Using Holographic 3D Imaging to Observe Virus-Induced Cytopathic Effects

Cytopathic effects (CPEs) are a hallmark of infection. Due to the phototoxicity of classical optical microscopy, CPEs are difficult to observe. This study reports different patterns of viral infection in live cells using Digital HoloTomographic Microscopy (DHTM). DHTM is label-free and records the phase shift of low-energy light passing through the sample on a transparent surface with minimal disturbance. DHTM measures the Refractive Index (RI) of the cells and calculates the Refractive Index Gradient (RIG), revealing the optical heterogeneity of the cells. The study found that infection with Vaccinia Virus (VACV), Herpes Simplex Virus (HSV), and Rhinovirus (RV) significantly increased RIG over time. VACV infection caused oscillations in cell volume, while all three viruses altered the dynamics of the plasma membrane and induced apoptotic features similar to those caused by Staurosporine. In summary, this study introduces DHTM as a quantitative, label-free microscopy technique for infection research, revealing virus type-specific changes and CPE in live cells with minimal disturbance.

Fig.1. Digital HoloTomographic Microscopy and Optical Microscopy Images of Fixed and Permeabilized Cells. (A) HeLa-ATCC

Cells were incubated with 2ml RPMI at 37°C for 8 hours, fixed in PBS with 4% PFA, and imaged with DHTM microscope and the accompanying fluorescence module. Under the control of early/late promoter (VACV E/L-GFP), cells were infected with GFP-labeled VACV at an MOI of 2 (top and middle images) and uninfected images (bottom image). The top image depicts VACV-GFP infected cells at the early infection stage. The middle image depicts a rounded cell, indicating the late stage of VACV infection. The bottom image shows an uninfected cell. RI is displayed in grayscale, GFP in green, and DAPI-stained nuclei in blue. Scale bar is 20μm. (B) Schematic of RI calculation. Cells are considered as gradient refractive index micro-lenses, changing their optical properties based on biochemical activity. (C) RIG is derived from RI, representing voxel measurements based on refractive index in 3D space. The equipped value of the intermediate voxel is represented as the intermediate blue box, calculated based on the difference with the light blue voxels in the 3D neighboring area. Note: The reference beam (curved green dashed line) does not pass through the sample. RI is based on the variation between the beam (vertical green dashed line) and the reference beam.

Fig.2. VACV Late Gene Expression Increases RIG in HeLa-ATCC Cells. (A) Representative images obtained by incubating with 2ml RPMI at 37°C for 8 hours and imaging with DHTM every 2 hours. In the presence of 10 M AraC, uninfected cells were infected with VACV-GFP at an MOI of 2, or infected with VACV-GFP at an MOI of 2. The perimeter of cells growing on the coverslip is outlined with a white dashed line. RIs are described as intensity values in the “thermal” lookup table. The obtained images are holograms and depicted as projections of maximum values along the z-axis of the 3D stack. Scale bar is 20μm. (B) Normalized quantitative RIG shown in image A. RIG values at 0 hours post-infection (pi) are normalized to 1. Bar graphs represent data from at least 10 cells collected at each condition and time point, with data presented as mean ± SEM. (C) Comparison of the “blebbing” phenotype frequency between VACV-GFP infected cells and those in RPMI with 10 M AraC. Data from at least 10 cells at each time point and condition were manually scored as blebbing or non-blebbing.

Fig.3. Observing Cell Morphology and VACV-GFP Transgene Expression Using Phase Contrast and Fluorescence Live Cell Time-Lapse Microscopy. HeLa-ATCC cells uninfected (left image) or infected (right image) with VACV-GFP using a cold adhesion scheme (30 minutes on ice, wash, transfer to 37°C). Cells were imaged every 5 minutes for 8 hours using a high-throughput wide-field microscope, observing cells with transmitted light and GFP fluorescence intensity color-coded (color from transparent to blue to white). Scale bar is 50μm. Also refer to the videos in supplementary materials S1.

Fig.4. Benchmarking Volume Measurement Using Specified Diameter Polystyrene Beads. (A) Specified diameter polystyrene beads (Tetraspeck; ThermoFisher) were diluted in PBS to settle at the bottom of the dish, imaged with the 3D Cell Explorer microscope. Small colored boxes show the digital RI staining of red beads on a black background. The large box displays the complete 3D visualized holographic image obtained. The 3D stack images of the beads were digitally stained (voxel segmentation), with RI covering at least 95% of the bead volume. Scale bar is 5μm. (B) Comparison of volume quantification through voxel summation using voxel counting in STEVE software or surface fitting in Imaris. The diameter of the bead was calculated based on voxel counts and voxel size and compared with the nominal diameter provided by the manufacturer.

