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LitCovid-sentences  

PMC:7473770 JSONTXT 19 Projects

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Id Subject Object Predicate Lexical cue
T1 0-104 Sentence denotes 3D virtual pathohistology of lung tissue from Covid-19 patients based on phase contrast X-ray tomography
T2 106-114 Sentence denotes Abstract
T3 115-377 Sentence denotes We present a three-dimensional (3D) approach for virtual histology and histopathology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal architecture of unstained lung tissue from patients who succumbed to Covid-19.
T4 378-643 Sentence denotes Based on this first proof-of-concept study, we propose multi-scale phase contrast x-ray tomography as a tool to unravel the pathophysiology of Covid-19, extending conventional histology by a third dimension and allowing for full quantification of tissue remodeling.
T5 644-862 Sentence denotes By combining parallel and cone beam geometry, autopsy samples with a maximum cross section of 8 mm are scanned and reconstructed at a resolution and image quality, which allows for the segmentation of individual cells.
T6 863-990 Sentence denotes Using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of 167 nm.
T7 991-1327 Sentence denotes We exemplify the capability of this approach by 3D visualization of diffuse alveolar damage (DAD) with its prominent hyaline membrane formation, by mapping the 3D distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment.
T8 1329-1341 Sentence denotes Introduction
T9 1342-1589 Sentence denotes Severe progression of the 2019 coronavirus disease (Covid-19) is frequently accompanied by the clinical acute respiratory distress syndrome (ARDS) and respiratory failure, an organ manifestation responsible for the majority of Covid-19 fatalities.
T10 1590-1843 Sentence denotes Lung injury associated with ARDS can be readily detected by radiographic chest imaging and clinical computed tomography (CT), which have assisted the diagnosis and management of Covid-19 patients (Lee et al., 2020; Shi et al., 2020; Chung et al., 2020).
T11 1844-2111 Sentence denotes Here, so-called peripheral lung ground-glass opacities are the main radiological hallmark of ARDS, and can be linked to the histological observation of diffuse alveolar damage (DAD) with edema, hemorrhage, and intraalveolar fibrin deposition (Ackermann et al., 2020).
T12 2112-2258 Sentence denotes These findings have also been reported for infections by Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and influenza viruses.
T13 2259-2507 Sentence denotes Distinctive features of pulmonary involvement of Covid-19 include severe endothelial injury associated with the presence of intracellular virions and inflammation, disrupted cellular membranes, as well as widespread thrombosis with microangiopathy.
T14 2508-2745 Sentence denotes As reported in Ackermann et al., 2020, alveolar capillary microthrombi were found to be nine times as prevalent in patients with Covid-19 as in patients with the also very aggressive H1N1 influenza A virus, also referred to as swine flu.
T15 2746-2958 Sentence denotes Importantly, in Covid-19 lungs, a specific variant of new vessel growth - intussusceptive angiogenesis - was significantly more prevalent, that is, 2.7 times as high as in lungs of patients with H1N1 influenza A.
T16 2959-3214 Sentence denotes Histomorphological assessment of formalin-fixed, paraffin-embedded (FFPE) tissue stained with haematoxylin and eosin still represents the gold standard in histological diagnostics of non-neoplastic lung diseases, including DAD and virus induced pneumonia.
T17 3215-3490 Sentence denotes In order to unravel the corresponding pathophysiology of the lungs, digitalization, visualization and quantification of the morphological changes associated with Covid-19 represent a key challenge, and require both high resolution and the capability to screen larger volumes.
T18 3491-3709 Sentence denotes For this reason, imaging the intricate three-dimensional (3D) tissue architecture of the lung and its pathological alterations on multiple length scales calls for 3D extensions of well-established histology techniques.
T19 3710-3914 Sentence denotes In this work, we want to demonstrate the potential of propagation-based phase contrast x-ray tomography as a tool for virtual 3D histology in general, and in particular for the histopathology of Covid-19.
T20 3915-4134 Sentence denotes Our work is based on the assertion that integration of 3D morphological information with well-established histology techniques can provide a substantial asset for unraveling the pathophysiology of SARS-Cov-2 infections.
T21 4135-4336 Sentence denotes To this end, we have collected x-ray tomography data from the same autopsies, which were previously studied by immunohistochemical analysis and measurements of gene expression (Ackermann et al., 2020).
T22 4337-4446 Sentence denotes We ask in particular, whether DAD and the morphology of blood vessels can be visualized and quantified in 3D.
T23 4447-4602 Sentence denotes This is a timely scientific question in Covid-19 research, especially in view of increased intussusceptive angiogenesis reported in Ackermann et al., 2020.
T24 4603-4719 Sentence denotes Here we present first results obtained from the postmortem lung samples of six patients who succumbed from Covid-19.
T25 4720-5049 Sentence denotes We exemplify the capability of this approach by 3D visualization of the DAD with hyaline membrane formation, by mapping the 3D distribution and density of angiocentric inflammation (perivascular T-cell infiltration), and by providing histograms of characteristic distances from the tissue interior to the closest air compartment.
T26 5050-5389 Sentence denotes In contrast to conventional histology based on thin sections, propagation-based x-ray phase-contrast tomography (PC-CT) offers a full 3D visualization with isotropic resolution and without destructive slicing of the specimen (Ding et al., 2019; Dejea et al., 2019; Saccomano et al., 2018; Töpperwien et al., 2018; Khimchenko et al., 2016).
T27 5390-5520 Sentence denotes The interaction of x-rays with the object is described by the continuous complex-valued index of refraction n⁢(𝐫)=1-δ⁢(𝐫)+i⁢β⁢(𝐫).
T28 5521-5843 Sentence denotes Phase contrast capitalizes on the fact that for hard x-rays, the real-valued decrement δ is several orders of magnitude higher in soft biological tissues than β, which accounts for absorption (Nugent, 2010) and that small contrast levels can be reconstructed by propagation imaging even at low fluence (Jahn et al., 2017).
T29 5844-6084 Sentence denotes Contrast is formed by transformation of the phase shifts into measurable intensity variations by self-interference of the exit wave during free-space propagation between sample and detector (Paganin and Nugent, 1998; Cloetens et al., 1999).
T30 6085-6246 Sentence denotes However, problems with phase retrieval, phase wrapping, insufficient coherence, or on the contrary strong phase gradients can render its application challenging.
T31 6247-6477 Sentence denotes For lung imaging it has already been demonstrated as an advantageous imaging technique up to macroscopic scales (Parsons et al., 2008; Stahr et al., 2016; Morgan et al., 2020), including live animal models of respiratory diseases.
T32 6478-6682 Sentence denotes But in contrast to other full-field phase contrast techniques, e.g. based on grating interferometry or analyzer crystals, it can also reach a resolution below optical microscopy (Khimchenko et al., 2018).
T33 6683-6955 Sentence denotes In this work we exploit both the high resolution capability of PC-CT and the fact that its phase sensitivity is high enough to probe the small electron density variations of unstained tissue, embedded in paraffin, ethanol, or even aqueous buffer (Töpperwien et al., 2019).
T34 6956-7099 Sentence denotes However, this requires careful optimization of photon energy, illumination function, and phase retrieval algorithms, as detailed further below.
T35 7100-7305 Sentence denotes The 3D virtual pathohistology approach for Covid-19 presented here was realized by implementing a novel multi-scale phase contrast x-ray tomography concept, with dedicated x-ray optics and instrumentation.
T36 7306-7541 Sentence denotes Overview and regions-of-interest (ROI) scans were recorded on the same paraffin-embedded sample, covering a maximum tissue cross section of 8 mm by stitching different tomograms, and with a minimum voxel size of 167 nm in certain ROIs.
T37 7542-7760 Sentence denotes Scale-bridging and dynamic ROI selection in close spatial and temporal proximity was implemented with dedicated instrumentation the GINIX endstation of the beamline P10/PETRA III (DESY, Hamburg) (Salditt et al., 2015).
T38 7761-7979 Sentence denotes Specifically, we combined two optical geometries, which has only been realized at different synchrotron beamlines before: (i) Parallel beam tomography, covering a large field of view (FOV), with a pixel size of 650 nm.
