LitCovid-sentences  

PMC:7473770 / 1329-43850 JSONTXT 11 Projects

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Id Subject Object Predicate Lexical cue
T8 0-12 Sentence denotes Introduction
T9 13-260 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 261-514 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 515-782 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 783-929 Sentence denotes These findings have also been reported for infections by Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and influenza viruses.
T13 930-1178 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 1179-1416 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 1417-1629 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 1630-1885 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 1886-2161 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 2162-2380 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 2381-2585 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 2586-2805 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 2806-3007 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 3008-3117 Sentence denotes We ask in particular, whether DAD and the morphology of blood vessels can be visualized and quantified in 3D.
T23 3118-3273 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 3274-3390 Sentence denotes Here we present first results obtained from the postmortem lung samples of six patients who succumbed from Covid-19.
T25 3391-3720 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 3721-4060 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 4061-4191 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 4192-4514 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 4515-4755 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 4756-4917 Sentence denotes However, problems with phase retrieval, phase wrapping, insufficient coherence, or on the contrary strong phase gradients can render its application challenging.
T31 4918-5148 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 5149-5353 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 5354-5626 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 5627-5770 Sentence denotes However, this requires careful optimization of photon energy, illumination function, and phase retrieval algorithms, as detailed further below.
T35 5771-5976 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 5977-6212 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 6213-6431 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 6432-6650 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 6651-7003 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 7004-7111 Sentence denotes Based on the geometrical magnification, the effective pixel size can be adjusted in the range 10 nm–300 nm.
T41 7112-7193 Sentence denotes The two imaging schemes are shown schematically in Figure 1c and d, respectively.
T42 7194-7467 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 7468-7659 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 7660-7862 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 7863-7872 Sentence denotes Figure 1.
T46 7874-7909 Sentence denotes Multi-scale x-ray tomography setup.
T47 7910-8090 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 8091-8448 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 8450-8457 Sentence denotes Results
T50 8458-8557 Sentence denotes In total, we investigated six postmortem lung samples from Covid-19 patients (Menter et al., 2020).
T51 8558-8680 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 8681-8822 Sentence denotes Information about age, gender, hospitalization, clinical, radiological and histological characteristics of all patients are shown in Table 2.
T53 8823-9011 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 9012-9223 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 9224-9279 Sentence denotes The datasets are denoted by patient I-VI, respectively.
T56 9280-9536 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 9537-9546 Sentence denotes Figure 2.
T58 9548-9645 Sentence denotes Overview of reconstruction volumes, exemplified for a punch biopsy of pulmonary autopsy sample I.
T59 9646-10120 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 10121-10223 Sentence denotes Scale bars: (a) 500⁢μ⁢m and (b,c) 100⁢μ⁢m. The entire reconstruction volume is visualized in Video 1. 
T61 10224-10232 Sentence denotes Video 1.
T62 10234-10302 Sentence denotes A video showing a fly-through the reconstruction volume of sample I.
T63 10303-10392 Sentence denotes First the parallel beam data set is shown, followed by the cone beam zoom reconstruction.
T64 10394-10424 Sentence denotes Reconstructed electron density
T65 10425-10541 Sentence denotes Next, we present representative slices through the reconstruction volumes of all samples for all acquisition scales.
T66 10542-10656 Sentence denotes Figure 3 presents the stitched reconstruction volumes, recorded under conditions of local tomography, see Table 3.
T67 10657-11028 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 11029-11218 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 11219-11302 Sentence denotes The results are presented in Figure 4, and validate the previous stitching results.
T70 11303-11417 Sentence denotes The 1 mm punches then also provided an appropriate size for the cone-beam recordings, which are shown in Figure 5.
T71 11418-11540 Sentence denotes Importantly, in each scan the previous level guided the choice for the next FOV and informed about the larger environment.
T72 11541-11640 Sentence denotes In the following, we briefly discuss the samples one-by-one, with regard to all acquisition scales.
T73 11641-11750 Sentence denotes A comparison of morphological features between conventional and virtual histology is shown in the Appendix 1.
T74 11751-11760 Sentence denotes Figure 3.
T75 11762-11835 Sentence denotes Stitched parallel-beam reconstructions for full pulmonary samples (I–VI).
