key: cord-0690889-lc9uhc8y
authors: Loconte, Valentina; Chen, Jian-Hua; Cortese, Mirko; Ekman, Axel; Le Gros, Mark A.; Larabell, Carolyn; Bartenschlager, Ralf; Weinhardt, Venera
title: Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells
date: 2021-10-28
journal: Cell Rep Methods
DOI: 10.1016/j.crmeth.2021.100117
sha: f8e14cc18d7daf6f10a52f3a95bb8c52a25ea854
doc_id: 690889
cord_uid: lc9uhc8y

High-resolution and rapid imaging of host cell ultrastructure can generate insights towards viral disease mechanism, for example for a severe acute respiratory 2 syndrome coronavirus-2 (SARS-CoV-2) infection. Here, we employ full-rotation soft X-ray tomography (SXT) to examine organelle remodeling induced by SARS-CoV-2 at the whole cell level with high spatial resolution and throughput. Most of the current SXT systems suffer from a restricted field of view due to use of flat sample supports and artifacts due to missing data. In this approach using cylindrical sample holders, a full-rotation tomogram of human lung epithelial cells is performed in less than 10 minutes. We demonstrate the potential of SXT imaging by visualizing aggregates of SARS-CoV-2 virions and virus-induced intracellular alterations. This rapid whole cell imaging approach allows us to visualize the spatiotemporal changes of cellular organelles upon viral infection in a quantitative manner.

To assure safe handling of viruses at the reduced biosafety level of the synchrotron, infected cells must be 71 neutralized or inactivated with fixatives prior to imaging. As SXT does not require chemical fixation of 72 cells, previous studies focused on characterizing the achievable contrast and quantitative nature of the X-73 ray absorption coefficient used to discriminate cellular organelles in unfixed cells (Ekman et To address this discrepancy, we characterized the soft X-ray linear absorption coefficient in native 77 compared with fixed human B cells. We were able to discriminate heterogeneous organelles in the 78 cytoplasm and changes in molecular composition, mainly carbon and nitrogen, in fixed cells that were 79 comparable to those in the native state. Our data shows that aldehyde fixation as performed in this study 80

does not interfere with quantitative X-ray imaging and, as with other microscopy techniques, many insights 81 into virus-induced cell remodelling can be studied on fixed cells with SXT. 82

Here, we illustrate the potential of SXT to characterize and quantify structural changes induced in cells 83 available to any laboratory and will provide the opportunity to visualize subcellular structures in whole cell 93 volumes without the need for complex and demanding imaging equipment. 94

The physical process resulting in a soft X-ray image is that of X-ray absorption, with a linear X-ray 97 absorption coefficient associated with each voxel of the reconstructed 3-D image. This is similar to medical 98

Computed Tomography (CT) where a linear attenuation coefficient, measured in Hounsfield units (HU), is 99 associated with each voxel of the patient's image. This is a quantitative way to distinguish density 100 differences between different tissues. For example, bone has a very different HU than lung tissue, and a 101 tumor has a different HU to normal tissue. For SXT, the linear absorption coefficient (LAC) is well 102 characterized in human B lymphocytes in hydrated and cryo-preserved cells (Larabell and Nugent, 2010; 103 Ma et al., 2020; Myllys et al., 2016; Weinhardt et al., 2020) . However, when working with infectious 104 pathogens cells must be neutralized with fixatives prior to imaging. In these cases, fixation protocols are 105 designed to ensure full inactivation of viruses and not optimized in terms of ultrastructural preservation. 106

To investigate whether the LAC of soft X-rays can be used to differentiate organelles in fixed cells, we 107 cultured, fixed with glutaraldehyde, then plunge froze the human B cells (cell line GM12878). The virtual 108 slices of fixed and "native-state" cells are shown in Figure 1A and Video S1. To the naked eye, X-ray 109 absorption contrast between organelles in native and fixed cells is comparable. In terms of gross 110 morphology, fixed human B cells show a convoluted shape with distorted cytoplasm. This cell blebbing is 111 a well-known effect of aldehydes on cell structure (Zhao et al., 2014) . Additionally, we found distorted 112 lipid droplets in some fixed cells (n=2/10, Figure S1 ), potentially due to known challenges in fixation and 113 permeabilization of lipid droplets (Ohsaki et al., 2005) . At the level of SXT resolution, we did not observe 114 significant differences in organelle morphology. 115