T39 7980-8332 Sentence denotes In this setting, a volumetric throughput on the order of 107μm/3s was achieved, while maintaining the ability to segment isolated cells in unstained tissue. (ii) Cone beam geometry for recording of highly magnified holograms, based on advanced x-ray waveguide optics, providing mode filtering, that is, enhanced spatial coherence and smooth wavefronts.
T40 8333-8440 Sentence denotes Based on the geometrical magnification, the effective pixel size can be adjusted in the range 10 nm–300 nm.
T41 8441-8522 Sentence denotes The two imaging schemes are shown schematically in Figure 1c and d, respectively.
T42 8523-8796 Sentence denotes Further, using this particular optics, together with appropriate choices of photon energy and geometric parameters, we can reach extremely small Fresnel numbers F of the deeply holographic regime, well below the typical range exploited at other nano-tomography instruments.
T43 8797-8988 Sentence denotes This offers the advantage of highest phase sensitivity sufficient to even probe the small electron density variations of unstained tissue, at relatively low dose (Hagemann and Salditt, 2018).
T44 8989-9191 Sentence denotes To exploit this sensitivity, we use advanced phase retrieval methods including non-linear generalizations of the CTF-method (Cloetens et al., 1999) based on Tikhonov regularization (Lohse et al., 2020).
T45 9192-9201 Sentence denotes Figure 1.
T46 9203-9238 Sentence denotes Multi-scale x-ray tomography setup.
T47 9239-9419 Sentence denotes (a) HE stain of a tissue micro-array paraffin block with samples of all six patients who succumbed to Covid-19. (b) Schematic representation of the sample preparation and mounting.
T48 9420-9777 Sentence denotes In a first step biopsy punches containing the entire individual lung tissues are transferred onto a holder for the parallel-beam local tomography acquisition, followed by a further reduction in size to a 1 mm biopsy punch, for cone-beam tomographic recordings. (c) Configuration for parallel-beam tomography. (d) Configuration for cone-beam holo-tomography.
T49 9779-9786 Sentence denotes Results
T50 9787-9886 Sentence denotes In total, we investigated six postmortem lung samples from Covid-19 patients (Menter et al., 2020).
T51 9887-10009 Sentence denotes A tissue micro-array paraffin block with samples of all six patients and the corresponding HE stain is shown in Figure 1a.
T52 10010-10151 Sentence denotes Information about age, gender, hospitalization, clinical, radiological and histological characteristics of all patients are shown in Table 2.
T53 10152-10340 Sentence denotes Before presenting the results for the six Covid-19 patients for each of the imaging levels (scales), we give an overview on the typical datasets for one exemplary sample (I), see Figure 2.
T54 10341-10552 Sentence denotes The typical FOVs, image quality, and appearance of the lung structure as well as the amount of data can be inferred, and inspected in the reconstructions provided online (https://doi.org/10.5281/zenodo.3892637).
T55 10553-10608 Sentence denotes The datasets are denoted by patient I-VI, respectively.
T56 10609-10865 Sentence denotes Gray values of the tomographic reconstruction represent phase shift per voxel with edge length v⁢x, the local electron density difference to the average paraffin can be computed by(1) Δρe(r)=φvx(r)vx⋅λ⋅r0,with wavelength λ and r0 classical electron radius.
T57 10866-10875 Sentence denotes Figure 2.
T58 10877-10974 Sentence denotes Overview of reconstruction volumes, exemplified for a punch biopsy of pulmonary autopsy sample I.
T59 10975-11449 Sentence denotes (a) Virtual section through the stitched reconstruction volume with indicated position for the 1 mm biopsy punch. (b) Virtual section through the reconstruction of the 1 mm biopsy punch volume with indicated position for the cone-beam dataset. (c) Virtual section through the cone beam dataset. (d) Image of an HE-stained histological section, which is in close proximity to the virtual slice in (a). (e) 3D views of the biopsy punch. (f) 3D views of the cone-beam datasets.
T60 11450-11552 Sentence denotes Scale bars: (a) 500⁢μ⁢m and (b,c) 100⁢μ⁢m. The entire reconstruction volume is visualized in Video 1. 
T61 11553-11561 Sentence denotes Video 1.
T62 11563-11631 Sentence denotes A video showing a fly-through the reconstruction volume of sample I.
T63 11632-11721 Sentence denotes First the parallel beam data set is shown, followed by the cone beam zoom reconstruction.
T64 11723-11753 Sentence denotes Reconstructed electron density
T65 11754-11870 Sentence denotes Next, we present representative slices through the reconstruction volumes of all samples for all acquisition scales.
T66 11871-11985 Sentence denotes Figure 3 presents the stitched reconstruction volumes, recorded under conditions of local tomography, see Table 3.
T67 11986-12357 Sentence denotes Conventional HE-stained histology images of all samples are shown in Appendix 1—figure 1. Since these volumes are computed from stitching up to 20 individual tomograms, the question arises to which extend the image quality is limited by potential artifacts of local tomography, that is, errors due to the fact that part of the sample is outside the reconstruction volume.
T68 12358-12547 Sentence denotes For this reason, 1 mm punches were taken after the stitched overview and rescanned in the parallel beam configuration, without local tomography conditions, since they fitted within the FOV.
T69 12548-12631 Sentence denotes The results are presented in Figure 4, and validate the previous stitching results.
T70 12632-12746 Sentence denotes The 1 mm punches then also provided an appropriate size for the cone-beam recordings, which are shown in Figure 5.
T71 12747-12869 Sentence denotes Importantly, in each scan the previous level guided the choice for the next FOV and informed about the larger environment.
T72 12870-12969 Sentence denotes In the following, we briefly discuss the samples one-by-one, with regard to all acquisition scales.
T73 12970-13079 Sentence denotes A comparison of morphological features between conventional and virtual histology is shown in the Appendix 1.
T74 13080-13089 Sentence denotes Figure 3.
T75 13091-13164 Sentence denotes Stitched parallel-beam reconstructions for full pulmonary samples (I–VI).
T76 13165-13270 Sentence denotes Representative virtual sections through the reconstruction volumes of full biopsies (I–VI), respectively.
T77 13271-13282 Sentence denotes Scale bars:
T78 13283-13291 Sentence denotes 500⁢μ⁢m.
T79 13292-13301 Sentence denotes Figure 4.
T80 13303-13364 Sentence denotes Parallel-beam reconstructions for 1 mm biopsy punches (I–VI).
T81 13365-13511 Sentence denotes Representative virtual sections through the reconstruction volumes of the 1 mm punches into the volumes of the full biopsies (I–VI), respectively.
T82 13512-13646 Sentence denotes The fact that the punches are isolated results in higher image quality, since the errors associated with local tomography are avoided.
T83 13647-13658 Sentence denotes Scale bars:
T84 13659-13667 Sentence denotes 100⁢μ⁢m.
T85 13668-13677 Sentence denotes Figure 5.
T86 13679-13824 Sentence denotes Cone-beam reconstructions for biopsy punches (I–VI), shown for approximately the same slices as in Figure 4 for the parallel beam reconstruction.
T87 13825-14024 Sentence denotes Virtual sections through the reconstruction volumes of the cone-beam recordings corresponding to sections in Figure 4, obtained by the parallel beam configuration, for biopsies (I–VI) , respectively.
T88 14025-14036 Sentence denotes Scale bars:
T89 14037-14045 Sentence denotes 100⁢μ⁢m.
T90 14047-14055 Sentence denotes Sample I
T91 14056-14330 Sentence denotes By conventional histopathological assessment, the peribronchial alveolar parenchyma of sample I showed DAD with focal formation of hyaline membranes adjacent to the epithelial lining, moderate lymphocytic interstitial pneumonia and singular thrombi in small pulmonary veins.
T92 14331-14552 Sentence denotes There is a moderate hypertrophy of the muscular media in smaller pre- and post-capillary blood vessels with desquamation of the endothelial cell layer as well as mild centrilobular emphysema (original magnification 100×).
T93 14553-14642 Sentence denotes In PC-CT, enlarged alveolar septa with pronounced lymphocytic inflammation are displayed.
T94 14643-14778 Sentence denotes The reconstruction volume contains a large artery filled with erythrocytes (Figure 3-I, lower left), which bifurcates into two vessels.
T95 14779-14844 Sentence denotes This area was then selected for the 1 mm biopsy punch extraction.
