Coherent Brightfield Microscopy Provides the Spatiotemporal

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Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution to Study Early Stage Viral Infection in Live Cells Yi-Fan Huang, Guan-Yu Zhuo, Chun-Yu Chou, Cheng-Hao Lin, Wen Chang, and Chia-Lung Hsieh ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b05601 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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

Yi-Fan Huang1, Guan-Yu Zhuo1, Chun-Yu Chou1, Cheng-Hao Lin1, Wen Chang2, Chia-Lung Hsieh1,*

1

Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan

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Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan

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Corresponding author: [email protected]

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 1

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cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image post-processing, 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 µm2/s) at microsecond timescales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Keywords: coherent brightfield microscopy; high-speed imaging; single-virus tracking; early-stage infection; virus-membrane interaction; diffusion; 3D localization; interferometric imaging; digital background removal; local delivery of virus particle.

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In viral infection, a virus particle needs to attach to a cell membrane, moves laterally, interacts with cellular receptors, and then enters the cell through endocytosis or plasma membrane fusion.1,2 Because of the complexity of cells, fates of identical virus particles are stochastically different and multiple pathways can be taken in parallel.3 Therefore, there is a need to develop tools that can directly visualize the dynamics of individual virus particles during infection at the single particle level.

Individual virus particles can be labeled with fluorescent markers in order to visualize them in cellular environments at the single particle level and in real time. After initial demonstration of this approach,4 various schemes involving tagging dyes,5-10 fluorescent proteins11-14 or nanoparticles15,16 have been developed. Using these approaches, we know that many virus particles migrate on the cell surface after attachment,5,8,13,17-19 and that they enter cells via different mechanisms including endocytosis and membrane fusion.9,17,18,20,21 After penetrating the cell plasma membrane, virus particles travel within cells and release their genomes for production.5,7,10,14-18,21

Although a lot has been learned about viral entry using optical microscopy, many facets of virus-host interaction remain elusive. Virus dynamics and interactions with host cells span a wide range of timescales. Fluorescence single-virus tracking is typically performed with a spatial localization precision of ~20 nm at video rate (< 50 Hz),7-9,14-17,22 which is suitable for monitoring processes of 3


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endocytosis and membrane fusion of virions over seconds to minutes.9,17,18,20,21 Other virus-host interactions take place at much shorter timescales. For example, viral fusion pore opening and the molecular conformational changes upon virus binding are estimated to occur within microseconds.23,24 The interplay of the virus particle with membrane receptors in nanoscopic heterogeneous plasma membrane structures is expected to last sub-milliseconds to milliseconds.25-27 Formation of the endocytic clathrin-coated pit takes only a few seconds.28 Due to the fine structures of cell plasma membranes, a spatial resolution down to molecular scale is desired in order to observe interactions between single virus particles and the complex cellular environment. Taken together, for the study of virus-membrane interactions, ideally the virus particle needs to be tracked with nanometer spatial precision in 3D and microsecond temporal resolution.

Tracking single virus particles via fluorescence labeling has been inhibited by fundamental limitations. The small sizes of virus particles limit the number and size of fluorescent tags that can be applied to a virion. A high density of labels can lead to crowding effects that interfere with viral function.3 Furthermore, increasing the labeling density can also cause self-quenching of the fluorescence signal.3 Consequently, the number of fluorescent tags, and therefore the available fluorescence signal, is limited for virus labeling. In addition, photobleaching of most fluorescent markers limits the total observation time before the tags become silent. Moreover, saturation of fluorescence due to the fluorescence lifetime limits the maximal number of signal photons per unit 4


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time. Due to these reasons, currently-available fluorescent markers struggle to provide sufficient signal to reveal fast virus-membrane interactions at the nanoscale, leaving some of the most basic questions unanswered. For example, what happens to a virus particle immediately after it contacts the cell surface? How does a virus particle migrate on the membrane to find receptors after attaching at a random site?

An alternative approach to probing and tracking single virus particles is to detect their intrinsic linear scattering light under illumination.29-36 The linear scattering signal is stable, without a photobleaching effect, thereby allowing for unlimited observation times. It has been previously demonstrated that single virus particles < 50 nm can be reliably detected29-31,34 and even tracked with nanometer precision using scattering-based approaches.30 Interferometric detection of the scattering signal is especially advantageous because a high signal-to-noise ratio (SNR) can be achieved.30-33 Previous scattering-based detection and tracking of single virus particles have been performed in artificial model systems where the background scattering from the environment is minimized as much as possible. In live cells, random background scattering of cell structures readily conceals the weak scattering signal of a virus particle. It was unclear whether high-precision and high-speed scattering-based detections could be extended to live cell imaging.

In this work, we demonstrate label-free, ultrahigh-speed, high-precision 3D tracking of native virus 5


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particles in live cells by coherent brightfield (COBRI) microscopy via scattering. The COBRI microscopy is an implementation of brightfield microscopy where a laser light source is used for illumination.