To quantitatively compare X-ray LAC, we segmented the major organelles in native (n=7) and fixed cells 116 (n=10), see Table S1 . Although aldehyde-based fixatives have been shown to alter biochemical content of . Thus, we conclude that the LAC of soft X-rays can be applied as a quantitative parameter to identify 123 and unambiguously segment organelles in SXT datasets for cells fixed as described in this study (see STAR 124

Methods). Additionally, due to the progressive increase in specimen thickness during rotation (Cinquin et al., 2014) , 135

visualizing cells from all angles is not possible and results in "missing wedge" artifacts. 136

To investigate the difference between flat and cylindrical sample holders, we reconstructed a native human 137 B cell, depicted in Figure 1A , using those projection images corresponding to the typical tilt angles of ±65° 138 rotation (Harkiolaki et al., 2018) , typically used for flat grids, see Figure 2C and Figure S2 . The missing 139 angular data results in streak artifacts over the virtual plane and, at the regions where X-ray projections are 140 missing, leads to distortions and decreased contrast between organelles. This simulation has the advantage 141 of examining the exact same cell but excludes the interference from the flat specimen holder itself that is 142 typically encountered. This "shadowing" of a sample holder shifts X-ray LAC values used for segmentation 143 and identification of organelles in SXT, see Figure S2A We employ an alternative method of sample handling and place cells in thin-wall glass capillaries, see 147 Figure 2A . This approach helps to match the SXT field of view with the diameter of a cell, ensures artifact-148 free imaging enabled by full 360° range of rotation and similar transmission for all angles, and generates a 149 reconstruction of the cell volume unobscured from the sample holder; this approach assures quantitative 150 LAC values. As projections are acquired over more rotation angles than in limited angle tomography, SXT 151 imaging in cylindrical sample holders could potentially introduce more radiation dose. Thus, we have 152 simulated X-ray radiation dose in SXT reconstructions for 360° rotation and limited ±65° angle tomography 153 acquired with the same sampling (1 projection image per 1° angle), see Figure 2D . As X-ray radiation dose 154 is linearly proportional to the number of X-ray photons per reconstructed voxel, the X-ray radiation dose 155 of 360° rotation tomography does not increase in comparison to limited angle tomography. On the contrary, 156 the progressive increase of exposure time required to compensate for the lower transmission of X-rays at 157 higher rotation angles results in higher radiation dose, which is distributed unevenly throughout the sample. 158

The distribution of X-ray dose in full 360° rotation tomography is more homogeneous for a cell than 159 tomography acquisitions with 180° rotation, and ±65° angle tomography, see Figure S2B and B. Moreover, 160

with the increasing rotation angle θ, the thickness of cells on flat specimen holders increases as cos(θ), such 161 that a 5 µm thick region at 0° becomes 11.8 µm thick at ±65°. The gradual increase in cell thickness results 162 in lower transmission of X-rays and is typically compensated by a comparable increase in exposure time 163 (Chiappi et al., 2016) , resulting in even higher X-ray dose in limited angle tomography ( Figure S2B ). 164

However, if positioned correctly (towards incoming X-ray beam) a flat support will act as a "shield" with 165 more homogeneous distribution of X-ray dose in ±65° angle tomography as well. For full rotation 166 tomography, the X-ray dose is about 0.5x10 8 Gy within the cell, which is below the previously reported 167 limit of 10 8 -10 9 Gy for X-ray radiation dose of cryogenically preserved specimens (Schneider et al., 1995) . An example of an HEK293T-ACE2 cell is shown in Figure 3A to differentiate ER, even though the X-ray absorption coefficient (LACER = 0.24±0.01µm -1 ) is very similar 222 to that of cytosol (LACCyt = 0.22±0.01µm -1 ). Thus, we hypothesize that the ER network of these particular 223 cell lines is in a different configuration than ER of B cells, and that there is an insufficient difference 224 between the X-ray LAC of ER and cytoplasm for unambiguous identification, at this resolution. 225