T96 14845-14932 Sentence denotes The cone-beam zoom tomogram was then centered around the perimeter of the blood vessel.
T97 14933-15074 Sentence denotes This volume is particularly well suited to investigate the connective tissue including elastic fibers and collagen, as well as smooth muscle.
T98 15076-15085 Sentence denotes Sample II
T99 15086-15292 Sentence denotes Histomorphological analysis shows peribronchial alveolar parenchyma with hyperemia of capillary and post-capillary blood vessels, as well as a moderate centrilobular emphysema (original magnification 100×).
T100 15293-15376 Sentence denotes On the level of blood vessels, both blood-filled and empty vessels are discernible.
T101 15377-15454 Sentence denotes It should be noted that septa with signs of parallel capillaries are visible.
T102 15455-15565 Sentence denotes In the reconstruction volume of the zoom tomogram, a single vessel can be easily tracked over large distances.
T103 15567-15577 Sentence denotes Sample III
T104 15578-15766 Sentence denotes The sample consists of peribronchial alveolar parenchyma showing prominent multifocal neutrophilic capillaritis as well as a moderate centrilobular emphysema (original magnification 100×).
T105 15767-15870 Sentence denotes In PC-CT, septa with again similar physiological size and distribution emerge, with moderate emphysema.
T106 15871-15952 Sentence denotes The bottom part of the sample contains a fibrous area near a larger blood vessel.
T107 15953-16030 Sentence denotes The zoom tomogram shows a single septum, a blood vessel and a fibrous region.
T108 16032-16041 Sentence denotes Sample IV
T109 16042-16242 Sentence denotes Histomorphological analysis shows peribronchial alveolar parenchyma with marked lymphocytic interstitial pneumonia, multifocal venous thrombi and focal intraalveolar fibrin deposition in terms of DAD.
T110 16243-16326 Sentence denotes Furthermore, there is a mild centrilobular emphysema (original magnification 100×).
T111 16327-16431 Sentence denotes In PC-CT, a network of thin septa, thrombi and emphysema, as well as a large empty blood vessel appears.
T112 16432-16486 Sentence denotes Electron-rich diffuse black granules are also visible.
T113 16487-16571 Sentence denotes The biopsy punch was selected to contain the empty blood vessel, some small thrombi.
T114 16572-16635 Sentence denotes It also includes thin septa and tissue embedded dirt-particles.
T115 16636-16751 Sentence denotes The zoom tomogram covered tissue with black granules as well as a band of inflammatory cells next to a septum wall.
T116 16753-16761 Sentence denotes Sample V
T117 16762-17066 Sentence denotes By conventional histological assessment, Sample V consists of peribronchial alveolar parenchyma showing massive lymphocytic interstitial pneumonia with ubiquitous hyaline membranes superimposed on the alveolar walls, neutrophilic capillaritis and multifocal post-capillary thrombi in terms of severe DAD.
T118 17067-17202 Sentence denotes Furthermore, bronchialized alveolar epithelial cells show cytopathic changes and multifocal desquamation, as does alveolar macrophages.
T119 17203-17356 Sentence denotes Focally, accumulation of intraalveolar neutrophilic granulocytes in the sense of a florid bronchopneumonia can be observed (original magnification 100×).
T120 17357-17531 Sentence denotes PC-CT data give rise to alterations of the overall morphology due to Covid-19, including substantial inflammation, pronounced hyaline membranes, and high load of lymphocytes.
T121 17532-17637 Sentence denotes The biopsy punch was chosen to include areas with increased presence of hyaline membrane and lymphocytes.
T122 17638-17766 Sentence denotes A blood vessel splitting into several smaller blood vessels is easily recognized when browsing through the reconstructed volume.
T123 17767-17882 Sentence denotes Noteworthy, different cell types as macrophages, T-cells or erythrocytes can be distinguished in the zoom tomogram.
T124 17884-17893 Sentence denotes Sample VI
T125 17894-18044 Sentence denotes Histomorphological analysis shows peribronchial alveolar parenchyma with lymphocytic interstitial pneumonitis and a singular thrombus in a small vein.
T126 18045-18119 Sentence denotes The interstitium of the alveolar septae is widened by myogenic metaplasia.
T127 18120-18183 Sentence denotes Adjacent, centrilobular emphysema and anthracosis are observed.
T128 18184-18334 Sentence denotes The bronchial mucosa shows varying degrees of lymphocytic inflammation in the sense of chronic bronchitis/bronchiolitis (original magnification 100×).
T129 18335-18489 Sentence denotes From PC-CT reconstructions, the sample consists of thin alveoli (Figure 3VI, upper left) evolving into compact, fibrotic tissue (Figure 3VI, lower right).
T130 18490-18545 Sentence denotes The amount of lymphocytes is rather low in this sample.
T131 18546-18621 Sentence denotes Black granules and some thrombi are embedded within the bulky tissue parts.
T132 18622-18784 Sentence denotes The biopsy punch covers the region of transition from alveoli to fibrotic tissue, containing also a thrombus and capillaries as identified from the zoom tomogram.
T133 18786-18848 Sentence denotes Visualization of pathologies, segmentation, and quantification
T134 18849-19014 Sentence denotes The overview scans of all paraffin embedded Covid-19 positive samples and one biopsy of a hydrated control lung were analyzed in terms of structural characteristics.
T135 19015-19126 Sentence denotes The top row of Figure 6 shows the workflow of the analysis on the example of the 3D reconstruction of sample V.
T136 19127-19240 Sentence denotes Based on the tissue mask the distances for each tissue voxel to the next voxel containing oxygen were determined.
T137 19241-19408 Sentence denotes For the analysis of the tissue it is mandatory to consider the three-dimensionality of the samples, similar to the analysis of porous structures (Müller et al., 2002).
T138 19409-19639 Sentence denotes More generally, we claim that 3D histology analysis based on x-ray tomography and light sheet microscopy (Power and Huisken, 2017) is required to quantitatively understand functional tissue properties based on its 3D architecture.
T139 19640-19788 Sentence denotes Here, this can also be seen in Figure 6d, which shows a zoom of the distance analysis around a small blood vessel which is marked with a yellow box.
T140 19789-19931 Sentence denotes While the wall thickness appears quite homogeneous in the 2d slice, the distance analysis reveals that the vessel is thicker on the top right.
T141 19932-19941 Sentence denotes Figure 6.
T142 19943-19983 Sentence denotes Tissue compactness and distance metrics:
T143 19984-20096 Sentence denotes The 3D reconstructions of the lung tissue were analyzed in terms of specific surface and characteristic lengths.
T144 20097-20157 Sentence denotes The workflow is sketched for sample V in the top part (a–g).
T145 20158-20214 Sentence denotes In a first step, the tissue was segmented using Ilastik.
T146 20215-20277 Sentence denotes A slice of the reconstructed electron density is shown in (a).
T147 20278-20514 Sentence denotes Based on the segmentation, the areas of air which are directly connected and potentially filled with oxygen (or blood) are masked out (b). (c) For each of the remaining tissue voxels the shortest possible distance to air was calculated.
T148 20515-20625 Sentence denotes Especially around vessels and larger alveoli, the distances are larger. (d) Zoom into an area around a vessel.
T149 20626-20944 Sentence denotes Further analysis is based on the distance distribution shown in (e). (f) Volume rendering of the reconstructed electron density, with (g) showing the corresponding 3D distance map. (h) Based on the tissue segmentation of all samples, the distance from the tissue interior to the closest air compartment was calculated.
T150 20945-21058 Sentence denotes In order to compare all samples, the count of voxels was normalized by the total volume of the respective sample.
T151 21059-21242 Sentence denotes The specific surface area SV (represented by the first value of each curve), the characteristic length Lc and the mean distance dO2¯ for each sample was calculated based on this data.
T152 21243-21312 Sentence denotes Double logarithmic scale, bin width of the distribution of distances:
T153 21313-21319 Sentence denotes 1 v⁢x.
T154 21320-21346 Sentence denotes Scale bars: (a–c) 100⁢μ⁢m.
T155 21347-21430 Sentence denotes The distribution of the distances obtained for a given slice is shown in Figure 6e.