T76 11836-11941 Sentence denotes Representative virtual sections through the reconstruction volumes of full biopsies (I–VI), respectively.
T77 11942-11953 Sentence denotes Scale bars:
T78 11954-11962 Sentence denotes 500⁢μ⁢m.
T79 11963-11972 Sentence denotes Figure 4.
T80 11974-12035 Sentence denotes Parallel-beam reconstructions for 1 mm biopsy punches (I–VI).
T81 12036-12182 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 12183-12317 Sentence denotes The fact that the punches are isolated results in higher image quality, since the errors associated with local tomography are avoided.
T83 12318-12329 Sentence denotes Scale bars:
T84 12330-12338 Sentence denotes 100⁢μ⁢m.
T85 12339-12348 Sentence denotes Figure 5.
T86 12350-12495 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 12496-12695 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 12696-12707 Sentence denotes Scale bars:
T89 12708-12716 Sentence denotes 100⁢μ⁢m.
T90 12718-12726 Sentence denotes Sample I
T91 12727-13001 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 13002-13223 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 13224-13313 Sentence denotes In PC-CT, enlarged alveolar septa with pronounced lymphocytic inflammation are displayed.
T94 13314-13449 Sentence denotes The reconstruction volume contains a large artery filled with erythrocytes (Figure 3-I, lower left), which bifurcates into two vessels.
T95 13450-13515 Sentence denotes This area was then selected for the 1 mm biopsy punch extraction.
T96 13516-13603 Sentence denotes The cone-beam zoom tomogram was then centered around the perimeter of the blood vessel.
T97 13604-13745 Sentence denotes This volume is particularly well suited to investigate the connective tissue including elastic fibers and collagen, as well as smooth muscle.
T98 13747-13756 Sentence denotes Sample II
T99 13757-13963 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 13964-14047 Sentence denotes On the level of blood vessels, both blood-filled and empty vessels are discernible.
T101 14048-14125 Sentence denotes It should be noted that septa with signs of parallel capillaries are visible.
T102 14126-14236 Sentence denotes In the reconstruction volume of the zoom tomogram, a single vessel can be easily tracked over large distances.
T103 14238-14248 Sentence denotes Sample III
T104 14249-14437 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 14438-14541 Sentence denotes In PC-CT, septa with again similar physiological size and distribution emerge, with moderate emphysema.
T106 14542-14623 Sentence denotes The bottom part of the sample contains a fibrous area near a larger blood vessel.
T107 14624-14701 Sentence denotes The zoom tomogram shows a single septum, a blood vessel and a fibrous region.
T108 14703-14712 Sentence denotes Sample IV
T109 14713-14913 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 14914-14997 Sentence denotes Furthermore, there is a mild centrilobular emphysema (original magnification 100×).
T111 14998-15102 Sentence denotes In PC-CT, a network of thin septa, thrombi and emphysema, as well as a large empty blood vessel appears.
T112 15103-15157 Sentence denotes Electron-rich diffuse black granules are also visible.
T113 15158-15242 Sentence denotes The biopsy punch was selected to contain the empty blood vessel, some small thrombi.
T114 15243-15306 Sentence denotes It also includes thin septa and tissue embedded dirt-particles.
T115 15307-15422 Sentence denotes The zoom tomogram covered tissue with black granules as well as a band of inflammatory cells next to a septum wall.
T116 15424-15432 Sentence denotes Sample V
T117 15433-15737 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 15738-15873 Sentence denotes Furthermore, bronchialized alveolar epithelial cells show cytopathic changes and multifocal desquamation, as does alveolar macrophages.
T119 15874-16027 Sentence denotes Focally, accumulation of intraalveolar neutrophilic granulocytes in the sense of a florid bronchopneumonia can be observed (original magnification 100×).
T120 16028-16202 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 16203-16308 Sentence denotes The biopsy punch was chosen to include areas with increased presence of hyaline membrane and lymphocytes.
T122 16309-16437 Sentence denotes A blood vessel splitting into several smaller blood vessels is easily recognized when browsing through the reconstructed volume.
T123 16438-16553 Sentence denotes Noteworthy, different cell types as macrophages, T-cells or erythrocytes can be distinguished in the zoom tomogram.