Using a spatially coherent laser light source, the COBRI microscopy can locally

illuminate an area of interest for high-speed imaging up to 100,000 frames per second. The laser illumination of high temporal coherence also increases the contrast of the virus particles for high-precision tracking. We characterize the sensitivity of COBRI microscopy and its capability for 3D localization of single vaccinia virus particles. We also present a procedure for image processing to remove cellular background from the signal of virus particles based on differences in their spatial and temporal fluctuating signatures. Combining COBRI imaging and digital background removal, we study early-stage infection of live cells by vaccinia virus. We capture the processes of single virus particles landing on the plasma membrane of live cells with a spatial precision of a few nanometers in 3D and a temporal resolution of 10 μs. With such ultrahigh spatiotemporal resolution, we reveal fast lateral diffusion and sub-millisecond transient nano-confinement of the virus particle on the membrane immediately after attachment. Ultrahigh-speed COBRI imaging opens the door to studying virus dynamics in live cells in a spatiotemporal regime of nanometers and microseconds.

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RESULTS Ultrahigh-speed 3D localization of vaccinia virus particles by COBRI microscopy The COBRI microscope works the same way as conventional brightfield microscopy except a coherent light source (a laser) is used for illumination (Figure 1, and METHODS). This modification offers many favorable features for high-speed tracking of small particles. The high spatial coherence of the laser allows for precise control of the beam shape and illumination area. This capability is especially critical for ultrahigh-speed imaging where sufficient light intensity is needed within each frame exposure of microseconds. Using lasers, we are able to focus light on the cell of interest, leaving the rest of the sample completely dark. By targeting the light dose in this way, photobleaching of fluorescent markers and phototoxicity are greatly reduced. We emphasize that the illumination intensity for ultrahigh-speed COBRI imaging is low; the typical excitation intensity at an acquisition rate of 100 kHz is only 10 µW/μm2 (illumination intensity is proportional to the acquisition rate), which is comparable to that required for single-molecule fluorescence imaging.37 Furthermore, compared to a white light source, the laser of high temporal coherence enhances the imaging sensitivity, which leads to stronger contrasts of small particles for detection and localization (see quantitative characterizations of COBRI microscopy and conventional brightfield microscopy in Supporting Information Figure S1 and S2).

Lasers have often been considered difficult to handle in scattering-based cell imaging because their 7


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high coherence can create speckles when the sample is highly scattering.38,39 We found that most speckles in the image can be reduced by avoiding multi-reflection and scattered light from the optical elements. Having a clean illumination beam profile is also helpful in minimizing speckles. This can be done by spatial filtering of the beam combined with high-speed beam-scanning using acousto-optic deflectors (Supporting Information Figure S3). Note that the COBRI microscope is compatible with fluorescence imaging. Future applications of COBRI microscopy together with simultaneous fluorescence imaging may provide complete dynamic and functional information for biological studies.

Vaccinia virus particles are brick-shaped and ~250 nm in diameter,40 and are clearly visible under the COBRI microscope when deposited on a clean coverslip (Figure 2a). We confirmed that the observed particles are indeed vaccinia virus particles by labeling the virus particle with mCherry fluorescent protein at its core and correlating the COBRI image with a fluorescence image (Figure 2b).13 Infectivity of the virus was not affected by mCherry labeling.13 In COBRI imaging, the well-defined phase difference between scattered light of the particle and transmitted light leads to height-sensitive interference signal of the particle in the axial direction. To characterize the height-dependent contrast, we immobilized virus particles on a clean coverslip and modulated the axial position of the sample relative to the microscope objective using a piezo stage (triangular function with amplitude of 1 µm peak-to-peak), during which a COBRI video was recorded (Supplementary Video 1). Snapshots of 8


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the virus particle at a few different axial positions are displayed in Figure 2c. Here the plane of z = 0 is defined as the height where the particle appears as the darkest spot. We quantitatively determined the contrast of the particle in every image by fitting the point spread function with a 2D Gaussian function (METHODS). For each virus particle, the calibration curve of contrast as a function of axial position is obtained by averaging measurements of 40 independent modulation cycles (Figure 2d). As a result of interference, the calibration curve between the darkest and the brightest spots can be well described with a sinusoidal function. Note that when the height of the particle moves beyond this range, the particle becomes out-of-focus and the height-dependent contrast can no longer be described by a sinusoidal function. We estimated the lateral and axial localization precision as a function of particle height from the accuracy of the Gaussian fitting (Figure 2e).41 Lateral localization precision remains almost unchanged as the height of the particle increases from 0 to 250 nm. Note that the axial localization precision is low when the particle is at z = 0 where the contrast is least sensitive to height variation. Although the localization precision depends on the height of the particle, precision better than 5 nm in 3D is achieved over an axial range of ~200 nm, which we define as our working range. The working range of 200 nm is relatively small considering the micron-thick biological cells. However, for the current study of viral dynamics on the flat plasma membrane over a length scale of a few microns, 200-nm observation depth is sufficient.

We benchmarked our 3D localization precision by tracking a single virus particle immobilized on a 9


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coverslip at 5 kHz. The standard deviations of measured particle positions in the x, y, and z directions are 2.3 nm, 1.7 nm and 2.3 nm, respectively (Figure 2f). These values correspond to the static localization precisions. Because of the short exposure time in high-speed imaging, the dynamic localization precisions of the data presented in the rest of this study are estimated to be almost identical (within 0.3%) to the static ones.42 This fact is verified by the comparable SNR of the virus particle in the dynamic measurements.