T156 21431-21616 Sentence denotes The swelling of the alveolar walls as well as the inflamed blood vessels can be identified by comparing the reconstructed electron density and the 3D-distance map (see Figure 6f and g).
T157 21617-21755 Sentence denotes Figure 6h shows the distribution of the tissue-air distances (histogram) for all samples, following the workflow illustrated in Figure 6e.
T158 21756-21809 Sentence denotes The binning of distances was set to one voxel length.
T159 21810-21923 Sentence denotes The figure underlines the high diversity of the tissue structure which could already be seen in the 3D histology.
T160 21924-22031 Sentence denotes Further, it directly informs about the specific surface SV, which is given by the first point of the graph.
T161 22032-22114 Sentence denotes The corresponding parameters and metrics are tabulated in Table 1 for all samples.
T162 22115-22219 Sentence denotes Additionally, the mean concentration of lymphocytes cl within the lung tissue is listed for all samples.
T163 22220-22318 Sentence denotes The values quantify the general structure of the tissue which is qualitatively discernible by eye.
T164 22319-22448 Sentence denotes Samples with a high amount of swollen, inflamed blood vessels and thick hyaline membranes exhibit a larger characteristic length.
T165 22449-22586 Sentence denotes Note, that the control lung was prepared in a hydrated environment and shrinking due to further preparation of the sample does not occur.
T166 22587-22667 Sentence denotes Hence, the results cannot be directly compared to the paraffin embedded samples.
T167 22668-22796 Sentence denotes Further, the analysis of the lymphocyte concentration was performed since no lymphocytes were found in the reconstructed volume.
T168 22797-22910 Sentence denotes The low values of Lc and dO2¯ for sample II correlate with the lack of ground-glass opacification in clinical CT.
T169 22911-23029 Sentence denotes Based on the extracted structural parameters, the degree of inflammation and swelling of lung tissue can be evaluated.
T170 23030-23150 Sentence denotes E.g. patient II has the highest surface area volume-ratios while sample I and VI have a relatively low specific surface.
T171 23151-23350 Sentence denotes Larger characteristic lengths may also be indicative of inflammation and the formation of hyaline membranes, which will be evaluated in the following based on ROI and high-resolution reconstructions.
T172 23351-23359 Sentence denotes Table 1.
T173 23361-23709 Sentence denotes Results of the analysis of tissue characteristics: specific surface area SV, characteristic length Lc and mean distance dO2¯ and standard deviation from all tissue voxels to air as well as the mean concentration of lymphocytes cl for all six Covid-19 positive samples as well as for one control sample. Colors match the distance graphs in Figure 6.
T174 23710-23721 Sentence denotes Patient no.
T175 23723-23779 Sentence denotes SV (%) Lc (μm) dO2¯ (μm) c l   ( 10 5 / m m 3 )
T176 23780-23808 Sentence denotes I 13.87 9.4 5.9±5.3 16.0
T177 23809-23838 Sentence denotes II 46.56 2.8 2.1±1.0 14.1
T178 23839-23868 Sentence denotes III 33.75 3.9 2.5±1.5 4.4
T179 23869-23897 Sentence denotes IV 25.90 5.0 3.2±2.3 7.1
T180 23898-23925 Sentence denotes V 19.28 6.7 3.6±2.1 4.8
T181 23926-23954 Sentence denotes VI 11.87 11.0 9.1±10 6.1
T182 23955-24084 Sentence denotes CTRL (hyd.) 20.04 6.5 5.0±5.1 - Figure 7 illustrates the aggregation of hyaline membrane in the vicinity of a single alveole.
T183 24085-24192 Sentence denotes Volumetric renderings in Figure 7a and b demonstrate particular attachment of fibrin to the alveolar walls.
T184 24193-24338 Sentence denotes In cases of severe hyaline membrane formation as for this patient, this pathological alteration can be tracked throughout the volume Figure 7c–e.
T185 24339-24409 Sentence denotes In Figure 7f, hyaline membranes of neighboring alveoles are indicated.
T186 24410-24586 Sentence denotes In the 3D-context, their locations with respect to blood vessels can be inspected, see Figure 7g, which exemplifies a direct connection of hyaline membranes to the vasculature.
T187 24587-24596 Sentence denotes Figure 7.
T188 24598-24703 Sentence denotes Rendering of hyaline membrane attached to alveolar walls (patient V, parallel beam-scan of a 1 mm punch).
T189 24704-24986 Sentence denotes The rendered subvolume was restricted to 1.15×1.10×0.56 mm3, to contain a single alveole foremost. (a) Volume rendering of the segmented hyaline membrane in same spatial orientation as (c)-(e), which show virtual slices through the (c) top, (d) center and (e) bottom of the alveole.
T190 24987-25266 Sentence denotes For a better spatial classification, (b) gives a combination of the volume in (a) and the slice in (d). (f) Volume rendering of the entire subvolume including neighboring alveolae. (g) Zoom-in onto a major blood vessel (red) which is directly connected with the hyaline membrane.
T191 25267-25293 Sentence denotes Scale bars: (c–e) 300⁢μ⁢m.
T192 25294-25501 Sentence denotes The severeness of hyaline membrane formation is case-specific, as the yellow rendering in Figure 8a,b,d demonstrates reduced amounts of deposits for patient I in a subvolume of parallel-beam reconstructions.
T193 25502-25614 Sentence denotes Further, lymphocytes (red) were identified based on the automated cell segmentation (see Materials and methods).
T194 25615-25743 Sentence denotes For clearer visualization, each cell is rendered as a sphere with a size corresponding to the mean cell volume in Figure 8a,b,d.
T195 25744-25940 Sentence denotes Based on convolution of the cell positions with a sphere of 100⁢μ⁢m in radius, the local cell density was calculated (Töpperwien et al., 2018) and presented as 3D-maps of cells/mm3 in Figure 8c,e.
T196 25941-26048 Sentence denotes This concept was then translated to a 3D-stitched volume of an entire tissue block as shown in Figure 8f,g.
T197 26049-26058 Sentence denotes Figure 8.
T198 26060-26221 Sentence denotes Rendering of alveolar wall with hyaline membrane and quantification of lymphocyte infiltration (unstained tissue, patient I, parallel beam scan of a 1 mm punch).
T199 26222-26449 Sentence denotes The illustrations in (a–e) show a subvolume of 0.60×0.48×0.26 mm3, (f and g) this concept applied to the full tissue block of 2.57×2.99×0.98 mm3. (a) Yellow contours mark the locations of hyaline membrane in an exemplary slice.
T200 26450-26693 Sentence denotes In same spatial orientation, (b, d and f) volume rendering of soft tissue (light pink), infiltrated by hyaline membrane (yellow) and lymphocytes (red) and (c, e and g) their local cell density among the lung tissue, including air compartments.
T201 26694-26827 Sentence denotes Next, the segmentation of blood vessels is demonstrated for the example of a splitting blood vessel in the zoom tomogram of sample V.
T202 26828-26868 Sentence denotes The segmentation was performed manually.
T203 26869-27015 Sentence denotes To give an impression of the separation of a single capillary, a series of virtual slices in the xy-plane (magnified views) is shown in Figure 9a.
T204 27016-27111 Sentence denotes The separation starts with the creation of a branch from the blood vessel (arrow in slice 336).
T205 27112-27209 Sentence denotes In slice 326, 1.7⁢μ⁢m above slice 336, this branch evolves into an empty and separated capillary.
T206 27210-27275 Sentence denotes Another 2⁢μ⁢m above, the capillary is entirely filled with cells.
T207 27276-27358 Sentence denotes Further 2.3⁢μ⁢m, the capillary is empty again and has a diameter of about 2.8⁢μ⁢m.
T208 27359-27485 Sentence denotes The segmentation of the blood vessel with all its separated branches is indicated for slice 336 in Figure 9b by the red lines.
T209 27486-27626 Sentence denotes In this slice, three capillaries have already separated from the main vessel, while the fourth starts to emerge, indicated by the red arrow.
T210 27627-27735 Sentence denotes The 3D shape of the blood vessel is illustrated by the 3D rendering of the segmentation, shown in Figure 9c.
T211 27736-27883 Sentence denotes This segmentation of the blood vessel shows the potential of the datasets, which may be fully exploited in future with more advanced segmentations.
T212 27884-27893 Sentence denotes Figure 9.