T124 16555-16564 Sentence denotes Sample VI
T125 16565-16715 Sentence denotes Histomorphological analysis shows peribronchial alveolar parenchyma with lymphocytic interstitial pneumonitis and a singular thrombus in a small vein.
T126 16716-16790 Sentence denotes The interstitium of the alveolar septae is widened by myogenic metaplasia.
T127 16791-16854 Sentence denotes Adjacent, centrilobular emphysema and anthracosis are observed.
T128 16855-17005 Sentence denotes The bronchial mucosa shows varying degrees of lymphocytic inflammation in the sense of chronic bronchitis/bronchiolitis (original magnification 100×).
T129 17006-17160 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 17161-17216 Sentence denotes The amount of lymphocytes is rather low in this sample.
T131 17217-17292 Sentence denotes Black granules and some thrombi are embedded within the bulky tissue parts.
T132 17293-17455 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 17457-17519 Sentence denotes Visualization of pathologies, segmentation, and quantification
T134 17520-17685 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 17686-17797 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 17798-17911 Sentence denotes Based on the tissue mask the distances for each tissue voxel to the next voxel containing oxygen were determined.
T137 17912-18079 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 18080-18310 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 18311-18459 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 18460-18602 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 18603-18612 Sentence denotes Figure 6.
T142 18614-18654 Sentence denotes Tissue compactness and distance metrics:
T143 18655-18767 Sentence denotes The 3D reconstructions of the lung tissue were analyzed in terms of specific surface and characteristic lengths.
T144 18768-18828 Sentence denotes The workflow is sketched for sample V in the top part (a–g).
T145 18829-18885 Sentence denotes In a first step, the tissue was segmented using Ilastik.
T146 18886-18948 Sentence denotes A slice of the reconstructed electron density is shown in (a).
T147 18949-19185 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 19186-19296 Sentence denotes Especially around vessels and larger alveoli, the distances are larger. (d) Zoom into an area around a vessel.
T149 19297-19615 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 19616-19729 Sentence denotes In order to compare all samples, the count of voxels was normalized by the total volume of the respective sample.
T151 19730-19913 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 19914-19983 Sentence denotes Double logarithmic scale, bin width of the distribution of distances:
T153 19984-19990 Sentence denotes 1 v⁢x.
T154 19991-20017 Sentence denotes Scale bars: (a–c) 100⁢μ⁢m.
T155 20018-20101 Sentence denotes The distribution of the distances obtained for a given slice is shown in Figure 6e.
T156 20102-20287 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 20288-20426 Sentence denotes Figure 6h shows the distribution of the tissue-air distances (histogram) for all samples, following the workflow illustrated in Figure 6e.
T158 20427-20480 Sentence denotes The binning of distances was set to one voxel length.
T159 20481-20594 Sentence denotes The figure underlines the high diversity of the tissue structure which could already be seen in the 3D histology.
T160 20595-20702 Sentence denotes Further, it directly informs about the specific surface SV, which is given by the first point of the graph.
T161 20703-20785 Sentence denotes The corresponding parameters and metrics are tabulated in Table 1 for all samples.
T162 20786-20890 Sentence denotes Additionally, the mean concentration of lymphocytes cl within the lung tissue is listed for all samples.
T163 20891-20989 Sentence denotes The values quantify the general structure of the tissue which is qualitatively discernible by eye.
T164 20990-21119 Sentence denotes Samples with a high amount of swollen, inflamed blood vessels and thick hyaline membranes exhibit a larger characteristic length.
T165 21120-21257 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 21258-21338 Sentence denotes Hence, the results cannot be directly compared to the paraffin embedded samples.
T167 21339-21467 Sentence denotes Further, the analysis of the lymphocyte concentration was performed since no lymphocytes were found in the reconstructed volume.
T168 21468-21581 Sentence denotes The low values of Lc and dO2¯ for sample II correlate with the lack of ground-glass opacification in clinical CT.
T169 21582-21700 Sentence denotes Based on the extracted structural parameters, the degree of inflammation and swelling of lung tissue can be evaluated.
T170 21701-21821 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 21822-22021 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 22022-22030 Sentence denotes Table 1.
T173 22032-22380 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 22381-22392 Sentence denotes Patient no.