We point out that COBRI microscopy shares with digital holographic microscopy the principle of interferometric detection of scattering signal for 3D localization.43,44 Unlike holographic microscopy, COBRI microscopy places the sample of small particles on the focal plane of the microscope objective (within 500 nm), thereby avoiding spread of the signal over many pixels on the camera. By doing so, COBRI microscopy generates a small particle image with the highest contrast in the raw image, without the need for holographic reconstruction. This convenient feature of COBRI imaging is particularly useful when recording rare and unpredictable events of small low-contrast particles, e.g., the virus-membrane interaction presented in this work, where real-time direct visualization of the particle is crucial to capture the event. Compared with other non-fluorescence interference optical microscopes,38,39,45 the COBRI microscope has the distinct advantages of high-speed imaging with optimal sensitivity and a low light dose.

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We characterized height-dependent contrast of 62 virus particles, from which the maximal contrast and the period of the fitted sinusoidal function were 0.24±0.02 and 1600±60 nm, respectively (Figure 2g). These two values are determined by the virus particle (size and density) and the optical setup (numerical aperture of the microscope objective). The small variation in maximal contrast indicates the high uniformity of the virus particles. We used the averaged values of the period and contrast to establish the calibration curve for reconstruction of the axial position of the vaccinia virus particle in the rest of the study. Discrepancy between the averaged and exact calibration curves of individual virus particles results in distortion of the trajectory in the axial direction. Considering the distributions of period and maximal contrast, we estimate the typical distortion is < 10 nm every 100-nm displacement within the working range. It should also be noted that when tracking virus particles away from the coverslip, e.g., in the aqueous buffer solution or on the cell membrane, distortion in the reconstruction arises from the index mismatch between the sample and the coverslip.46 Based on previous studies,47 we estimate this distortion is < 15 nm every 100-nm displacement.

Digital cellular background removal We applied COBRI microscopy to 3D tracking of single vaccinia virus particles on the plasma membranes of live cells. The combination of nanometer spatial precision and microsecond temporal resolution provided the opportunity to visualize single virus particles after landing on the membrane 11


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at a random location. Note that in the raw COBRI image, the scattering signal of the virus particle is embedded in a heterogeneous background due to scattering by the cell structures (Figure 3). The presence of this cellular background impedes high precision localization of the virus particle. Several strategies have been demonstrated for noise reduction and background subtraction in scattering-based imaging.30,48,49 Where possible, capture of a background image without the signal and then subtracting it from the raw image is the most straightforward approach.30 Filtering and smoothing via digital image processing have also been used to reduce speckles.48 It has also been demonstrated that the static signal can be distinguished from the speckle noise based on their different stochastic properties.49 Here, we explore the possibility of differentiating the signal of the virus particle from the cellular background based on their distinct spatial and temporal behaviors via digital image processing. We show that although the contrasts of the particle and cellular background are comparable, their spatial and temporal fluctuating behaviors are very different in high-speed imaging (acquisition rate > 5 kHz). In most cases, the cellular background varies slowly, whereas the signal of virus particles fluctuates rapidly over space as a result of diffusion. By recording a high-speed COBRI video for a few seconds, we were able to calculate a background image of the relatively stationary cell structures (Figure 3 and METHODS). After removing this background from the raw images frame by frame, we obtained background-free COBRI images of the particle for precise localization (Figure 3). The virus dynamics reported in this work were mostly recorded on the plasma membrane at the cell periphery where the normalized residual background fluctuation 12


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after background removal is low, typically < 8×10-3 (standard deviation, see data in Supporting Information Figure S4). Such fluctuation is dominated by our photon shot-noise fluctuation, indicating that the residual cellular background is lower than our imaging sensitivity (see Supporting Information Figure S4 for details). We note that the strength of residual fluctuation depends on the thickness and condition of the cell. Our method is generally suitable for tracking small-sized objects because their motions are inherently diffusive and thus very different from the motions of micron-sized cellular structures. All results presented in this work use one background image to correct one video of ~ 1 second. For longer observation times during which the cell background changes, the background can be dynamically calculated and corrected.50,51 Our current background removal approach fails when the particle of interest is immobile because the signal becomes indistinguishable from the background.

3D nanoscopic motion of a single virus particle on the cell plasma membrane Using COBRI microscopy and digital background removal, we successfully captured in a continuous manner the process of a single virus particle initially diffusing in the buffer solution and then landing on the plasma membrane (Supplementary Video 2). In the experiments, the virus particles were locally introduced through a glass micropipette to live HeLa cells cultured on a coverslip (METHODS). The detected particle was confirmed to be a virus particle through colocalization based on simultaneous COBRI and fluorescence imaging. The 3D position of the virus particle was 13