T213 27895-27964 Sentence denotes Segmentation of the blood vessel network, exemplified for biopsy (V).
T214 27965-28078 Sentence denotes (a) Series of slices in z-direction illustrate the separation of a capillary (≈5μm diameter) from a blood vessel.
T215 28079-28120 Sentence denotes Red arrow marks the separating capillary.
T216 28121-28311 Sentence denotes In slice 314, the capillary is entirely filled with erythrocytes. (b) Red lines in the entire virtual slice (336) represent the contours of a manual segmentation of the blood vessel network.
T217 28312-28508 Sentence denotes Three capillaries already separated from the main vessel, while the fourth starts to emerge, indicated by the red arrow. (c) Segmentation illustrating the 3D structure of the blood vessel network.
T218 28509-28520 Sentence denotes Scale bars:
T219 28521-28528 Sentence denotes 50⁢μ⁢m.
T220 28530-28540 Sentence denotes Discussion
T221 28541-28767 Sentence denotes In summary, we have demonstrated that multi-scale x-ray phase contrast tomography can firstly augment pathohistology of the lung to full three dimensions, and secondly provide a link between microscopic and macroscopic scales.
T222 28768-29025 Sentence denotes For the first time, characteristic morphological changes associated with DAD such as hyaline membranes and pronounced inflammation have been imaged in three dimensions, in particular in Covid-19, the most severe pandemic which mankind has faced for decades.
T223 29026-29158 Sentence denotes Moreover, we conducted our study in FFPE material, a fixation method established for well over a century and ubiquitously available.
T224 29159-29336 Sentence denotes The advantage of 3D histology compared to conventional histology relates to the simple fact that features can be visualized and investigated in their full native dimensionality.
T225 29337-29520 Sentence denotes This enables the accurate determination of cell densities, the quantification of morphological parameters such as the distance metrics shown in Figure 6, and tracing of blood vessels.
T226 29521-29761 Sentence denotes Standard histology and in particular immunohistochemical staining, on the other hand, can inform us more precisely on the biomolecular origin of the structure, while immunostaining coupled with x-ray contrast agents is not yet within reach.
T227 29762-29888 Sentence denotes Importantly, since PC-CT is nondestructive, histology can be performed on well-chosen sections after the scan, see Appendix 1.
T228 29889-30199 Sentence denotes On the side of imaging technology and exploitation, there is still ample room for improvement: higher resolution could be achieved by further geometric zooms, waveguide optics with higher numerical aperture, in combination with pixel detector technology, and further improvements in holographic reconstruction.
T229 30200-30349 Sentence denotes Equally important to extend the length scales covered to small scales is the further upscaling of the FOV based on stitching different sub-tomograms.
T230 30350-30491 Sentence denotes At the same time the scales should be extended by including more than two zoom levels, based on different detector and illumination settings.
T231 30492-30564 Sentence denotes In this way, one could bridge scales to cover the entire lobe of a lung.
T232 30565-30771 Sentence denotes To this end, optimized recording and data flow is a larger bottleneck than photons and optics, in particular since 4th generation synchrotron radiation sources are about to deliver unprecedented brilliance.
T233 30772-30942 Sentence denotes Finally, in view of exploitation and information gain, specific labels for different cell types and 3D immunostaining coupled to radiocontrast agents should be developed.
T234 30943-31046 Sentence denotes Here, lung offers an advantage over other tissue in view of volume accessibility and label diffusivity.
T235 31047-31196 Sentence denotes Most importantly, efforts have to be directed to exploit the full information contained in the reconstruction volumes based on advanced segmentation.
T236 31197-31355 Sentence denotes Tracing of capillaries is an obvious important next step, in view of unraveling the role of intussusceptive angiogenesis in Covid-19 (Ackermann et al., 2020).
T237 31356-31575 Sentence denotes The current data, which are made fully available at https://doi.org/10.5281/zenodo.3892637, are likely to already provide such clues once that more advanced segmentation approaches based on machine learning are applied.
T238 31576-31710 Sentence denotes More generally, it may advance our understanding of DAD in the particular case of Covid-19, as an intrinsically volumetric phenomenon.
T239 31711-31836 Sentence denotes Beyond the current data, further studies could shed further light on the differences between moderate and severe progression.
T240 31837-31940 Sentence denotes Possibly, phase contrast tomography of lung biopsies could in future also help diagnosis and treatment.
T241 31942-31963 Sentence denotes Materials and methods
T242 31965-32016 Sentence denotes Autopsy, clinical background and tissue preparation
T243 32017-32115 Sentence denotes In total we investigated six postmortem lung samples from Covid-19 patients (Menter et al., 2020).
T244 32116-32238 Sentence denotes A tissue micro-array paraffin block with samples of all six patients and the corresponding HE stain is shown in Figure 1a.
T245 32239-32380 Sentence denotes Information about age, gender, hospitalization, clinical, radiological and histological characteristics of all patients are shown in Table 2.
T246 32381-32505 Sentence denotes All patients suffered from hypertension and were treated with RAAS (renin-angiotensin-aldosterone-system) interacting drugs.
T247 32506-32718 Sentence denotes Heterogeneous ground glass and consolidation were observed in all patients clinical CT scans and the cause of death was also related to respiratory failure in each patient (patient IV cardio-respiratory failure).
T248 32719-32834 Sentence denotes Additionally, tumor-free lung samples from partial resections of pulmonary carcinomas were analyzed as a reference.
T249 32835-32843 Sentence denotes Table 2.
T250 32845-32876 Sentence denotes Sample and medical information.
T251 32877-33095 Sentence denotes Age and gender, clinical presentation with hospitalization and treatment; NIV: non-invasive ventilation, I: immunosuppression, S: smoker, GGO: ground-glass opacification, C: consolidation, DAD: diffuse alveolar damage.
T252 33096-33106 Sentence denotes Sample no.
T253 33108-33200 Sentence denotes Age group, gender Hospitalization, clinical, radiological, and histological characteristics
T254 33201-33229 Sentence denotes I 60–70, F 5-10d, GGO, DAD
T255 33230-33260 Sentence denotes II 80–90, M 5-10d, C, I, DAD
T256 33261-33294 Sentence denotes III 90–100, M 1-4d, GGO, C, DAD
T257 33295-33334 Sentence denotes IV 70–80, M 1-4d, NIV, S, GGO, C, DAD
T258 33335-33374 Sentence denotes V 60–70, M 5-10d, NIV, S, GGO, C, DAD
T259 33375-33409 Sentence denotes VI 70–80, M 1-4d, S, GGO, C, DAD
T260 33410-33538 Sentence denotes CTRL 20–30, M - From each of the six Covid-19 patients, two tissue samples with edge lengths of about 4 mm each were analyzed.
T261 33539-33667 Sentence denotes To one sample of each patient, a metal containing stain (uranium acetate, UA) was applied, the other samples remained unstained.
T262 33668-33783 Sentence denotes Separated for their stain, six tissue samples were dehydrated and embedded in the same multi-sample paraffin block.
T263 33784-33960 Sentence denotes The size of the postmortem tissue samples made available for the study varied between the different patients (I-VI), with maximum cross-section of about 4 mm after dehydration.
T264 33961-34078 Sentence denotes From all six samples, biopsy punches were taken by either a 8 mm or a 3.5 mm punch, depending on the individual size.
T265 34079-34319 Sentence denotes The punches were then transferred onto a holder for the parallel-beam local tomography acquisition, followed by a further reduction in size (after measurement of the entire sample) to a 1 mm biopsy punch, for further tomographic recordings.
T266 34320-34377 Sentence denotes A sketch of the sample preparation is shown in Figure 1b.
T267 34378-34619 Sentence denotes The control lung sample was first mounted in an Eppendorf tube for parallel beam acquisitions, and a 1 mm biopsy punch was then transferred into a polyimide tube similar to the paraffin-embedded ones, but scanned in fixative buffer solution.
T268 34621-34646 Sentence denotes Phase contrast tomography
T269 34647-34767 Sentence denotes For Covid-19 lung tissue, the scans were recorded at the GINIX endstation of the PETRA III storage ring (DESY, Hamburg).