T175 22394-22450 Sentence denotes SV (%) Lc (μm) dO2¯ (μm) c l   ( 10 5 / m m 3 )
T176 22451-22479 Sentence denotes I 13.87 9.4 5.9±5.3 16.0
T177 22480-22509 Sentence denotes II 46.56 2.8 2.1±1.0 14.1
T178 22510-22539 Sentence denotes III 33.75 3.9 2.5±1.5 4.4
T179 22540-22568 Sentence denotes IV 25.90 5.0 3.2±2.3 7.1
T180 22569-22596 Sentence denotes V 19.28 6.7 3.6±2.1 4.8
T181 22597-22625 Sentence denotes VI 11.87 11.0 9.1±10 6.1
T182 22626-22755 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 22756-22863 Sentence denotes Volumetric renderings in Figure 7a and b demonstrate particular attachment of fibrin to the alveolar walls.
T184 22864-23009 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 23010-23080 Sentence denotes In Figure 7f, hyaline membranes of neighboring alveoles are indicated.
T186 23081-23257 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 23258-23267 Sentence denotes Figure 7.
T188 23269-23374 Sentence denotes Rendering of hyaline membrane attached to alveolar walls (patient V, parallel beam-scan of a 1 mm punch).
T189 23375-23657 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 23658-23937 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 23938-23964 Sentence denotes Scale bars: (c–e) 300⁢μ⁢m.
T192 23965-24172 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 24173-24285 Sentence denotes Further, lymphocytes (red) were identified based on the automated cell segmentation (see Materials and methods).
T194 24286-24414 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 24415-24611 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 24612-24719 Sentence denotes This concept was then translated to a 3D-stitched volume of an entire tissue block as shown in Figure 8f,g.
T197 24720-24729 Sentence denotes Figure 8.
T198 24731-24892 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 24893-25120 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 25121-25364 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 25365-25498 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 25499-25539 Sentence denotes The segmentation was performed manually.
T203 25540-25686 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 25687-25782 Sentence denotes The separation starts with the creation of a branch from the blood vessel (arrow in slice 336).
T205 25783-25880 Sentence denotes In slice 326, 1.7⁢μ⁢m above slice 336, this branch evolves into an empty and separated capillary.
T206 25881-25946 Sentence denotes Another 2⁢μ⁢m above, the capillary is entirely filled with cells.
T207 25947-26029 Sentence denotes Further 2.3⁢μ⁢m, the capillary is empty again and has a diameter of about 2.8⁢μ⁢m.
T208 26030-26156 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 26157-26297 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 26298-26406 Sentence denotes The 3D shape of the blood vessel is illustrated by the 3D rendering of the segmentation, shown in Figure 9c.
T211 26407-26554 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 26555-26564 Sentence denotes Figure 9.
T213 26566-26635 Sentence denotes Segmentation of the blood vessel network, exemplified for biopsy (V).
T214 26636-26749 Sentence denotes (a) Series of slices in z-direction illustrate the separation of a capillary (≈5μm diameter) from a blood vessel.
T215 26750-26791 Sentence denotes Red arrow marks the separating capillary.
T216 26792-26982 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 26983-27179 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 27180-27191 Sentence denotes Scale bars:
T219 27192-27199 Sentence denotes 50⁢μ⁢m.
T220 27201-27211 Sentence denotes Discussion
T221 27212-27438 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 27439-27696 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 27697-27829 Sentence denotes Moreover, we conducted our study in FFPE material, a fixation method established for well over a century and ubiquitously available.
T224 27830-28007 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 28008-28191 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 28192-28432 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 28433-28559 Sentence denotes Importantly, since PC-CT is nondestructive, histology can be performed on well-chosen sections after the scan, see Appendix 1.
T228 28560-28870 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 28871-29020 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 29021-29162 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 29163-29235 Sentence denotes In this way, one could bridge scales to cover the entire lobe of a lung.
T232 29236-29442 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 29443-29613 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 29614-29717 Sentence denotes Here, lung offers an advantage over other tissue in view of volume accessibility and label diffusivity.
T235 29718-29867 Sentence denotes Most importantly, efforts have to be directed to exploit the full information contained in the reconstruction volumes based on advanced segmentation.
T236 29868-30026 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 30027-30246 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 30247-30381 Sentence denotes More generally, it may advance our understanding of DAD in the particular case of Covid-19, as an intrinsically volumetric phenomenon.
