34 research outputs found
Micromirror total internal reflection microscopy for high-performance single particle tracking at interfaces
Single particle tracking has found broad applications in the life and
physical sciences, enabling the observation and characterisation of nano- and
microscopic motion. Fluorescence-based approaches are ideally suited for
high-background environments, such as tracking lipids or proteins in or on
cells, due to superior background rejection. Scattering-based detection is
preferable when localisation precision and imaging speed are paramount due to
the in principle infinite photon budget. Here, we show that micromirror-based
total internal reflection dark field microscopy enables background suppression
previously only reported for interferometric scattering microscopy, resulting
in nm localisation precision at 6 s exposure time for 20 nm gold
nanoparticles with a 25 x 25 m field of view. We demonstrate the
capabilities of our implementation by characterizing sub-nm deterministic flows
of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach
the optimal combination of background suppression, localisation precision and
temporal resolution achievable with pure scattering-based imaging and tracking
of nanoparticles at regular interfaces.Comment: 27 pages, 4 figure
Mass Photometry of Membrane Proteins
Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramersāin contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation
Quantifying the heterogeneity of macromolecular machines by mass photometry
Data set for the manuscript of the same title. Contains mass photometry raw data in the form of either movie files (in the National Instruments .tdms file format or Refeyn LTD .mp file format) and extracted values used for generating the figures (generally .txt, .dat or .xlsx files). Explanatory notes on the arrangement of the data within these files are provided, also as .txt files
Micromirror total internal reflection microscopy for high-performance single particle tracking at interfaces
Single particle tracking has found broad applications in the life and
physical sciences, enabling the observation and characterisation of nano- and
microscopic motion. Fluorescence-based approaches are ideally suited for
high-background environments, such as tracking lipids or proteins in or on
cells, due to superior background rejection. Scattering-based detection is
preferable when localisation precision and imaging speed are paramount due to
the in principle infinite photon budget. Here, we show that micromirror-based
total internal reflection dark field microscopy enables background suppression
previously only reported for interferometric scattering microscopy, resulting
in nm localisation precision at 6 s exposure time for 20 nm gold
nanoparticles with a 25 x 25 m field of view. We demonstrate the
capabilities of our implementation by characterizing sub-nm deterministic flows
of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach
the optimal combination of background suppression, localisation precision and
temporal resolution achievable with pure scattering-based imaging and tracking
of nanoparticles at regular interfaces