Fig.5. Morphological and Volume Dynamics of VACV-GFP Infected Cells Observed Using Label-Free Real-Time DHTM. Following the cold adhesion scheme, HeLa-ATCC cells were infected with VACV-GFP. Holograms of cells were collected every minute for a total duration of 8 hours. RI is displayed as a grayscale image. Volume measurement was conducted using Imaris software through surface fitting and 3D rendering (see the lower left corner of each frame). The bottom right image shows the relative volume normalized to 0 minutes pi (in arbitrary units [a.u.]). The red dashed line corresponds to the time point of the respective hologram. Scale bar is 20μm. Also refer to the videos in supplementary materials S2 and S3.

Fig.6. HSV-1 and RV Increase Cell RIG in Late Infection. (A) Representative images of uninfected and infected cells taken at specified time points by DHTM. HeLa-ATCC cells infected with HSV-1-GFP (MOI of 10), HeLa-Ohio cells infected with RV-A1a (MOI of 50). The perimeter of the cells growing on the coverslip is outlined with a white dashed line, RIs represented by intensity in the “thermal” lookup table. The obtained images are holograms and depicted as projections of maximum values along the z-axis of the 3D stack. Scale bar is 20μm. (B) Normalized quantitative RIG shown in image A. Bar graphs represent data from at least 10 cells collected at each condition and time point, with data presented as mean ± SEM.

Fig.7. Drug Treatment Can Inhibit RIs Changes in RV-A1a Infected Cells. Representative images of HeLa Ohio cells simulated infection (top image) or RV-A1a infected (MOI of 50) (bottom three images). Cells were treated with PIK93 (5 M) or MLN9708 (10 M) and imaged using DHTM every minute for 8 hours. Cell membranes are labeled in green, and high RI and RIG regions are shown in red. The images in the left column display central z-axis slices of the reconstructed hologram, and the images in the right column are 3D reconstructions of the hologram. Also refer to videos S4 to S7. Scale bar is 10μm.

Fig.8. Staurosporine-Induced Apoptosis Rapidly Increases Cell RIG. (A) Representative DHTM images of HeLa-Ohio cells incubated with Staurosporine (10 M) at 37°C for 5 hours. The perimeter of cells on the glass coverslip is outlined with a white dashed line, RIs represented by intensity in the “thermal” lookup table. The obtained images are holograms and depicted as projections of maximum values along the z-axis of the 3D stack. Scale bar is 20μm. (B) Normalized quantitative RIG shown in image A. RIG values at 0 hours post-infection (pi) are normalized to 1. Bar graphs represent data from at least 10 cells collected at each condition and time point, with data presented as mean ± SEM.

Conclusion:This study introduces label-free Digital HoloTomographic Microscopy (DHTM) and the measurement of Refractive Index Gradient (RIG) in live virus-infected cells, using DHTM to describe virus type-specific cytopathic effects, including cyclical volume changes in Vaccinia Virus infection distinct from apoptotic cells, and cytoplasmic condensation in Herpes and Rhinovirus infections. This work first demonstrates that DHTM is suitable for observing cells infected with viruses and distinguishing virus type-specific features under non-invasive conditions. It lays the foundation for future research where related fluorescence microscopy of cell and virus structures can annotate different RIG values derived from DHTM.

This article titled “Label-Free Digital Holo-tomographic Microscopy Reveals Virus Induced Cytopathic Effects in Live Cells” was published in November 2018 in mSphere.

Paper Link:

https://msphere.asm.org/content/3/6/e00599-18

The cell imaging part of the article utilized Nanolive’s 3D cell imaging technology. Nanolive’s label-free 3D imaging, also known as live cell micro-CT, successfully breaks through the optical diffraction limit of 167nm through multi-angle diffraction field tomography technology, achieving a lateral resolution of up to 75nm, making it very suitable for nanoscale observation of live cell structures, such as various organelles. In 1994, super-resolution fluorescence microscopy technology (awarded the Nobel Prize in Chemistry in 2014) first broke the optical diffraction limit of 200nm, and Nanolive is currently the only second optical imaging technology to break the 167nm limit, and the only technology capable of non-invasive, label-free live cell nanoscale imaging. This technology was published in Nature, Cotte Y. et al., Marker-free phase nanoscopy, Nature Photonics, 2013. The 3D Cell Explorer series is an exploratory tool that does not conflict with any existing technology. Since the phenomena revealed by the 3D Cell Explorer represent the most authentic and essential changes in cells (the 3D Cell Explorer is currently the only technology capable of constructing label-free live cell 3D cell structures at the 167nm scale), it allows researchers to explore from a new perspective, aiding in the discovery of new cellular activities and can be combined with traditional experimental methods for deeper research, including various high-end confocal, electron microscopy, atomic force, nuclear magnetic resonance, mass spectrometry, etc.

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Nanolive’s real-time label-free 3D microscope series imaging system is a newly developed technology in recent years, which has been applied in several fields and is continuously expanding:

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