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73 results for “multiplexing imaging”
Minimal dataset to test multiplexed DNA imaging (Hi-M) software pipelines
<p>This is a dataset of nuclei (DAPI), and 3 multiplexed DNA imaging cycles to test and validate processing software packages, such as pyHiM (https://github.com/marcnol/pyHiM). This dataset was acquired in a nc14 Drosophila embryo.</p> <p>File contents:</p> <p>scan_001_RT27_001_ROI_converted_decon_ch00.tif barcode 27, fiducial <br> scan_001_RT27_001_ROI_converted_decon_ch01.tif barcode 27<br> scan_001_RT29_001_ROI_converted_decon_ch00.tif barcode 29, fiducial <br> scan_001_RT29_001_ROI_converted_decon_ch01.tif barcode 29 <br> scan_001_RT37_001_ROI_converted_decon_ch00.tif barcode 37, fiducial <br> scan_001_RT37_001_ROI_converted_decon_ch01.tif barcode 37 <br> scan_006_DAPI_001_ROI_converted_decon_ch00.tif DAPI <br> scan_006_DAPI_001_ROI_converted_decon_ch01.tif DAPI, fiducial <br> scan_006_DAPI_001_ROI_converted_decon_ch02.tif RNA</p> <p> </p> <p>To test this dataset please refer to <a href="https://github.com/marcnol/pyHiM">pyHiM documentation page</a>.</p>
Multiplexed DNA-FISH imaging dataset, drosophila embryos, nuclear cycles 11-14
<p>Multiplexed DNA-FISH imaging dataset from Drosophila embryos at nuclear cycles 11-14.</p> <p>Examples on how to load and use this dataset can be found at this <a href="https://github.com/NollmannLab/Goetz_etal">GitHub repository</a>.</p> <p><strong>Data processing details</strong></p> <p>Barcodes were segmented using a neural network (<a href="https://github.com/stardist/stardist"><em>stardist</em></a>) specifically trained for the detection of 3D diffraction limited spots produced by our microscope. To extract the position of the barcode with sub-pixel accuracy, a subsequent 3D Gaussian fit of the regions segmented by <em>stardist</em> was performed with Big-FISH (<a href="https://github.com/fish-quant/big-fish">https://github.com/fish-quant/big-fish</a>). Barcode localizations with intensities lower than 1.5 times that of the background were filtered out.</p> <p>Nuclei were segmented from projected DAPI images using <em><a href="https://github.com/stardist/stardist">stardist</a> </em>with a neural network trained for detection of nuclei from <em>Drosophila</em> embryos under our imaging conditions. Barcodes were then attributed to single nuclei by using the XY coordinates of the barcodes and the DAPI masks of the nuclei. Finally, pairwise distance matrices were calculated for each single nucleus.</p> <p><strong>Processed data in Figures</strong></p> <p>This new version of the dataset contains the raw data for each of the figures in the manuscript:</p> <p><strong>Associated publication</strong></p> <p><strong>Multiple parameters shape the 3D chromatin structure of single nuclei at the doc locus in </strong><em>Drosophila</em>.</p> <p>Markus Götz, Olivier Messina, Sergio Espinola, Jean-Bernard Fiche, Marcelo Nollmann</p> <p>Nature Communications (2022).</p>
Multiplexed fluorescence imaging based on cycles, raw and processed data.