T270 34768-34970 Sentence denotes The projections were acquired at two different photon energies Eph, 8⁢keV and 13.8⁢keV, using the first and third harmonic of the 5 m P10 undulator and a Si(111) channel-cut monochromator, respectively.
T271 34971-35065 Sentence denotes Data are shown here only for 8⁢keV, which gave highest contrast for the unstained lung tissue.
T272 35066-35151 Sentence denotes Two tomography configurations were combined to cover a larger range of length scales:
T273 35153-35170 Sentence denotes (1–parallel beam)
T274 35171-35351 Sentence denotes Recordings with the unfocused quasi-parallel beam illumination and a high resolution microscope detection system, resulting in in a FOV of 1.6 mm sampled at a pixel size of 650 nm.
T275 35353-35366 Sentence denotes (2–cone beam)
T276 35367-35653 Sentence denotes Holographic recordings with the divergent and coherence filtered beam emanating from a compound focusing system composed of a Kirkpatrick-Baez (KB) mirrors system and an x-ray waveguide, resulting in a FOV of 0.4 mm sampled at a pixel size of about 167 nm (depending on exact geometry).
T277 35654-35801 Sentence denotes Both configurations were implemented side-by-side, using the same fully-motorized tomography stage and mounting, as detailed in Frohn et al., 2020.
T278 35802-35856 Sentence denotes A sketch of both configurations is shown in Figure 1 .
T279 35857-35956 Sentence denotes First, parallel-beam overview scans were acquired of the entire tissue volume embedded in paraffin.
T280 35957-36185 Sentence denotes To this end a 3.5 mm biopsy punch was taken from the multi-sample tissue block, and then scanned in a stitching-mode yielding a large overview reconstruction, composed of up to 20 individual tomograms (depending on tissue size).
T281 36186-36446 Sentence denotes To increase image quality and avoid artifacts related to region-of-interest (ROI) or local tomography, a selected 1 mm punch was then taken from the already scanned larger tissue cylinder, and re-scanned, first in the parallel- and then the cone-beam geometry.
T282 36447-36533 Sentence denotes Experimental and acquisition parameters used for the data shown are listed in Table 3.
T283 36534-36542 Sentence denotes Table 3.
T284 36544-36621 Sentence denotes Data acquisition parameters for x-ray phase-contrast tomography measurements.
T285 36622-36654 Sentence denotes Cone geometry Parallel geometry
T286 36655-36693 Sentence denotes FOV 0.4 mm × 0.35 mm 1.6 mm × 1.4 mm
T287 36694-36720 Sentence denotes Pixel size 167 nm 650 nm
T288 36721-36736 Sentence denotes z 01 125 mm -
T289 36737-36766 Sentence denotes z 12 4975 mm 10 mm − 100 mm
T290 36767-36803 Sentence denotes Regime holographic direct contrast
T291 36804-36836 Sentence denotes Rotation start-stop continuous
T292 36837-36862 Sentence denotes Exposure 2 s 0.035 ⁢ s
T293 36863-36899 Sentence denotes Total exposure ≃ 63 m i n ≃ 75 s
T294 36900-37041 Sentence denotes Volumetric flowrate 1.16 × 10 4 μ m / 3 s 3.75 × 10 7 μ m / 3 s The configuration for (1–parallel beam) is depicted in Figure 1c.
T295 37042-37312 Sentence denotes The high-resolution microscope detection system (Optique Peter, France) was based on a 50⁢μ⁢m thick LuAG:Ce scintillator imaged with a 10× magnifying microscope objective onto a sCMOS sensor (pco.edge 5.5, PCO, Germany), resulting in an effective pixel size of 0.65⁢μ⁢m.
T296 37313-37473 Sentence denotes The high photon flux density allowed for image acquisition with continuous motor movement, short acquisition time of 35 ms per frame, and a framerate of 20 fps.
T297 37474-37603 Sentence denotes Single-distance tomogram recordings with about 1500 projections, and flat images before and after the scan, took less than 2 min.
T298 37604-37801 Sentence denotes For these scans, the focusing optics (KB-mirrors and waveguide) as well as the fastshutter (Cedrat technologies) were moved out of the beam, and beam size was adjusted by the upstream slit systems.
T299 37802-37892 Sentence denotes To avoid detector saturation, the beam was attenuated by 4× single crystal silicon wafers.
T300 37893-37954 Sentence denotes The configuration for (2–cone beam) is depicted in Figure 1d:
T301 37955-38010 Sentence denotes The beam was focused by the KB-mirrors to about 300 nm.
T302 38011-38466 Sentence denotes To further reduce the secondary source size, to increase coherence, and to achieve a smooth wavefront for holographic illumination, an x-ray waveguide formed by 1 mm long lithographic channels in silicon with a cross-section of about 100 nm was positioned in the focal plane of the KB-mirrors, resulting in an exit flux of 1–4⋅109 photon/s (depending on alignment and storage ring), as measured with the single photon counting detector (Pilatus, Dectris).
T303 38467-38603 Sentence denotes The sample was positioned at variable (defocus) distances behind the focus (waveguide exit), typically z01=125mm for the first distance.
T304 38604-38805 Sentence denotes The geometrically magnified holograms were recorded by a fibre-coupled sCMOS sensor (Zyla HF 5.5 detector, Andor Technologies) with a customized 15⁢μ⁢m thick Gadox scintillator, and 6.5⁢μ⁢m pixel size.
T305 38806-38985 Sentence denotes The detector position at about z02=5100mm behind the focus resulted in a magnification of about M=41 (for the first defocus distance), and hence an effective pixel size of 167 nm.
T306 38986-39069 Sentence denotes 1442 projections were recorded, with a typical exposure time of 2 s per projection.
T307 39071-39126 Sentence denotes Phase retrieval, image reconstruction, and segmentation
T308 39128-39162 Sentence denotes Phase retrieval and reconstruction
T309 39163-39511 Sentence denotes Phase retrieval was performed from dark and empty beam corrected holograms, using both linearised single step CTF-approach (Cloetens et al., 1999; Turner et al., 2004), and non-linear generalizations of the CTF-method based on Tikhonov regularization (NL-CTF), using our code package HoloTomoToolbox as described and deposited (Lohse et al., 2020).
T310 39512-39633 Sentence denotes Importantly, both CTF and NL-CTF implementations can be augmented by imposing support and range constraints, when needed.
T311 39634-39792 Sentence denotes When available, projections recorded at several defocus distances were first aligned with sub-pixel accuracy and then used for multi-distance phase retrieval.
T312 39793-39961 Sentence denotes For these purposes, the HoloTomoToolbox provides auxiliary functions, which also help to refine the Fresnel number or to correct for drift in the illumination function.
T313 39962-40266 Sentence denotes After phase reconstruction of all projections, the tomographic reconstruction was carried out with the MATLAB implemented iradon-function (Ram-Lak filter) for the parallel geometry and with the FDK-function of the ASTRA toolbox (van Aarle et al., 2015; van Aarle et al., 2016) for the cone beam geometry.
T314 40267-40594 Sentence denotes Hot pixel and detector sensitivity variations as well as strong phase features in the parallel beam illumination resulting from upstream window materials, which persist after empty beam correction, can all result in ring artifacts in the tomographic reconstruction, in particular as these flaws can increase by phase retrieval.
T315 40595-40763 Sentence denotes To correct for this, the extra information provided by 360° scans was used to mask out the corresponding pixels and replace them with values of the opposing projection.
T316 40764-40871 Sentence denotes Stitching of reconstructions from different tomographic scans was performed using (Miettinen et al., 2019).
T317 40872-40939 Sentence denotes Resolution estimates were obtained by FSC analysis, see Appendix 2.
T318 40941-40978 Sentence denotes Image segmentation and quantification
T319 40979-41080 Sentence denotes For each patient the stitched overview scans were analyzed with regard to structural characteristics.
T320 41081-41303 Sentence denotes The 3D-reconstructions were first binned (2 × 2 × 2), and the tissue was then segmented from the surrounding paraffin using the segmentation software Ilastik (Berg et al., 2019), which was then further refined with MATLAB.
T321 41304-41442 Sentence denotes In order to exclude single macrophages or detached tissue, only voxels connected to the tissue block were considered for the distance map.
T322 41443-41558 Sentence denotes Further, the areas of the paraffin which represent air compartments were linked to the outside of the tissue block.