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determined from the background-free COBRI image through 2D Gaussian fitting and prior calibration. Figure 4 plots a few representative 3D-reconstructed trajectories of single virus particles landing on plasma membranes, recorded at 5 kHz. Our data show that the virus particles are locally confined on the membrane within hundreds of nanometers after landing (Figure 4a and 4b). The moment when a virus attaches to the membrane can be determined from the abrupt change in diffusion characteristics and particle height (Figure 4c). Interestingly, immediately after landing, the virus particle was not completely immobile. Based on our observations, it almost always migrated laterally on the membrane over hundreds of nanometers, interacting transiently with nanoscopic zones spanning tens of nanometers, before finally being strongly confined (for at least a few seconds of our observation time) (Figures 4b, 4d and 4e). The entire process from landing to strong confinement typically happens within 1 second. Between those nanoscopic confinements, the virus explores the membrane with a large lateral diffusion coefficient of 0.5–1 μm2/s (determined by the step sizes at the shortest timescale of 0.2 ms). We also found that some of the nano-confinement zones on the membrane are stable for at least hundreds of milliseconds because they can be revisited (Figure 4e and Supplementary Video 3). These zones could be clusters of attachment factors or membrane receptors. More virus landing and confinement trajectories can be found in Supporting Information Figure S5. In comparison, when virus particles attached to a coverslip without cell membranes, they became immediately immobile after binding (Supporting Information Figure S6).

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To further resolve the fast virus-membrane interaction, we increased the acquisition rate to 100 kHz (corresponding to a frame time of 10 μs). Increasing the acquisition rate in COBRI imaging does not deteriorate the SNR as long as the illumination intensity is increased accordingly, thanks to the unsaturated scattering signal and the shot-noise-limited interferometric detection.52 At such high spatiotemporal resolution, we again observed that the virus particle diffused rapidly on the membrane with a diffusion coefficient of 1.1 µm2/s (determined by the step sizes at a timescale of 10 μs) immediately after it adsorbed on the membrane (Figure 5a and Supplementary Video 4). This diffusion rate is close to the free diffusion rate of a 250-nm spherical particle in water, indicating that the initial interaction of the virus particle with the membrane is surprisingly weak. Figure 5b displays the mean-square displacement (MSD) of the lateral diffusion trajectory in Figure 5a, whereby weak anomalous diffusion (anomalous diffusion exponent α = 0.95±0.01) is observed in the short timescales from 10 μs to 1 ms (see METHODS for the MSD analysis). The non-Brownian diffusion indicates complex interplay between the virus particle and the plasma membrane. No apparent change in diffusion characteristics was observed within a second after binding to the membrane (see the transient diffusion coefficient as a function of time after landing in Supporting Information Figure S7). Figure 5c is the 3D reconstructed trajectory of the virus particle while it explored the plasma membrane, showing a spatially-varying height distribution in the trajectory. Figure 5c could partly reflect the 3D topography of the cell membrane. It is important to note that the plasma membrane may fluctuate over a few nanometers even in microseconds.53 Also, because of the 15


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elongated shape of the vaccinia virus particle, rotation and tumbling of the virus particle on the membrane could lead to changes in the measured axial position (of the center of mass of the virus particle). Further investigation is needed to distinguish these effects. Despite these uncertainties, we conclude that the virus particle is truly associated with the membrane because displacement in the axial direction is much smaller than that in the lateral directions (Figure 5d). To investigate the cause of the measured anomalous subdiffusion, we analyzed the transient confinement zones (TCZs) in the trajectory (METHODS). Figure 5e plots the trajectory, with detected TCZs highlighted in red, showing a total of 59 TCZs. The typical diameter of TCZs is 10 to 20 nm with a particle residence time of submilliseconds. A high temporal resolution is necessary to detect these transient nano-confinements. Figure 5f plots three segments of trajectories with TCZs. Some TCZs were revisited in the trajectory, indicating that the TCZs are stable for at least sub-milliseconds. We verified that the detected TCZs are not statistical fluctuations by comparing the numbers of detected TCZs and their residence times with those of simulated trajectories of Brownian motion (Figure 5g, see METHODS for the simulation parameters). We detected three times more TCZs with longer lifetimes (45 μs versus 25 μs, fitted by exponential functions) from the virus trajectory. The amount of time in TCZs for the experimental data was twice that of the simulated Brownian motion (2% versus 1% of the total observation time). All these analyses indicate that the detected TCZs in the viral trajectory are real traps, not statistical fluctuations.

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DISCUSSION In most of the previous single-virus tracking experiments, the virus particles were already attached to the cell membrane when imaging was performed. Therefore, it was not possible to know the behavior of the virus particle immediately after it landed. In order to probe the initial virus-membrane interaction, we delivered the virus particles locally with a micropipette and captured the virus landing event in a continuous manner. A few studies have captured landing events by chance using fluorescence, but the temporal resolution was too low (2 Hz in ref. 17 and 20 Hz in ref. 8) to reveal initial exploration of the membrane as captured in our study. Based on our observations, within a second immediately after landing, the virus particles diffused laterally with a very high diffusion coefficient, almost as fast as their diffusion in water. These observations imply that there is a weak virus-membrane interaction that attracts the virus to the membrane whilst also allowing virus particles to explore the membrane laterally (with a diffusion rate of 0.5–1 μm2/s). During lateral exploration, we often observed transient confinements within zones of tens of nanometers, which may be clusters of membrane receptors. Previous studies have shown that vaccinia virus particles bind to cell surface glycosaminoglycans (GAGs) and laminin and subsequently to the raft-associated membrane proteins integrin β1 and CD98 on the plasma membrane, which triggers endocytosis into cells.54,55 The TCZs detected in our ultrahigh-speed data could be the result of transient association of the virus particle with these membrane proteins within the raft membrane nanodomains. Future investigation is required to resolve the factors responsible 17


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for these observed TCZs.