<p>This dataset was created from a larger acquisition in order to provide an example of reasonnable size, as a companion data set to the F1000Research paper preprint DOIXXX.</p> <ul> <li>The original raw data including metadata files are included in <strong>Microscope_Output.zip.</strong></li> <li><strong>Experiment.json</strong> and<strong> channelnames.txt </strong>are the ones generated by the acquisition software. They are the only files needed when starting from one of the processed data set below.</li> <li>The deconvolution obtained with the commercial software Microvolution is also provided in <strong>bu_deconvolution.zip.</strong> To start from Step 1(Extended Depth of Field) instead of Step 0 (deconvolution), unzip this file in your output directory and rename the folder bu_deconvolution to out.</li> <li>The extended field of view 2D images created from step 0 to step 2, provided for convenince in <strong>edfonly.zip</strong></li> <li>The final files generated by trhe Multiplex processor, including the segmentation mask , are provided in<strong> finaloutput.zip</strong>. These files can be used in a specific analysis software.</li> </ul> <p> </p>
Image-based & machine learning-guided multiplexed serology test for SARS-CoV-2
<p>Single-cell extracted imaging features created in project "Image-based & machine learning-guided multiplexed serology test for SARS-CoV-2". The dataset includes train (with annotations) and test features used in the manuscript. Four SARS-CoV-2 antigens (S, N, R, M) were imaged separately with serum samples presenting IgG, IgA and IgM antibodies.</p>
Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes
<p>Raw multichannel and/or Z-stack source data from time series images in TIF format to accompany publication of:</p> <p><strong>Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes</strong></p> <p>Jina Ko<sup>1</sup>, Martin Wilkovitsch<sup>2</sup>, Juhyun Oh<sup>1</sup>, Rainer Kohler<sup>1</sup>, Evangelia Bolli<sup>1,3</sup>, Mikael J. Pittet<sup>1,3,4,5</sup>, Claudio Vinegoni<sup>1</sup>, David B. Sykes<sup>6,7</sup>, Hannes Mikula<sup>2</sup>, Ralph Weissleder<sup>1,8</sup>*, Jonathan C. T. Carlson<sup>1,7</sup>*</p> <p><sup>1 </sup>Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, MA 02114 </p> <p><sup>2</sup> Institute of Applied Synthetic Chemistry, TU Wien, 1060 Vienna, Austria </p> <p><sup>3</sup> Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland</p> <p><sup>4</sup> Ludwig Institute for Cancer Research, Lausanne Branch, Switzerland</p> <p><sup>5</sup> AGORA Cancer Center, Lausanne, Switzerland</p> <p><sup>6</sup> Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA</p> <p><sup>7 </sup>Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA</p> <p><sup>8 </sup>Department of Systems Biology, Harvard Medical School, 200 Longwood Ave, Boston, MA 02115</p>
OMAP-23: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of vermiform appendix (FFPE) with MICS on MACSima
<p><strong>Description</strong></p> <p> OMAP-23 was designed for MICS (MACSima imaging cyclic staining) imaging of FFPE human vermiform appendix sample. Tissue fixation and antigen retrieval is described in (<a href="https://www.biorxiv.org/content/biorxiv/early/2023/11/07/2023.10.27.564191.full.pdf">Spatial protein and RNA analysis on the same tissue section using MICS technology</a>). The MACSima technology is described in detail in the following publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors</a>). All, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region, removing Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing non-specific binding. The use of human IgG1 recombinant antibodies allows for the addition of uncoupled monoclonal antibodies from other species followed by a fluorescence labelled secondary reagent specific for the species of the monoclonal antibody. The multiplex system has been described already for OMAP-10 and OMAP-21. A new dye VioB515 is used for one reagents. The fluorescence is removed by cleavage. The panel contains 28 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for six anatomical structures and most cell types present in the vermiform appendix (link to ASCT+B table added). OMAP-23 follows OMAP-21, with fewer antibodies but adding antibodies for non-immune cells.</p> <p>All reagents are from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation between lots. For this reason, lot information is not included in this table. Analysis was performed by an accompanied software package MACSIQ View Analysis also described in the MACSima publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">https://doi.org/10.1038/s41598-022-05841-4</a>). The MACSima system is continuously evolving, this is the third OMAP for the MACSima system. A representative dataset created using OMAP-23 can be found here 10.5281/zenodo.14008816 .The AVRs for the dataset can be found here (to be added).</p>
Vectra Polatis image of human colorectal cancer (CRC1) from: A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially resolved tissue phenotyping at single-cell resolution.