T323 41559-41654 Sentence denotes Individual self-contained areas of paraffin (not connected to air) were excluded from the mask.
T324 41655-41772 Sentence denotes Based on this segmentation, the distance to the nearest voxel containing oxygen was calculated for each tissue voxel.
T325 41773-41845 Sentence denotes The tissue volume V is given by the sum of all voxels containing tissue.
T326 41846-41936 Sentence denotes The surface area SA is defined by all tissue voxels with a distance of 1 pixel to the air.
T327 41937-42042 Sentence denotes From this information we calculated the specific surface(2) SV=SA/Vwith S⁢A/V surface area volume -ratio.
T328 42043-42151 Sentence denotes Further a characteristic length(3) Lc=V/SA⋅vxwith v⁢x edge length of a voxel was determined for each sample.
T329 42152-42287 Sentence denotes Additionally, the mean distance (dO2¯) from all tissue voxels to the closest air compartment and its standard deviation was calculated.
T330 42288-42426 Sentence denotes Hyaline membranes and capillary networks were extracted using semi-manual segmentation functions in Avizo (Thermo Fisher Scientific, USA).
T331 42427-42582 Sentence denotes Beside hyaline membranes and the capillary network, different cell types can be readily identified based on the 3D reconstructions of the electron density.
T332 42583-42693 Sentence denotes In particular, inflammatory cell subpopulations - that is, macrophages and lymphocytes - can be distinguished.
T333 42694-42807 Sentence denotes To this end, an automatic and parameter-controlled algorithm denoted as BlobFinder was used (arivis AG, Germany).
T334 42808-42951 Sentence denotes Based on its segmentation output, the amount and position of the lymphocytes in the 3D-reconstructions from parallel beam scans was calculated.
T335 42952-43024 Sentence denotes The algorithm is able to identify roundish structures with a given size.
T336 43025-43108 Sentence denotes For the segmentation of the lymphocytes, we chose a characteristic size of 6.5⁢μ⁢m.
T337 43109-43207 Sentence denotes The structures identified in this step also include macrophages and parts of the capillary system.
T338 43208-43356 Sentence denotes For the unbinned datasets, a distinction between lymphocytes and the nuclei of the macrophages was made based on the difference in electron density.
T339 43357-43465 Sentence denotes In the tomographic reconstructions, the lymphocytes appear denser compared to the nuclei of the macrophages.
T340 43466-43606 Sentence denotes Further, nuclei from endothelial cells and parts of the capillaries filled with blood residues were excluded based on their elongated shape.
T341 43607-43672 Sentence denotes Only structures with a sphericity higher than 0.55 were included.
T342 43673-43850 Sentence denotes Based on the segmentation of lymphocytes, the total number of lymphocytes Nl was obtained and the mean concentration of lymphocytes within the lung tissue cl=Nl/V was estimated.
T343 43852-43871 Sentence denotes Funding Information
T344 43872-43921 Sentence denotes This paper was supported by the following grants:
T345 43922-44029 Sentence denotes http://dx.doi.org/10.13039/501100002347Bundesministerium für Bildung und Forschung 05K19MG2 to Tim Salditt.
T346 44030-44121 Sentence denotes http://dx.doi.org/10.13039/100010663H2020 European Research Council 771883 to Danny Jonigk.
T347 44122-44189 Sentence denotes Max-Planck Schools Matter to Life to Marius Reichardt, Tim Salditt.
T348 44190-44297 Sentence denotes http://dx.doi.org/10.13039/501100001659Deutsche Forschungsgemeinschaft EXC 2067/1-390729940 to Tim Salditt.
T349 44298-44365 Sentence denotes Botnar Research Center for Child Health BRCCH to Alexandar Tzankov.
T350 44367-44383 Sentence denotes Acknowledgements
T351 44384-44677 Sentence denotes We thank Maximilian Ackermann and Florian Länger for their helpful suggestions, Patrick Zardo for providing control specimen, Emily Brouwer for help in sample preparation, Bastian Hartmann and Jan Goemann for technical help with instrumentation and IT, and Jakob Koch for help in segmentation.
T352 44678-44792 Sentence denotes It is also our pleasure to acknowledge DESY photon science management for the Covid-19 beamtime call and beamtime.
T353 44794-44816 Sentence denotes Additional information
T354 44817-44836 Sentence denotes Competing interests
T355 44837-44869 Sentence denotes No competing interests declared.
T356 44870-44890 Sentence denotes Author contributions
T357 44891-45018 Sentence denotes Data curation, Software, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing.
T358 45019-45146 Sentence denotes Data curation, Software, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing.
T359 45147-45274 Sentence denotes Data curation, Software, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing.
T360 45275-45342 Sentence denotes Software, Investigation, Methodology, Writing - review and editing.
T361 45343-45366 Sentence denotes Resources, Methodology.
T362 45367-45390 Sentence denotes Resources, Methodology.
T363 45391-45413 Sentence denotes Resources, Validation.
T364 45414-45455 Sentence denotes Validation, Investigation, Visualisation.
T365 45456-45504 Sentence denotes Resources, Validation, Writing - original draft.
T366 45505-45606 Sentence denotes Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing - original draft.
T367 45607-45810 Sentence denotes Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing.
T368 45811-45817 Sentence denotes Ethics
T369 45818-45833 Sentence denotes Human subjects:
T370 45834-45965 Sentence denotes The study was approved by and conducted according to requirements of the ethics committees at the Hannover Medical School (vote Nr.
T371 45966-45982 Sentence denotes 9022 BO K 2020).
T372 45984-46000 Sentence denotes Additional files
T373 46001-46027 Sentence denotes Transparent reporting form
T374 46029-46046 Sentence denotes Data availability
T375 46047-46124 Sentence denotes All datasets were uploaded to zenodo: https://doi.org/10.5281/zenodo.3892637.
T376 46125-46161 Sentence denotes The following dataset was generated:
T377 46162-46416 Sentence denotes Salditt T Frohn J Eckermann M Reichardt M Osterhoff M Westermeier F Sprung M Tzankov A Kühnel M Jonigk D 2020 3d Virtual Patho-Histology of Lung Tissue from Covid19 Patients based on Phase Contrast X-ray Tomography Zenodo 10.5281/zenodo.3892637
T378 46418-46428 Sentence denotes Appendix 1
T379 46429-46474 Sentence denotes Medical information and correlative histology
T380 46475-46638 Sentence denotes Before recording tomographic scans, tissue sections of 2.5⁢μ⁢m thickness were cut from the top, stained by HE (hematoxylin and eosin) and imaged with a microscope.
T381 46639-46703 Sentence denotes Appendix 1—figure 1 shows the histological slice of each sample.
T382 46704-46811 Sentence denotes The imaged section is just above the upper plane of the 3D PC-CT reconstruction volume, but not part of it.
T383 46812-46832 Sentence denotes Appendix 1—figure 1.
T384 46834-46911 Sentence denotes Microscopic images of HE-stained histological sections of all samples (I–VI).
T385 46912-47033 Sentence denotes Histological slices show comparable morphologies to the virtual slices in Figure 3, which represent different z-position.
T386 47034-47045 Sentence denotes Scale bars:
T387 47046-47054 Sentence denotes 400⁢μ⁢m.
T388 47055-47279 Sentence denotes An overview of different morphological features identified by conventional HE histology and virtual 3D histology is presented in Appendix 1—figure 2, while Appendix 1—figure 3 presents a direct comparison for the same slice.
T389 47280-47356 Sentence denotes For this purpose, the sample was sectioned and stained after the PC-CT scan.
T390 47357-47545 Sentence denotes Artery lumen, artery wall, erythrocytes, thrombus, alveolar septum, marcophage, hyaline membrane and black granules (anthracosis) are shown in Appendix 1—figure 2 for both imaging methods.
T391 47546-47657 Sentence denotes Contrast of the hyaline membrane is homogenous in both modalities, facilitating idetification and segmentation.
T392 47658-47810 Sentence denotes Erythrocytes are easily recognized by eye in the conventional histology image, due to the HE staining, but less well distinguished by virtual histology.
T393 47811-47971 Sentence denotes This results in a difficult differentiation between thrombus and blood stasis as well as a difficult identification of blood capillaries in the alveolar septum.