From our data, almost all virus particles were strongly confined in these

TCZs within a few seconds after landing. Although strong confinement within seconds after landing was observed, the virus particle can still migrate slowly on the membrane in the timescale of seconds to minutes (Supporting Information Figure S8).

Previous single-virus experiments have shown that virus particles undergo slow diffusion (~0.01–0.1 μm2/s)8,17 and directional “surfing” on the plasma membrane56,57 during which the virus particle searches for endocytic sites. We stress that our findings of rapid diffusion over short timescales (microseconds) are not contradictory to these previous results of slow diffusion over longer timescales (sub-seconds). From our ultrahigh-speed measurements, representing long trajectories spanning over five orders of magnitude in time (Figure 5a), we calculated the apparent diffusion coefficient as a function of observation timescale, from 10 μs to 100 ms (see METHODS and Supporting Information Figure S9). It appears that the apparent diffusion coefficient is almost constant at 1 µm2/s in the sub-millisecond regime (as the anomalous diffusion is weak with an exponent of 0.95), and it starts to decrease in the millisecond timescale. At the longest timescale of 100 ms, the apparent diffusion coefficient is estimated to be 0.27±0.06 µm2/s, which is much lower than that for the microsecond timescale. The decreased apparent diffusion coefficient over the longer timescale is the result of spatial confinement at the landing site. Our data extends our understanding of virus-membrane interactions to a spatiotemporal regime of short timescales (microseconds) and 18


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small length scale (nanometers), revealing rapid diffusion and nanoscopic confinement. These events could not have been detected with low temporal sampling or low localization precision.

In COBRI imaging, the strength of signal is highly size-dependent. For small particles (Rayleigh scatters), their COBRI contrasts are expected to be proportional to the volume of the particles.58 Varying the particle diameter by a factor of 10 changes the COBRI contrast by three orders of magnitude. Such strong size dependency is convenient for imaging biological particles, including viruses, in complex live cellular environments. While biological particles of > 100 nm in diameter are clearly visible under COBRI microscopy, smaller entities such as protein complexes or macromolecules, generate much weaker signal with negligible perturbation in the image. We stress that this does not imply that small entities are not detectable by COBRI microscopy since the sensitivity can be increased by increasing the effective illumination intensity.50,51 This can be done by integrating many images into one image, thereby reducing the normalized shot-noise fluctuation. By integrating 100 frames into one frame, 80 nm silica nanoparticles with a COBRI contrast of -5×10-3 can be clearly observed (Supporting Information Figure S10). When the sensitivity is improved, it is the random residual cellular background that sets the limit for the smallest particle that can be detected. While we demonstrate COBRI imaging of non-absorbing biological particles via scattering, COBRI microscopy takes extinction images that are particularly suitable for imaging small absorptive particles or molecules whose extinction signal is much stronger than the pure scattering 19


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signal. For example, small gold nanoparticles have an absorption cross-section that is much larger than their scattering cross-section.59 Indeed, with sufficient illumination intensity and proper noise cancellation, it has been demonstrated that single absorptive molecules can be detected via extinction.60

We point out that interferometric scattering (iSCAT) microscopy also uses coherent light sources and interferometric detection of scattering signal of small particles.61-63 iSCAT microscopy has been repeatedly demonstrated as a powerful imaging method with high sensitivity and high spatiotemporal resolution.64-67 In iSCAT microscopy, the light is projected through a high numerical aperture microscope objective and the backscattered light is collected by the same objective. The reference beam of iSCAT microscopy comes from the reflection from an optical interface in the system (usually the coverslip holding the sample). COBRI microscopy can be considered the transmission counterpart of iSCAT microscopy. Through interference, the sensitivities of COBRI and iSCAT microscopy are both shot-noise-limited. In principle, they have the same sensitivity under the same illumination intensity. From a technical point of view, COBRI microscopy uses the transmitted non-scattered light as the reference beam, avoiding the necessity of a nearby optical interface to create the reflective reference that may not always be available. Furthermore, by detecting in transmission geometry, the calibration for localization in the axial direction becomes convenient, as the transmitted reference beam is nearly unchanged when the axial position of the particle is 20


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displaced by moving the whole sample for calibration. Finally, when imaging adherent cells on a coverslip, we found that the iSCAT image is often dominated by the reflected signal from the cell plasma membrane, similar to what has been previously observed by reflection interference contrast microscopy.39 In contrast, under transmission of COBRI microscopy, the signal of the plasma bilayer membrane is not apparent.