<p>Two 4 µm thick serial sections were cut from CRC1 FFPE block using a microtome. The first slide was dewaxed and rehydrated before carrying out HIER with Antigen Retrieval Reagent-Basic (R&D Systems). The tissue was then blocked and incubated with the anti-CD3 antibody (Dako, Supplementary Table 2) followed by horseradish peroxidase (HRP) conjugated anti-rabbit antibody (Dako) and stained with 3,3' diaminobenzidine (DAB) substrate (Abcam) and haematoxylin. Areas with CD3<sup>+</sup> infiltration in the proximity of the tumour invasive margin were identified by a clinical pathologist (M. R-J.)</p> <p>The second slide was stained with a panel of six antibodies (CD8, PD1, Ki67, PDL1, CD68, GzB, Supplementary Table 2), Opal fluorophores and 4’,6-diamidino-2-phenylindole (DAPI) on a Ventana Discovery Ultra automated staining platform (Roche). Expected expression and cellular localisation of each marker as well as fluorophore brightness were used to minimise fluorescence spillage upon antibody-Opal pairing. Following a one-hour incubation at a 60°C, the slide was subjected to an automated staining protocol on an autostainer. The protocol involved deparaffinisation (EZ-Prep solution, Roche), HIER (DISC. CC1 solution, Roche) and seven sequential rounds of: one hour incubation with the primary antibody, 12 minutes incubation with the HRP-conjugated secondary antibody (DISC. Omnimap anti-Ms HRP RUO or DISC. Omnimap anti-Rb HRP RUO, Roche) and 16 minute incubation with the Opal reactive fluorophore (Akoya Biosciences). For the last round of staining, the slide was incubated with Opal TSA-DIG reagent (Akoya Biosciences) for 12 minutes followed by Opal 780 reactive fluorophore for our hour (Akoya Biosciences). A denaturation step (100°C for 8 minutes) was introduced between each staining round in order to remove the primary and secondary antibodies from the previous cycle without disrupting the fluorescent signal. The slide was counterstained with DAPI (Akoya Biosciences) and coverslipped using ProLong Gold antifade mounting media (Thermo Fisher Scientific). The Vectra Polaris automated quantitative pathology imaging system (Akoya Biosciences) was used to scan the labelled slide. Six fields of view, within the area selected by the pathologist, were scanned at 20x and 40x magnification using appropriate exposure times and loaded into inForm{Kramer, 2018 #23} for spectral unmixing and autofluorescence isolation using the spectral libraries. After spectral unmixing and merging of six 20x fields of view for a total of >5mm<sup>2</sup> ROI (Table 2), one single-tiff image was extracted for each marker and its intensity was rescaled from 0 to 1 with custom R scripts.</p>
OMAP-10: Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MACSima v1.0
<p> </p> <p>OMAP-10 was designed for MACSima (MACSima imaging cyclic staining) imaging of <em>paraformaldehyde</em> (PFA)-fixed human tonsil samples. The MACSima technology is described in detail in the following publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">https://doi.org/10.1038/s41598-022-05841-4</a>). Most, but not all, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region. The described mutation removes the Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing non-specific binding of human antibodies on human tissues. Highly multiplexed imaging is achieved through cycles of immunolabeling with FITC, PE, and APC conjugated antibodies and photobleaching to eliminate fluorescence signal between imaging cycles. The panel contains 30 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for all anatomical structures and most cell types present in the ASCT+B tonsil table, v1.0 (submitted for review). OMAP-10 follows closely OMAP-1 described for human lymph nodes (<a href="https://hubmapconsortium.github.io/ccf-releases/v1.3/docs/omap/omap-1-human-lymph-node-ibex.html">https://hubmapconsortium.github.io/ccf-releases/v1.3/docs/omap/omap-1-human-lymph-node-ibex.html</a>). The initial dataset associated with OMAP-10 can be found in this dataset. All reagents were obtained from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation between lots. For this reason, lot information is not included in the table below. Analysis was performed by an accompanied software package MACSIQ View Analysis also described in the MACSima publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">https://doi.org/10.1038/s41598-022-05841-4</a>). The plan was to also include the data analysis in the uploaded dataset. However mixing the original data with analyses data would lead to confusion. Therefore only the original image files are included here. This is in brief the image analysis pipeline: the software processes the raw images of the MACSima run, generates stitched images, and then allows downstream analysis including cell segmentation, cell gating, data normalization, dimension reduction plots (tSNE, UMAP), heat maps, distance analyses and cluster analyses, all of which are interactively linked together. The MACSima system is continuously evolving, and this is the first OMAP dataset generated by using the MACSima system (Instrument, Reagents and Software).