Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells

Abstract

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/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans

“…It can be hypothesized that the mobile virions can take advantage of differences in GAG types and charge gradients induced by burst-like appearances of highly sulfated stretches of the HS/CS chains in the glycocalyx (20) to ''stochastically roll'' toward the cell surface while relying on its ability to break and reform single GAG-bonds. Our findings further suggest that the initial attachment of HSV-1 to GAGs exposed on the cell membrane could be a dynamic process in which viruses move in a two-dimensional plane in search of secondary receptors before firmly attaching to the membrane and proceeding with viral entry, as reported for other viruses (70)(71)(72). This hypothesis and the importance of this motion behavior during viral entry will be the subject of further investigation involving live-cell experiments.…”

Interferometric Scattering (iSCAT) Microscopy and Related Techniques

“…Interestingly, interferometric microscopies are also flourishing in the general context of label-free imaging of cells and membranes even if nanoparticles are not at the center of attention [75,76,90,[92][93][94][95][96][97][98][99][100][101][102][103][104][105][106]. The underlying physics of these methods remains the same although a plethora of acronyms such as interference reflectance imaging sensing (IRIS) [77], rotating coherent scattering (ROCS) [98], interference plasmonic imaging (iPM) [88], coherent bright-field imaging (COBRI) [107], stroboscopic interference scattering imaging (stroboSCAT) [108], interferometric scattering mass spectrometry (iSCAMS) [109] are on the rise. In what follows, we bring all these techniques under the umbrella of iSCAT, emphasizing the two central concepts and mechanisms of interference and scattering as the basis for recording the extinction signal (nano-shadow) generated by nanoparticles.…”

“…In the following years, iSCAT was extended in our laboratory to different illumination and detection conditions [114,115] and used to detect single unlabeled viruses [116,117], semiconductor quantum dots [115], lipid vesicles [118,119] and unlabeled proteins [120]. In more recent years, several other groups have also successfully applied different illumination/detection variants of iS-CAT to detect single proteins [109,121,122], single viruses [83,107], lipids [123,124] and other nanoparticles [82], and even charge carriers [108].…”

“…While iSCAT is predestined for addressing this issue, its ultrahigh sensitivity virtue is accompanied by the vice of a large speckle-like pattern originating from the cellular membrane and corpus, thus making cellular iSCAT particle tracking a challenge. Nevertheless, first attempts in tackling this issue have been successfully reported [107,139,185] (see Fig. 16).…”

Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane

“…Refs. [ 3,28 ]), but in our experiments it was assessed for each frame. We also emphasize that inclusion of the positional uncertainty σ is important for a correct assessment of the MSD 26 , and especially in high-speed imaging where the mean square displacement becomes comparable to the localization precision.…”