The mitochondria, however, are clearly visible and appear as a large tubular network, occupying clearly visible due to the higher LAC (0.33 ± 0.03 µm -1 ) than that of mitochondria (LAC: 0.31 to 0.32 µm -263 1 ) and typical "ring-like" morphology ( Figure 4C and Figure S4B ). The DMVs were often found with 264 physical contacts to mitochondria, suggesting their involvement in the cross-talk between mitochondria and 265 endoplasmic reticulum as suggested by others (Singh et al., 2020) . An example of a cell with highly 266

convoluted CMs that are easily distinguishable from DMVs can be seen in Figures 4C and D.  267 The DMVs are about 366±37 nm in diameter, which is in good agreement with previously reported sizes Among cells at 24 hpi, we found one cell containing a double nucleus, see Figure 4E , Video S4. In this 286 single giant cell, we found both DMVs and CMs often located at the periphery of nuclei. Such 287 multinucleated cells, also known as syncytium, is a result of cell-to-cell cytoplasmic fusion to spread the 288 virus (Sattentau, 2008) , recently reported for SARS-CoV-2 infected cells as well (Sanders et al., 2020) . 289

Taking advantage of rapid whole-cell imaging with SXT, we performed a quantitative analysis of the 291 morphological remodelling of SARS-CoV-2 infected HEK293T-ACE2 cells at 6 and 24 hpi, see Figure 5 . 292

lipid bodies, vesicles, and virus-associated organelles. The total volume of mitochondria networks increased 294 at 6 hpi by 21% and remained unchanged at 24 hpi. Recent observations via cryoFIB/SEM have reported 295 that SARS-CoV-2 leads to disruption of the mitochondria network (Mendonca et al., 2020) . We observed 296 only local alterations of mitochondria in proximity of viral replication organelles (see Figure 4D ), consistent 297 with a recent report (Cortese et al. 2020 ). On a whole cell level (see Figure 5A ), we did not find any 298 significant difference in the volume of mitochondria (mock: 3.81 ± 1.52%, 6 hpi: 4.61±0.94%, 24 hpi: 299 3.78±1.01%), X-ray linear absorption coefficient (mock: 0.30±0.01 µm -1 , 6 hpi: 0.32±0.01 µm -1 , 24 hpi: 300 0.32±0.02 µm -1 ), number of isolated mitochondria per cell volume (mock: 5.36±1.47*10 -6 , 6 hpi: 301 5.97±1.24*10 -6 , 24 hpi: 4.08±2.29*10 -6 ) or changes in shape, measured as surface to volume ratio (mock: 302 0.29±0.06%, 6 hpi: 0.39±0.13%, 24 hpi: 0.21±0.06%). Similarly, we did not observe differences in the 303 nucleus volume or DNA packing within the nucleus in infected cells (see Figure S5 ). 304

We observed an increase in the total volume of lipid bodies in cells at 24 hpi (0.044±0.070%, total volume 305 normalized by cell volume) compared to 6 hpi (0.010±0.004%) and mock (0.012±0.007%) cells with up to 306 n=75 lipid bodies in individual cell. Together with total volume, we observed an increase in average radius 307 of the bodies (mock: 154±38 nm; 6 hpi: 155±33 nm; 184±45 nm), but no changes in the chemical 308 composition are detected (X-ray absorption coefficient in mock: 0.48±0.06 µm -1 ; 6 hpi: 0.50 ± 0.04 µm -1 ; 309 24 hpi: 0.50±0.05 µm -1 ; Figure 5B ). Such an increased accumulation of lipid bodies at later infection stages 310 are signs of a possible inflammatory response, which was recently described in SARS-CoV-2 infected 311 monocytes (Dias et al., 2020) . 312

Compared to lipid bodies, the total volume of vesicles in infected cells at first (6 hpi) increased and then 313 dropped at 24 hpi as compared to mock cells (mock: 1.01±0.51*10 -3 %, 6hpi: 1.88±1.22*10 -3 %, 24hpi: 314 1.26±1.22*10 -3 %, total vesicle volume normalize by cell volume), see Figure 5B . Additionally, we 315 observed a gradual increase in chemical density (mock: 0.36±0.04 µm -1 , 6hpi: 0.37±0.04 µm -1 , 24hpi: Here, we demonstrate that soft X-ray microscopy is a rapid imaging technique that can visualize SARS-341