</p> <p>The images enclosed are in OME-tif format (16 bit depth). The optical resolution is 0.17 micron/pixel. The imaged area size is 1.6 mm x 1.3 mm. This Data is currently under review by the OMAP community and changes are still possible in the follow up version. The image contains stichted images of 9 fields of view. The complete runtime on the MACSima for this image dataset was about 12 hours on the instrument. The OMAP description also links to the ASCT+B table for the palatine tonsil.</p> <p><strong>Anatomical Structures, Cell Types, plus Biomarkers (ASCT+B) table for Palatine Tonsil v1.0</strong></p> <p><strong>Description</strong></p> <p><a href="https://hubmapconsortium.github.io/ccf/pages/ccf-anatomical-structures.html">Anatomical Structures, Cell Types, plus Biomarkers (ASCT+B) tables</a> aim to capture the nested <em>part of</em> structure of anatomical human body parts, the typology of cells, and biomarkers used to identify cell types. The tables are authored and reviewed by an international team of experts. The Palatine Tonsil ASCT+B table is derived from published literature, public datasets, and unpublished studies from table authors. The Palatine Tonsil is part of the tonsiluar ring of Waldeyer network. In comparison to other Tonsils the Palatine Tonsil has an enlarged lymphoid tissue.</p> <p>The gene biomarkers are primarily derived from a preprint on an Atlas of Cells of the human tonsil (<a href="https://www.biorxiv.org/content/10.1101/2022.06.24.497299v1">Ramon Massoni-Badosa et al 2022</a>). The tonsil azimuth data set can be explored <a href="https://azimuth.hubmapconsortium.org/references/human_tonsil/">here</a> .Cell phenotypes, especially for antibody-based assays like the MACSima are very complex and in its first version, only the basic cell types are listed with many more to be included in the next iteration of the ASCT+B table. The correlation between protein detection and RNA expression data at the single cell level needs to be established. In total, this table reports 13 anatomical structures, 17 cell types, and 30 biomarkers. </p> <p>The following list contains the file name and the target name of the antibody used in a given staining:</p> <p>ACTIN_REAL650/ACTA2</p> <p>Bcl2_REA872/BCL2</p> <p>CD11c_REAL235/ITGAX</p> <p>CD138_REA929/SDC1</p> <p>CD15_VIMC6/FUT4</p> <p>CD19_REAL106/CD19</p> <p>CD1c_REA694/CD1C</p> <p>CD209_REAL1087/CD209</p> <p>CD20_REA1087/MS4A1</p> <p>CD21_REA940/CR2</p> <p>CD274_PDL1/CD274</p> <p>CD279_REAL531/PDCD1</p> <p>CD27_REA499/CD27</p> <p>CD39_REA739/ENTPD1</p> <p>CD3_REAL1097/CD3E</p> <p>CD44_REA690/CD44</p> <p>CD4_REA1307/CD4</p> <p>CD68_REA1306/CD68</p> <p>CD79a_REA1142/CD79A</p> <p>CD8_REA734/CD8A</p> <p>CollagenIV_REAL1212/COL4A1</p> <p>Cytokeratin_CK36H5/KRT7,KRT8,KRT18,KRT19</p> <p>FoxP3_REA1253/FOXP3</p> <p>HLADR_REAL550/HLA-DRA</p> <p>IgD_REA740/IGHD</p> <p>IgM_REAL689/IGHM</p> <p>Ki67_REA183/MKI67</p> <p>PlasmaCell_REA908/CKAP4</p> <p>Vimentin_REA409/VIM</p>
Multiplexed Staining Dataset - OMAP 5 - Liver-Lanthanides-conjugated antibodies and C60-secondary ion mass spectrometry imaging
<p>This dataset contains images of multiplexed antibody panel on a human pediatric liver section including the nuclear marker and antibodies conjugated with lanthanides tags. The dataset is one example of serial experiments of multiplexed antibody staining and imaging. The antibody panel targets the major cell types and tissue structures in the liver tissue. Data acquisition was performed using single multiplexing imaging by C60-secondary ion mass spectrometry.</p> <p> </p>
OMAP-8: Multiplexed Antibody-Based Imaging of Placenta with Imaging Mass Cytometry (IMC), v1.0
<p>OMAP-8 was designed for Imaging Mass Cytometry (IMC) (<a href="https://pubmed.ncbi.nlm.nih.gov/24584193/">https://pubmed.ncbi.nlm.nih.gov/24584193/</a>) of formalin-fixed paraffin-embedded (FFPE) human term-placenta samples. The tissue slides were prepared with a two-step antigen retrieval process (pH 6 and pH 9, as described <a href="https://dx.doi.org/10.17504/protocols.io.bpwumpew">https://dx.doi.org/10.17504/protocols.io.bpwumpew</a>). OMAP antibodies validated by immunohistochemistry and IMC were conjugated to polymers containing metal isotopes. Conjugated antibodies were used to stain processed human term-placenta tissue simultaneously. Regions of the processed tissue were then acquired on the imaging mass cytometer (Hyperion; Standard BioTools) by laser ablation and visualized. The panel contains 26 antibodies conjugated to unique metal isotopes and iridium marks the DNA. This OMAP provides a spatial context for key placenta cell types in the <a href="https://doi.org/10.48539/HBM446.WGLG.755">ASCT+B v.1.0 table</a>. Single-cell RNA sequencing data were used to guide marker selection for multiplexed tissue imaging. For example, ASCL2, HLA-G, PD-L1, CD68 and LYVE1 allow functionally specialized cell types to be visualized and quantified in the placenta. Note that one of our core antibodies is to LYVE1 but, unlike in other tissues where it is used to mark lymphatic vasculature, here we use it to mark the macrophage of the placenta (Hofbauer cells) – there should be no lymphatics in the placenta.</p>