CONCLUSIONS In summary, COBRI microscopy and background-removal image processing have allowed us to resolve the 3D nanoscopic dynamics of vaccinia virus particles in live cells via scattering. The ultrahigh spatiotemporal resolution may provide the opportunity to visualize highly dynamic interactions of single virus particles with cell membrane receptors68 in a spatiotemporal regime that has not been available before. The capability for continuous visualization of 3D single-particle dynamics is also valuable for many other applications, such as studying the delivery of nanoparticles into cells as nano-biomedicine69 and vesicle transport in neurons.70 A combination of COBRI microscopy and fluorescence superresolution microscopy presents an exciting prospect, whereby COBRI imaging offers dynamic information on single particles (virus particles, nanoparticles, cell vesicles and organelles, etc.) and superresolution imaging shows the surrounding cellular structures with nanometer resolution. We foresee that COBRI microscopy will open an avenue for investigating cell biology at the nanoscale. 21


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METHODS COBRI microscopy COBRI microscopy detects forward scattering light from nano-sized particles via imaging-based interferometry (see schematics in Figure 1). The light source is a continuous wave laser of 532-nm wavelength (Finesse Pure, Laser Quantum). The laser beam is spatially filtered by a single-mode fiber. The beam is rapidly scanned at 220–330 kHz by a two-axis acousto-optic deflector (AOD, DTSXY-400, AA Opto Electronic), synchronized with the acquisition of COBRI images via a high-speed CMOS camera (Phantom v711, Vision Research).30 The beam scanning produces uniform illumination over a desirable area, which allows visualization of weak scattering signal in the raw COBRI image during the measurement without further image processing. The deflected beam is projected to the back focal plane of lens L3 (f3 = 6 cm) via relay lenses L1 (f1 = 30 cm) and L2 (f2 = 30 cm). The sample is placed at the front focal plane of L3. Without beam scanning, the illuminating Gaussian spot size is ~ 3 μm in diameter. Uniform illumination over a larger area can be realized when scanning. The achievable uniform illumination area depends on the time allowed for beam scanning, i.e., the exposure time. At the highest image acquisition rate of 100 kHz (exposure time = 9.6 μs), the scanning beam can create uniform illumination over an area of ~6 × 6 μm2, sufficient for the measurements presented in this work. At a lower speed of 1 kHz, uniform illumination over 25 × 25 μm2 can be obtained. The sample position can be adjusted in three dimensions by the combination of an XY motorized stage (MLS203, Thorlabs) and a Z piezo stage (MZS500-E, Thorlabs). A 22


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custom-made top-stage incubator (Live Cell Instrument) is placed on top of the piezo stage, which maintains the sample at 37°C and with 5% CO2. The COBRI signal and the fluorescence signal are collected by an oil-immersion microscope objective (UPLSAPO 100XO, NA1.4, Olympus) in transmission geometry. The COBRI and fluorescence signal are separated by a dichroic mirror (DM, FF560-Di01, Semrock), and projected to the CMOS camera and an EMCCD camera (iXon Ultra 897, Andor) via lenses L4 (f4 = 75 cm) and L5 (f5 = 30 cm), respectively. A long-pass filter (BLP01-532R-25, Semrock) is placed right in front of the EMCCD camera to remove the residual excitation light. The pixel resolutions of COBRI imaging and fluorescence imaging are 48×48 nm2 and 96×96 nm2 per pixel, respectively. The working range in the axial direction for continuous tracking is ~200 nm, determined by the depth of focus of the microscope objective. Interferometric detection circumvents the problem of readout noise of the camera, enabling shot-noise-limited sensitivity at high speed.52

Preparation of virus particles and cells The mCherry-labeled vaccinia mature virus was constructed by fusion of mCherry to the N terminus of A4 core protein as previously described.13 Mature virus particles were harvested in BSC 40 cells and purified by 36% sucrose cushion and subsequent CsCl gradient centrifugation as previously described.71 HeLa cells were grown in DMEM (HyClone, Thermo Fisher Scientific Inc. Waltham, MA, USA) with 10% fetal bovine serum (FBS, Gibco-BRL, Rockville, MD, USA), 100 units/ml 23


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penicillin-streptomycin (Corning Inc., Corning, NY, USA) at 37°C in 5% CO2. For live cell imaging, cells were plated on a coverglass-bottomed chamber (NuncTM Lab-TekTM II Chambered Coverglass, ThermoFisher Scientific) 24-hours before imaging.

3D localization by COBRI microscopy To localize a virus particle in 3D, the COBRI image of the particle is fitted by a 2D Gaussian function. The fitting is performed on the center part of the point spread function (9×9 pixels) where the outer concentric rings are avoided. The lateral position is given by the center of the fitted Gaussian function. The axial position is determined by the amplitude of the fitted Gaussian function and a calibration curve of height-dependent COBRI contrast of the particle that is measured separately. Note that the COBRI contrast is defined as the normalized difference in intensity caused by the presence of the particle.

Background extraction and correction Tracking nano-sized virus particles via intrinsic scattering in cellular environments is complicated by the presence of heterogeneous background scattering of the cell structures. Moderate background scattering of the cytoskeleton deteriorates the localization accuracy, and large background scattering of the cell nucleus overwhelms the signal of the particle of interest. Proper background correction and image processing is key to single-particle tracking with nanometer spatial precision. In this work, 24


