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Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm 2 /s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Polarization-resolved second harmonic generation (P-SHG) microscopy is commonly used to analyze the second-order susceptibility, χ(2), tensor, which enables the calculation of the molecular structure of harmonophores. However, despite extensive research on type I collagen, the measured χ(2) ratios vary considerably among published values, which raises the question of whether P-SHG imaging is universally applicable to all tissues containing harmonophores. In this work, we propose that the deviation of χ(2) ratios is primarily due to ignoring the molecular tilt angle and chirality in image analysis. To confirm our hypothesis, we present an analytical model based on C6 symmetry that takes into account these two factors. We also introduce an imaging scheme that splits SHG into X- and Y-polarized image components for χ(2) tensor analysis. Our approach effectively improves the precision of determining χ(2) ratios, depending on how much the two factors affect the P-SHG signals. [ABSTRACT FROM AUTHOR]