Accompanying dataset for: "IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues"
<p>Mouse datasets were acquired using the manual IBEX multiplex imaging protocol and accompany the manuscript “IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues”, A. Radtke <em>et al.</em>, 2020, PNAS.</p> <p>All image data are stored using the <a href="https://imaris.oxinst.com/support/imaris-file-format">Imaris file format</a>. To view these multi-channel images, you can either use one of these free viewers, <a href="https://imaris.oxinst.com/imaris-viewer">Imaris viewer</a>, <a href="https://imagej.net/Fiji">Fiji</a>.</p> <p>Each experiment has an associated imaging meta-data file in xlsx format and the resulting image in Imaris format.</p> <p><strong>Mouse spleen (Manual)</strong></p> <p>Dataset is a 16 parameter IBEX experiment performed on a mouse spleen section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse thymus (Manual)</strong></p> <p>Dataset is a 26 parameter IBEX experiment performed on a mouse thymus section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse lung (Manual)</strong></p> <p>Dataset is a 23 parameter IBEX experiment performed on a mouse lung section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.379 µm), y (0.379 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse small intestine (Manual)</strong></p> <p>Dataset is a 20 parameter IBEX experiment performed on a mouse small intestine section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse liver (Manual)</strong></p> <p>Dataset is an 18 parameter IBEX experiment performed on a liver section from a LysM-tdtomato reporter mouse labeled with antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse naive lymph node (Manual)</strong></p> <p>Dataset is a 41 parameter IBEX experiment performed on a mouse lymph node section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p> <p><strong>Mouse immunized lymph node (Manual)</strong></p> <p>Dataset is a 41 parameter IBEX experiment performed on a mouse lymph node section labeled with the nuclear marker JOJO-1 and antibodies directed against the indicated markers. Images were acquired using an inverted Leica TCS SP8 X confocal microscope equipped with a 40X objective (NA 1.3), 4 HyD and 1 PMT detectors, a white light laser that produces a continuous spectral output between 470 and 670 nm as well as 405, 685, and 730 nm lasers. All images were captured at an 8-bit depth, with a line average of 3, and 1024x1024 format with the following pixel dimensions: x (0.284 µm), y (0.284 µm), and z (1 µm). Images were tiled and merged using the LAS X Navigator software (LAS X 3.5.5.19976).</p>
Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging
<p>MELC (Multi-epitope ligand cartography) multiplex imaging data of our neuroblastoma cohort supporting the publication " Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging". The zip folders contain raw image data of one to four fields of view (FoV). The folder "RoI" contains the masks of user-selected regions. "marker_status.csv" is used for normalization with RESTORE. "MELC_single_cell_data.csv" contains the normalized single-cell data with cell type assignments.</p>
Multiplexed imaging mass cytometry reveals distinct tumor-immune microenvironments linked to immunotherapy responses in melanoma
<p><strong>- melanoma_IMC_data.zip</strong></p> <p>The zip file contains the raw IMC images (in the raw_tiff folder) and corresponding single cell masks (in the mask folder) associated with the paper "Multiplexed imaging mass cytometry reveals distinct tumor-immune microenvironments linked to immunotherapy responses in melanoma". The MCD files by CyTOF IMC were exported to a multi-channel TIFF file including 41 channels, and the order of the channel was provided in the <strong>Melanoma_panel.csv</strong>. </p> <p><strong>- Melanoma_code_data.zip</strong></p> <p>The zip file contains the 4 folders described as follows: </p> <ul> <li>Folder ”data“: the processed data for the result shown in paper<br> - Folder "input": <br> - sc_data.csv: the single cell protein expression data;<br> - Folder "abundance": the cell type abundance files;<br> - Folder "clidata": the response and survival data for 4 melanoma datasets used in the paper; <br> - Folder "hc_result": TME archetypes annotation for each sample/ROI from hierarchical clustering;<br> - Folder "ICB": data for ICB analysis (presented in FigS3);<br> - Folder "meta": panel file for clustering;<br> - Folder "RNAseq_data": the RNAseq data for 4 melanoma datasets used in the paper;<br> - Folder "RNAseq_deconv": the result of cell type deconvolution from bulk RNAseq; <br> - Folder "spatial": data for neighbourhood analysis (presented in Fig3, FigS4).