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we always extract and remove the heterogeneous background due to the inevitable non-uniform illumination and scattering from the cell structures before localization. The successful background subtraction relies on the fact that the background is relatively stationary compared to the mobile particles of interests. This condition is generally valid in high-speed imaging where the cellular background is almost unchanged within the total observation time of a few seconds. In contrast, motions of small particles are inherently dynamic due to the diffusion driven by thermal fluctuation. The virus dynamics reported in this work, i.e., landing and diffusion of the virus particles on the plasma membrane, are highly rapid events. Therefore, the signal of the particle has distinct spatial and temporal signatures compared with the cellular background. To find the background, we examined the statistics of the fluctuating intensity for a recorded image series pixel by pixel. If the intensity fluctuation of a pixel was comparable to its shot-noise fluctuation (within a factor of two), we simply used the median value of the recorded intensities as the background value of that pixel. If the fluctuation exceeded that threshold, we assumed that the change in intensity was due to transient occupation of that pixel by the mobile particle, and those large fluctuations should be avoided when determining the background value. In the calibration measurements where the particles were fixed on a clean coverslip, we captured a background image separately by imaging an empty area near to the particles by laterally translating the sample. Once the stationary background had been obtained, we used it to correct all images of the recorded video. The detected intensity of the raw image is the sum of the background, the signal, and their interference, which can be written as 25


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= || + | | + 2||| |cosφ

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(1)

where , , and are the transmitted background, the forward scattering signal of the particle, and their relative phase difference, respectively. In our case, the forward scattering intensity of the biological particle was much weaker than the transmitted background intensity, so ≅ || + 2||| |cosφ

(2)

When provided with the extracted stationary background || , the background-free signal | |cosφ can be obtained by first subtracting || from followed by dividing by ||. In practice, we found that directly dividing by || gives similar localization results.

Local delivery of virus particles by glass micropipette In order to increase the success rate of capturing landing events of single virus particles, a local delivery system was implemented. A high density of virus particles (~1010 virions/mL) was loaded into a glass micropipette with an opening of ~ 4 μm in diameter. The micropipette was mounted on a motorized micromanipulator (TransferMan4, Eppendorf) and the pressure was controlled by an electronic microinjector (FemtoJet 4, Eppendorf). The opening of the micropipette was placed a few microns above the cell membrane. Through precise pressure control (precision of 0.01 psi), virus particles could be gently delivered to the volume above the cell. The virus particles underwent Brownian motion in solution after injection, and some virus particles attached to the cell membrane.

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Capture of rare events with a post trigger In high-speed imaging, capturing rare biological events (e.g., attachment of a virus particle on the cell membrane) becomes challenging because the total available recording time is very limited due to the large amount of memory required per unit time. An on-board memory of 10 GB only allows for a continuous recording time of < 4 seconds at 100 kHz with a resolution of 128×128. In order to capture rare events at high speed, we exploited the post trigger function of the high-speed CMOS camera. In operation, the camera continuously records and stores images into a running buffer memory. When we observed an event taking place, we manually sent a trigger signal to the camera and a specified number of frames before and after receiving the trigger signal were recorded. We set an adequate number of recorded frames before the camera received the trigger to compensate for the operator’s response time. With post trigger, we were able to successfully capture very rare events at will.

Calculation of MSD and apparent diffusion coefficient The time-averaged MSD as a function of the time interval ∆t was calculated according to:

!"jδt + nδt − "jδt%& , MSDΔt = nδt = ∑ '(

(3)

where δt is the frame time, "· is the lateral position of the particle, N is the total number of frames of the trajectory, and n and j are positive integers. In the model of anomalous diffusion, MSD is written as:42 27


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MSDΔt = D, Δt , + ε.//012 ,

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(4)

where α is the anomalous exponent, D, is the anomalous diffusion coefficient, and ε.//012 is the offset due to the localization error. We fit the MSD with the model described in Eq. (4) with three fitting parameters: α, D, , and ε.//012 . The apparent diffusion coefficient D344 Δt is calculated as: D344 Δt = !MSDΔt − ε.//012 %/4Δt.

(5)

Detection of TCZs We used the method described by Simson et al.72 with slight modifications for detection of TCZs. The trajectory was divided into segments with a sliding window of 30 steps. For each segment, the sum of squared displacement between all 29 adjacent steps was calculated. When motion is confined locally, the value of the sum of squared displacement decreases. When the value was lower than a threshold (0.0015 μm2), we defined it as transient confinement. No smoothing was used in our calculations. We note that, for ultrahigh-speed imaging, diffusion length at the shortest time interval is small (√2DΔt = 4.7 nm for D = 1.1 μm2/s and Δt = 10 μs), comparable to our localization error. In order to reduce the effect of localization error on TCZ detection, we chose to use the total displacement (instead of the maximal displacement)72 as the parameter, which provides more reliable results. In the simulation of Brownian motion, we used the apparent diffusion coefficient measured in the submillisecond timescale (1.1 µm2/s) because the transient confinements occurred in the submillisecond timescale. The length of the simulated trajectory is the same as the experimental data. 28


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To ensure a fair comparison, the experimental localization error of 2 nm (standard deviation) was included in the simulated trajectory by adding a Gaussian error to every step.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.xxxxxxx. Comparison of COBRI imaging and conventional brightfield imaging of live biological cells and nano-sized particles; contrasts of dielectric and metallic particles under different illuminations of varying spatial and temporal coherence; illumination patterns with and without acousto-optic deflector (AOD) beam scanning; residual cellular background fluctuation after digital cellular background removal; trajectories of single virus particles landing on the cell plasma membranes; trajectories of virus particles attaching to a coverslip without cell membranes; transient lateral diffusion coefficient of the virus particle immediately after binding to the plasma membrane; trajectory of a virus particle slowly migrating and surfing on cell membrane; apparent diffusion coefficient as a function of observation timescale, COBRI image of 80 nm silica particles (PDF) COBRI imaging of single vaccinia virus particle at continuously varying axial positions (AVI) Simultaneous observation of single virus particle landing on cell plasma membrane via fluorescence imaging and COBRI imaging (AVI) 29