CoV-2 induced cellular remodelling at the whole cell level. We show that SXT is capable of imaging To conclude, we believe that the combination of minimal sample preparation, quantitative identification of 371 cellular organelles in native and fixed cells, and rapid imaging of whole cells will meet the need for 372 systematic analysis of virus-induced cellular remodelling with statistically significant sample sizes. The colorbar is from 1.5x10 7 to 1.5x10 8 Gy. Scale bars are 1 m. 410 

Further information and requests for resources and reagents should be directed to and will be fulfilled by 443 the lead contact, Venera Weinhardt (venera.weinhardt@cos.uni-heidelberg.de). 444  Any additional information required to reanalyze the data reported in this paper is available from 451 the lead contact upon request 452

Cell culture 454

The human B lymphocytes (GM12878) were purchased from the NGIMS Human Genetics Cell Repository, conditions, all data were normalized and transferred to linear absorption coefficient by accounting for pixel 506

Simulations 508

To simulate the effect of limited rotation angle on SXT data, we have used part of 2D projection images 509 obtained for the B cell shown in Figure 1A , corresponding to the ±65° (total 130°) tilt angles. To include a 510 "shadowing effect" of the flat support, each 2D projection was multiplied by the transmission function of 511 a flat grid: Img_plate(θ)=Img(θ)*exp[-µt(θ)]. Here, µ is the absorption coefficient of a support grid 512 (formvar with carbon coating), and t is the thickness of the support for each projection angle θ. 513

The X-ray dose calculations are done as in (Weiß et al., 2000) bu cumulatively adding local dose from each 514 illumination angle and by propagating the intensity obtained from the flat field image through the 515 reconstructed sample. We used X-rays with an energy of 543 eV, and a Fano factor of 0.11 for the CCD. 516

The calculated photon count is scaled by the optical transmission of the microscope to account for the 517 reduction of dose between the sample and the CCD. For this, an X_ray objective lens, with transmission of 518 0.125 and a Si3N4 membrane with transmission of 0.67863 have been considered. Similar to 3D 519 reconstructions, X-ray dose calculations were performed for different rotation angles, that is full rotation 520 of 360°, 180°, ±65° and ±65° rotation with gradual increase in exposure time, see Figure S2C . bodies were identified setting the threshold between 0.48 and 0.90 µm -1 , with average value set at about 529 0.58 µm -1 and by thanks to their elongated shape; vesicles where label according to the lower X-ray 530 absorption, with threshold sets between 0.25-0.48 µm -1 , and average value around 0.38 µm -1 . The large 531 apoptotic body was identified by its natural contrast and the presence of an external rim in contact with the 532 cytosol; whereas the CMs where recognized due to their curved and hollow shape. DMVs were outlined 533 based on X-ray absorption values (typically higher than mitochondria) and typical "donut"-like shape. 534

Aggregates of virions were found at the cell surface and were segmented based on X-ray absorption values. 535

Finally, the ER was manually segmented by outlining features in each virtual slice. 536

To visualize ultrastructure of large compartments, we applied series of Laplacian filters on the images. We 538 used the Laplace of the Gaussian susceptible to spherical structures between 100 and 200 nm in radius. The 539 maximum of the scale-normalized Laplacian operator t(Lxx+Lyy+Lzz), where t is the standard deviations of 540 the Gaussian filter was used to visualize the substructure of the compartment, as shown in Figure S4C . 541

All statistical analyses were undertaken using Prism (version 8.0, GraphPad Software). t-test and analysis 543 of variance (ANOVA) analyses were performed to determine statistical significance among two or three 544 conditions, respectively. Post hoc analysis was performed to identify difference in means for specific 545 conditions. 546

Video S1. Related to Figure 1 infected HEK293T cell. Each organelle is color coded as: nucleus -blue, mitochondria -green, lipid 557

bodies -yellow, vesicles -cyan, virions -magenta, DMVs -red, CMs -orange, and large compartment -558 grey. 559 560

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