T394 47972-48128 Sentence denotes Importantly, however, feature identification can be confirmed by correlative 2d and 3D histology on the same section, as exemplified by Appendix 1—figure 3.
T395 48129-48149 Sentence denotes Appendix 1—figure 2.
T396 48151-48244 Sentence denotes Comparison of morphological features between conventional HE histology and virtual histology.
T397 48245-48509 Sentence denotes Artery lumen, artery wall, erythrocytes, thrombus, alveolar septum, macrophage, hyaline membrane and anthracotic pigments (i.e. the black granules) are presented on exemplary slices of different samples (I–VI) for conventional (left) and virtual histology (right).
T398 48510-48550 Sentence denotes Scale bars: left 200⁢μ⁢m, right 100⁢μ⁢m.
T399 48551-48571 Sentence denotes Appendix 1—figure 3.
T400 48573-48650 Sentence denotes Direct comparison of virtual and HE histology for an identical slice/section.
T401 48651-48779 Sentence denotes (top left) Region of interest of the parallel beam tomogram (Sample II). (bottom left) Corresponding HE stained histology slice.
T402 48780-48850 Sentence denotes Thrombi and erythrocytes can be identified in both imaging modalities.
T403 48851-48963 Sentence denotes The red square marks the position of the zoom tomogram, for which the corresponding slice is shown on the right.
T404 48964-48975 Sentence denotes Scale bars:
T405 48976-48983 Sentence denotes 50⁢μ⁢m.
T406 48985-48995 Sentence denotes Appendix 2
T407 48996-49006 Sentence denotes Resolution
T408 49007-49138 Sentence denotes Resolution estimates are challenging for tissue reconstructions due to the absence of sharp edges or features of well defined size.
T409 49139-49209 Sentence denotes Here, we follow the approach known as Fourier-shell correlation (FSC).
T410 49210-49373 Sentence denotes Accordingly, an upper bound for the resolution is be obtained in the following way: the CT scan is split into two, and from each half a 3D volume is reconstructed.
T411 49374-49553 Sentence denotes After registry, that is, mutual alignment of the volumes, the correlation between the two independent reconstructions in Fourier space is plotted as function of spatial frequency.
T412 49554-49709 Sentence denotes This correlation must not necessarily reflect the pure system resolution, but instead the range of spatial frequencies over which the results are reliable.
T413 49710-49834 Sentence denotes As such it is not only affected by the system resolution but also by the sample contrast and the noise of the specific scan.
T414 49835-50007 Sentence denotes Further, it represents an average over all structures, not taking into account that features with stronger/weaker contrast can correspondingly show higher/lower resolution.
T415 50008-50099 Sentence denotes Here the Fourier operation for FSC was implemented with a Kaiser-Bessel window of 7 pixels.
T416 50100-50245 Sentence denotes For the parallel-beam data, a central volume of 650 × 650 × 650 voxels was correlated, for the cone-beam data a volume of 685 × 680 × 250 voxels.
T417 50246-50380 Sentence denotes These sub-volumes were selected to obtain the average values for the tissue while minimizing contributions from paraffin-filled holes.
T418 50381-50437 Sentence denotes The correlation curves are shown in Appendix 2—figure 1.
T419 50438-50561 Sentence denotes The intersection of the curve with the half-bit threshold yields the resolution estimate, indicated with dashed black line.
T420 50562-50706 Sentence denotes Correspondingly, a half-period resolution of 0.71⁢μ⁢m and 0.39⁢μ⁢m (or better) is obtained for the parallel and cone beam dataset, respectively.
T421 50707-50917 Sentence denotes However, since the splitted dataset resulted in only 721 angles for the reconstruction, i.e. the resolution estimate is severely affected by under-sampling artifacts, and hence can only serve as an upper bound.
T422 50918-50963 Sentence denotes Appendix 2—figure 1 illustrates the analysis.
T423 50964-50984 Sentence denotes Appendix 2—figure 1.
T424 50986-51050 Sentence denotes Quantification of the 3D-resolution for the example of sample V.
T425 51051-51160 Sentence denotes FSC analysis was carried out for two independent reconstructions, each from half the projections of the scan.
T426 51162-51172 Sentence denotes Appendix 3
T427 51173-51189 Sentence denotes Further datasets
T428 51190-51199 Sentence denotes Controls:
T429 51200-51290 Sentence denotes The imaging workflow was also applied to hydrated and/or healthy lung tissue as a control.
T430 51291-51355 Sentence denotes First, overview scans covering the entire samples were recorded.
T431 51356-51538 Sentence denotes Then, hydrated 1 mm biopsy punches (two for CTRLI, one for CTRLII and CTRLIII, where CTRLII and CTRLIII are from the same patient) were recorded in (1 - parallel beam) configuration.
T432 51539-51621 Sentence denotes Biopsy punches from CTRLII and CTRLIII were also examined in (2 - cone beam) mode.
T433 51622-51822 Sentence denotes Appendix 3—figure 1 presents (a) the rendering of the hydrated control, and (b,c) virtual slices through the reconstruction volume, showing the lung parenchymal architecture in healthy control tissue.
T434 51823-51842 Sentence denotes UA-stained samples:
T435 51843-51947 Sentence denotes For all Covid-19 patients, autopsies were also treated by UA-staining in order to increase the contrast.
T436 51948-52044 Sentence denotes Stitched overview scans in (1 - parallel beam) configuration were recorded, similar to Figure 3.
T437 52045-52185 Sentence denotes From the UA-labeled tissue blocks of patients I, III, IV and V, 1 mm, biopsy punches were than scanned in the same configuration (Figure 4).
T438 52186-52339 Sentence denotes Using the (2 - cone beam) setup, these samples from I, III and IV were imaged at 8.0⁢keV x-rays, while V was examined at 13.8⁢keV, as shown for Figure 5.
T439 52340-52374 Sentence denotes Variation of propagation distance:
T440 52375-52562 Sentence denotes Scans of the unstained tissue block from patient II were recorded at different propagation distances (z12=50, 100 and 125keV) and different x-ray energies (13.3, 13.8, 14.3 and 14.8 keV).
T441 52563-52664 Sentence denotes In cone-beam configuration, the unstained biopsy punch from patient I was scanned at 13.8⁢keV x-rays.
T442 52665-52683 Sentence denotes Compact μCT scans:
T443 52684-53042 Sentence denotes Prior to the synchrotron experiment, some of the samples have been examined with a laboratory phase-contrast μCT-setup in large mm2-sized FOV-configuration (Liquid metal jet source, Kα=9.5keV, pxeff=5μm, z12=1.7m, 1200 projections of 1 s exposure time with a flat panel CMOS detector with 150⁢μ⁢m Gadox-scintillator, PerkinElmer, USA) (Bartels et al., 2013).
T444 53043-53252 Sentence denotes Metal-staining (here UA) of the lung tissue helped to achieve sufficient contrast, similar to previous μCT-studies of other biological tissues (Müller et al., 2017; Busse et al., 2018; De Clercq et al., 2019).
T445 53253-53339 Sentence denotes The resulting overview scans could also be correlated well with histological sections.
T446 53340-53360 Sentence denotes Appendix 3—figure 1.
T447 53362-53410 Sentence denotes Illustrations of control lung tissue (hydrated).
T448 53411-53624 Sentence denotes (a) Volume rendering of the tissue block (0.97×1.00×0.73 mm3) and (b) slice through the volume, examined in PB-configuration. (c) Slice from the cone-beam scan, arrows indicating the structure of a healthy septum.
T449 53625-53675 Sentence denotes Particularly, macrophages and erythrocytes emerge.
T450 53676-53716 Sentence denotes Scale bars: (b) 100⁢μ⁢m and (c) 300⁢μ⁢m.
T451 53717-53737 Sentence denotes Appendix 3—figure 2.
T452 53739-53792 Sentence denotes Screening with a laboratory phase-contrast μCT-setup.
T453 53793-54000 Sentence denotes (UA-stained tissue block, patient I). (a) Histological and (b) correlative virtual slice from laboratory phase-contrast tomography. (c) Volume rendering of the entire tissue block from a similar perspective.
T454 54001-54011 Sentence denotes Scale bar:
T455 54012-54017 Sentence denotes 1 mm.