T239 30382-30507 Sentence denotes Beyond the current data, further studies could shed further light on the differences between moderate and severe progression.
T240 30508-30611 Sentence denotes Possibly, phase contrast tomography of lung biopsies could in future also help diagnosis and treatment.
T241 30613-30634 Sentence denotes Materials and methods
T242 30636-30687 Sentence denotes Autopsy, clinical background and tissue preparation
T243 30688-30786 Sentence denotes In total we investigated six postmortem lung samples from Covid-19 patients (Menter et al., 2020).
T244 30787-30909 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 30910-31051 Sentence denotes Information about age, gender, hospitalization, clinical, radiological and histological characteristics of all patients are shown in Table 2.
T246 31052-31176 Sentence denotes All patients suffered from hypertension and were treated with RAAS (renin-angiotensin-aldosterone-system) interacting drugs.
T247 31177-31389 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 31390-31505 Sentence denotes Additionally, tumor-free lung samples from partial resections of pulmonary carcinomas were analyzed as a reference.
T249 31506-31514 Sentence denotes Table 2.
T250 31516-31547 Sentence denotes Sample and medical information.
T251 31548-31766 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 31767-31777 Sentence denotes Sample no.
T253 31779-31871 Sentence denotes Age group, gender Hospitalization, clinical, radiological, and histological characteristics
T254 31872-31900 Sentence denotes I 60–70, F 5-10d, GGO, DAD
T255 31901-31931 Sentence denotes II 80–90, M 5-10d, C, I, DAD
T256 31932-31965 Sentence denotes III 90–100, M 1-4d, GGO, C, DAD
T257 31966-32005 Sentence denotes IV 70–80, M 1-4d, NIV, S, GGO, C, DAD
T258 32006-32045 Sentence denotes V 60–70, M 5-10d, NIV, S, GGO, C, DAD
T259 32046-32080 Sentence denotes VI 70–80, M 1-4d, S, GGO, C, DAD
T260 32081-32209 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 32210-32338 Sentence denotes To one sample of each patient, a metal containing stain (uranium acetate, UA) was applied, the other samples remained unstained.
T262 32339-32454 Sentence denotes Separated for their stain, six tissue samples were dehydrated and embedded in the same multi-sample paraffin block.
T263 32455-32631 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 32632-32749 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 32750-32990 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 32991-33048 Sentence denotes A sketch of the sample preparation is shown in Figure 1b.
T267 33049-33290 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 33292-33317 Sentence denotes Phase contrast tomography
T269 33318-33438 Sentence denotes For Covid-19 lung tissue, the scans were recorded at the GINIX endstation of the PETRA III storage ring (DESY, Hamburg).
T270 33439-33641 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 33642-33736 Sentence denotes Data are shown here only for 8⁢keV, which gave highest contrast for the unstained lung tissue.
T272 33737-33822 Sentence denotes Two tomography configurations were combined to cover a larger range of length scales:
T273 33824-33841 Sentence denotes (1–parallel beam)
T274 33842-34022 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 34024-34037 Sentence denotes (2–cone beam)
T276 34038-34324 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 34325-34472 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 34473-34527 Sentence denotes A sketch of both configurations is shown in Figure 1 .
T279 34528-34627 Sentence denotes First, parallel-beam overview scans were acquired of the entire tissue volume embedded in paraffin.
T280 34628-34856 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 34857-35117 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 35118-35204 Sentence denotes Experimental and acquisition parameters used for the data shown are listed in Table 3.
T283 35205-35213 Sentence denotes Table 3.
T284 35215-35292 Sentence denotes Data acquisition parameters for x-ray phase-contrast tomography measurements.
T285 35293-35325 Sentence denotes Cone geometry Parallel geometry
T286 35326-35364 Sentence denotes FOV 0.4 mm × 0.35 mm 1.6 mm × 1.4 mm
T287 35365-35391 Sentence denotes Pixel size 167 nm 650 nm
T288 35392-35407 Sentence denotes z 01 125 mm -
T289 35408-35437 Sentence denotes z 12 4975 mm 10 mm − 100 mm
T290 35438-35474 Sentence denotes Regime holographic direct contrast
T291 35475-35507 Sentence denotes Rotation start-stop continuous
T292 35508-35533 Sentence denotes Exposure 2 s 0.035 ⁢ s
T293 35534-35570 Sentence denotes Total exposure ≃ 63 m i n ≃ 75 s
T294 35571-35712 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 35713-35983 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 35984-36144 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 36145-36274 Sentence denotes Single-distance tomogram recordings with about 1500 projections, and flat images before and after the scan, took less than 2 min.