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Single-virus trajectory showing transient confinements in nano-zones on the cell plasma membrane immediately after landing (AVI) Ultrahigh-speed (100 kHz) direct visualization of rapid diffusion of single vaccinia virus particle on cell plasma membrane immediately after landing (AVI)

ACKNOWLEDGEMENTS This work is supported by the Nano Program of Academia Sinica and the Ministry of Science and Technology, Taiwan (grants no. 102-2112-M-001-002-MY3). Y.F.H. acknowledges financial support from the Academia Sinica Postdoctoral Research Fellowship. We thank L.-W. Tu at the Computer Center of the Institute of Molecular Biology, Academia Sinica for her kind support on preparing the Table of Contents graphic.

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Figure 1. Schematics of COBRI microscopy. A continuous wave laser beam at 532 nm is spatially filtered by a single mode fiber (SM). The beam is rapidly scanned by a two-axis acousto-optic deflector (AOD), and the deflected beam is projected to the back focal plane of lens L3 via relay lenses L1 and L2. The sample is placed at the front focal plane of L3. The biological sample is immersed in aqueous buffer solution supported on a coverslip. The COBRI signal and fluorescence signal are collected by an oil-immersion microscope objective (OBJ) in transmission geometry. The COBRI and fluorescence signal are separated by a dichroic mirror (DM), and projected to a high-speed CMOS camera and an EMCCD camera via lenses L4 and L5 respectively. Inset: close-up schematics of the sample under illumination.

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Figure 2. Direct visualization and high-precision tracking of single virus particles by COBRI microscopy. (a)(b) COBRI image (a) and fluorescence image (b) of single virus particles on a coverslip. (c) COBRI images of a virus particle at different z positions. The contrast changes from dark to bright as the particle moves in the z plane. (d) Measured contrast of the virus particle as a function of its z position. The error bars correspond to one standard deviation. The red curve is a sinusoidal calibration function that fits the measured data. The gray area marks the working range within which localization precision better than 5 nm in 3D can be facilitated. (e) Estimated localization precision in lateral and axial directions as a function of the z position of a virus particle. (f) 3D localization of a single virus particle immobilized on a coverslip at an acquisition rate of 5 kHz. Localization precisions (standard deviations) in x, y, z directions are 2.3 nm, 1.7 nm and 2.3 nm, respectively. (g) Calibration curves (gray) measured from 62 different virus particles and their average (red).

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Figure 3. Digital cellular background extraction and removal. The relatively stationary background is calculated from a series of images (see text for more details). Removal of this background from raw images gives background-free images of the virus particle on the plasma membrane of live cells.

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Figure 4. Fast exploration and transient nano-confinement of a single virus particle on the plasma membrane immediately after landing. (a) Reconstructed 3D trajectory of a single virus particle landing on the plasma membrane. The virus particle was locally confined in zones of tens of nanometers within 1 second after landing. (b)(c) Lateral (x-y) (b) and orthogonal (x-z) (c) projections of the 3D trajectory shown in (a). After landing on the plasma membrane, the virus particle diffused across the membrane and was transiently confined in zones < 100 nm. (d) Lateral (x-y) plot of a virus landing trajectory. It shows fast exploration on the membrane for < 0.1 second immediately after landing. The virus was transiently trapped in a nano-zone of ~50 nm in diameter for ~0.3 seconds and migrated to a tight nano-confinement of 20 nm nearby. Inset: close-up view of the tight confinement. (e) Lateral (x-y) plot of a virus landing trajectory, showing escape from and revisit to a 50-nm confined zone (see Supplementary Video 3). All data in this figure were recorded at 5 kHz. 34


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Figure 5. Ultrahigh-speed 3D tracking of a single vaccinia virus particle on a cell plasma membrane immediately after landing. All data in this figure were captured at 100 kHz. (a) Rapid exploration of a virus particle on the cell membrane (see also Supplementary Video 4). (b) MSD analysis of the trajectory in (a), displaying anomalous subdiffusion in the timescale from 10 μs to 1 ms with an anomalous exponent of 0.95. (c) Reconstructed 3D trajectory, showing spatially-varying height distribution across the membrane with nanoscopic details. (d) Displacements of the virus particle in three directions (x (blue), y (red), and z (black)) as a function of time. Lateral displacements (x, y) are clearly larger than axial displacement, implying the virus is attached to the membrane. (e) 35


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Trajectory (gray) with TCZs highlighted in red. (f) Three representative segments of a trajectory exhibiting TCZs (highlighted in red). (g) Histograms of the residence times in TCZs detected from an experimental trajectory (red) and simulated Brownian motion (blue). Solid lines are exponential fits, giving residence lifetimes of 45 μs and 25 μs for the virus and simulated trajectories, respectively. Three times more TCZs with longer residence times are detected from the virus trajectory than from the simulated Brownian motion.

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Coherent Brightfield Microscopy Provides the Spatiotemporal

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