Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10 µm either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.
Ultrahigh-speed imaging of rotational diffusion on a lipid bilayer
Mahdi Mazaheri, Jens Ehrig, Alexey Shkarin, Vasily Zaburdaev, Vahid Sandoghdar
We studied the rotational and translational diffusion of a single gold nanorod linked to a supported lipid bilayer with ultrahigh temporal resolution of two microseconds. By using a home-built polarization-sensitive dark-field microscope, we recorded particle trajectories with lateral precision of three nanometers and rotational precision of four degrees. The large number of trajectory points in our measurements allows us to characterize the statistics of rotational diffusion with unprecedented detail. Our data show apparent signatures of anomalous diffusion such as sublinear scaling of the mean-squared angular displacement and negative values of angular correlation function at small lag times. However, a careful analysis reveals that these effect stem from the residual noise contributions and confirms normal diffusion. Our experimental approach and observations can be extended to investigate diffusive processes of anisotropic nanoparticles in other fundamental systems such as cellular membranes or other two-dimensional fluids.
Long-range three-dimensional tracking of nanoparticles using interferometric scattering (iSCAT) microscopy
Tracking nanoparticle movement is highly desirable in many scientific areas, and various imaging<br>methods have been employed to achieve this goal. Interferometric scattering (iSCAT) microscopy has<br>been particularly successful in combining very high spatial and temporal resolution for tracking small<br>nanoparticles in all three dimensions. However, previous works have been limited to an axial range<br>of only a few hundred nanometers. Here, we present a robust and efficient strategy for localizing<br>nanoparticles recorded in high-speed iSCAT videos in three dimensions over tens of micrometers. We<br>showcase the performance of our algorithm by tracking gold nanoparticles as small as 10 nm diffusing<br>in water while maintaining 5 μs temporal resolution and nanometer axial localization precision. Our<br>results hold promise for applications in cell biology and material science, where the three-dimensional<br>motion of nanoparticles in complex media is of interest
Max-Planck-Zentren und -Schulen
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