T298 36275-36472 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 36473-36563 Sentence denotes To avoid detector saturation, the beam was attenuated by 4× single crystal silicon wafers.
T300 36564-36625 Sentence denotes The configuration for (2–cone beam) is depicted in Figure 1d:
T301 36626-36681 Sentence denotes The beam was focused by the KB-mirrors to about 300 nm.
T302 36682-37137 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 37138-37274 Sentence denotes The sample was positioned at variable (defocus) distances behind the focus (waveguide exit), typically z01=125mm for the first distance.
T304 37275-37476 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 37477-37656 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 37657-37740 Sentence denotes 1442 projections were recorded, with a typical exposure time of 2 s per projection.
T307 37742-37797 Sentence denotes Phase retrieval, image reconstruction, and segmentation
T308 37799-37833 Sentence denotes Phase retrieval and reconstruction
T309 37834-38182 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 38183-38304 Sentence denotes Importantly, both CTF and NL-CTF implementations can be augmented by imposing support and range constraints, when needed.
T311 38305-38463 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 38464-38632 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 38633-38937 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 38938-39265 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 39266-39434 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 39435-39542 Sentence denotes Stitching of reconstructions from different tomographic scans was performed using (Miettinen et al., 2019).
T317 39543-39610 Sentence denotes Resolution estimates were obtained by FSC analysis, see Appendix 2.
T318 39612-39649 Sentence denotes Image segmentation and quantification
T319 39650-39751 Sentence denotes For each patient the stitched overview scans were analyzed with regard to structural characteristics.
T320 39752-39974 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 39975-40113 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 40114-40229 Sentence denotes Further, the areas of the paraffin which represent air compartments were linked to the outside of the tissue block.
T323 40230-40325 Sentence denotes Individual self-contained areas of paraffin (not connected to air) were excluded from the mask.
T324 40326-40443 Sentence denotes Based on this segmentation, the distance to the nearest voxel containing oxygen was calculated for each tissue voxel.
T325 40444-40516 Sentence denotes The tissue volume V is given by the sum of all voxels containing tissue.
T326 40517-40607 Sentence denotes The surface area SA is defined by all tissue voxels with a distance of 1 pixel to the air.
T327 40608-40713 Sentence denotes From this information we calculated the specific surface(2) SV=SA/Vwith S⁢A/V surface area volume -ratio.
T328 40714-40822 Sentence denotes Further a characteristic length(3) Lc=V/SA⋅vxwith v⁢x edge length of a voxel was determined for each sample.
T329 40823-40958 Sentence denotes Additionally, the mean distance (dO2¯) from all tissue voxels to the closest air compartment and its standard deviation was calculated.
T330 40959-41097 Sentence denotes Hyaline membranes and capillary networks were extracted using semi-manual segmentation functions in Avizo (Thermo Fisher Scientific, USA).
T331 41098-41253 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 41254-41364 Sentence denotes In particular, inflammatory cell subpopulations - that is, macrophages and lymphocytes - can be distinguished.
T333 41365-41478 Sentence denotes To this end, an automatic and parameter-controlled algorithm denoted as BlobFinder was used (arivis AG, Germany).
T334 41479-41622 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 41623-41695 Sentence denotes The algorithm is able to identify roundish structures with a given size.
T336 41696-41779 Sentence denotes For the segmentation of the lymphocytes, we chose a characteristic size of 6.5⁢μ⁢m.
T337 41780-41878 Sentence denotes The structures identified in this step also include macrophages and parts of the capillary system.
T338 41879-42027 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 42028-42136 Sentence denotes In the tomographic reconstructions, the lymphocytes appear denser compared to the nuclei of the macrophages.
T340 42137-42277 Sentence denotes Further, nuclei from endothelial cells and parts of the capillaries filled with blood residues were excluded based on their elongated shape.
T341 42278-42343 Sentence denotes Only structures with a sphericity higher than 0.55 were included.
T342 42344-42521 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.