of light scattering can provide information on individual macromolecules and complexes. Young et al. used a light-scattering approach for accurate mass determination proteins as small 20 kDa (see the Perspective by Lee Klenerman). Movies protein complex association dissociation were analyzed to extract biophysical parameters from single molecules assemblies without labeling. Using this approach, authors determined in vitro kinetics fibril...
Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and diffraction-limited area which light can be confined in far field. We show that a partially transmissive spatial mask placed near back focal plane of high numerical aperture microscope objective enhances contrast scatterer an interface approximately T-1/2, where T transmissivity mask. Numerical-aperture-based differentiation background from scattered represents...
Abstract Sample purity is central to in vitro studies of protein function and regulation, the efficiency success structural using techniques such as x-ray crystallography cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize heterogeneity a sample minimal material with high resolution within matter minutes. To benchmark our approach, use negative stain electron (nsEM), popular method for EM screening. We include typical workflows developed...
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating chaotic cellular environment, potential disorder on the track, and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy reveal myosin strides 22 34 actin...
Interferometric scattering microscopy (iSCAT) is an extremely sensitive imaging method based on the efficient detection of light scattered by nanoscopic objects. The ability to, at least in principle, maintain high contrast independent exposure time or cross section object allows for unique applications single-particle tracking, label-free (dis)assembly, and quantitative single-molecule characterization. We illustrate these capabilities areas as diverse mechanistic studies motor protein...
Abstract The quantification of membrane-associated biomolecular interactions is crucial to our understanding various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition fluorescent labels, which complicates involved stoichiometries and dynamics because low temporal resolution inherent limitations associated with labeling efficiency, photoblinking photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking...
Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found detached head occupies loosely fixed position to one side from which it rebinds in controlled manner while executing step. Improving sub-nm regime provided evidence for an ångstrom-level structural transition domain associated power stroke. Simultaneous tracking both...
Abstract Protein–protein and protein–substrate interactions are critical to function often depend on factors that difficult disentangle. Herein, a combined biochemical biophysical approach, based electrically switchable DNA biochips single‐molecule mass analysis, was used characterize the binding protein oligomerization of transcription factor, forkhead box P2 (FOXP2). FOXP2 contains domains commonly involved in nucleic‐acid oligomerization, such as C 2 H ‐zinc finger (ZF), leucine zipper...
Cortactin (CTTN), first identified as a major substrate of the Src tyrosine kinase, actively participates in branching F-actin assembly and cell motility invasion. CTTN gene is amplified its protein overexpressed several types cancer. The phosphorylated form cortactin (pTyr421) required for cancer In this study, we demonstrate that majority tested primary colorectal tumor specimens show greatly enhanced expression pTyr421-CTTN, but no change at mRNA level compared to healthy subjects, thus...
The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that remodeling acto-myosin network modifies local organization, but molecular details are only partly understood because difficulties with experimentally accessing relevant time length scales. Here, we use interferometric scattering microscopy to investigate minimal linked supported lipid bilayer membrane. Using magnitude contrast, which is...
Single particle tracking has found broad applications in the life and physical sciences, enabling observation characterization of nano- microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as lipids or proteins on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision imaging speed paramount principle infinite photon budget. Here, we show that micromirror-based total internal...
Abstract Sample purity is central to in vitro studies of protein function and regulation, as well the efficiency success structural requiring crystallization or computational alignment individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking multi-step...
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast the majority of macroscopic machines, they need navigate chaotic cellular environment, potential disorder in track and Brownian motion. Nevertheless, decades nanometer-precise optical studies suggest that myosin-5a, one prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin Here, we use high-resolution interferometric scattering (iSCAT)...
Abstract Protein–protein and protein–substrate interactions are critical to function often depend on factors that difficult disentangle. Herein, a combined biochemical biophysical approach, based electrically switchable DNA biochips single‐molecule mass analysis, was used characterize the binding protein oligomerization of transcription factor, forkhead box P2 (FOXP2). FOXP2 contains domains commonly involved in nucleic‐acid oligomerization, such as C 2 H ‐zinc finger (ZF), leucine zipper...
Abstract The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural dynamic heterogeneity, despite being key to protein drug function, continues pose a fundamental challenge existing analytical structural methodologies used study these associations. Here, we use interferometric scattering microscopy mass-image single biomolecules in solution with <2% mass error, up 19-kDa resolution 1-kDa precision. Thereby, resolve oligomeric distributions at...
Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and diffraction limited area which light can be confined in far field. We show that a partially transmissive spatial mask placed near back focal plane of high numerical aperture microscope objective enhances contrast scatterer an interface approximately $T^{-1/2}$, where T transmissivity mask. Numerical based differentiation background from scattered represents...
Abstract We introduce dynamic mass photometry, a method for label-free imaging, tracking and measurement of membrane-associated proteins. Our enables quantitative studies their mobility, membrane affinity interactions at the single molecule level. Application to remodelling GTPase dynamin1 reveals heterogeneous mixtures oligomers suggesting that fundamental building block oligomerisation is dimer, challenging current tetramer-centric models. Dynamic photometry has ability transform our...
Extract HTML view is not available for this content. However, as you have access to content, a full PDF via the ‘Save PDF’ action button. Extended abstract of paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4,