<br> - Folder "output": intermediate result for analysis.</li> <li>Folder "Rscript": R scripts for reproducing results in the paper.<br> - generate_Figs.Rmd: ploting figures presented in the paper;<br> - functions.R: functions used for analysis;<br> - Clustering.Rmd: determining cell types based on marker intensities;<br> - Spatial_analysis.Rmd: neighbourhood analysis to get significant interction/avoidance cell relationships.</li> <li>Folder "Figs": figures presented in paper.</li> <li>Folder "HE_figs": the H&E image and the ROIs distribution for each sample.</li> </ul>
Multi-modal image analysis for large scale cancer tissue studies within IMMUcan: multiplex immunofluorescence images
<p>In cancer research, multiplexed imaging has enabled the in-depth characterization of the tumor microenvironment (TME) and how it relates to patient prognosis. However, standardized, multi-modal data from large numbers of patients to identify robust biomarkers is missing. To provide such data across five cancer indications, the IMMUcan consortium performs broad molecular and cellular spatial profiling of thousands of cancer samples. Two reproducible and scalable workflows have been developed for whole slide multiplexed immunofluorescence (mIF) and imaging mass cytometry (IMC) to overcome challenges of reproducibility and scalability. For mIF we developed IFQuant, a web-based tool optimized for user-friendliness and reproducibility. This Zenodo record contains the mIF images and IFQuant settings to reproduce the results presented in the referenced publication. The companion IMC dataset is available as a joint Zenodo record.</p>
OMAP-21: Organ Mapping Antibody Panel (OMAP) for Multiplexed Antibody-Based Imaging of Human Palatine Tonsil with MICS on MACSima 1.5
<p>OMAP-21 was designed for MICS (MACSima imaging cyclic staining) imaging of FFPE human tonsil samples. Tissue fixation and antigen retrieval is described in (<a href="https://www.biorxiv.org/content/biorxiv/early/2023/11/07/2023.10.27.564191.full.pdf">Spatial protein and RNA analysis on the same tissue section using MICS technology</a>). The MACSima technology is described in detail in the following publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors</a>). Most, but not all, antibodies in this panel are recombinant antibodies with a mutated human IgG1 constant region. The described mutation removes the Fc receptor binding capacity of human IgG1, eliminating the need for additional blocking steps and reducing non-specific binding of human antibodies on human tissues. Highly multiplexed imaging is achieved through cycles of immunolabeling with FITC, PE, and APC conjugated antibodies and photobleaching to eliminate fluorescence signal between imaging cycles. The panel contains 47 antibodies and the nuclear marker DAPI for image alignment and nuclear segmentation. This OMAP provides a spatial context for all anatomical structures and most cell types present in the human palatine tonsil. OMAP-21 follows closely OMAP-1 described for human lymph nodes (<a href="https://cdn.humanatlas.io/hra-releases/v1.4/docs/omap/omap-1-human-lymph-node-ibex.html">omap-1-human-lymph-node-ibex</a>) and OMAP-10 (<a href="https://cdn.humanatlas.io/hra-releases/v1.4/docs/omap/omap-10-palatine-tonsil-macsima.html">omap-10-palatine-tonsil-macsima</a>).</p> <p>All reagents were obtained from Miltenyi Biotec and have been rigorously tested through an internal quality control system to have minimal variation between lots. For this reason, lot information is not included in this table. Analysis was performed by an accompanied software package MACSIQ View Analysis also described in the MACSima publication (<a href="https://doi.org/10.1038/s41598-022-05841-4">https://doi.org/10.1038/s41598-022-05841-4</a>). The result of the analysis is included in the uploaded dataset. In brief, the software processes the raw images of the MACSima run, generates stitched images, and then allows downstream analysis including cell segmentation, cell gating, data normalization, dimension reduction plots (tSNE, UMAP), heat maps, distance analyses and cluster analyses, all of which are interactively linked together. The MACSima system is continuously evolving, this is the second OMAP for the MACSima system. A representative dataset created using OMAP-21 can be found here:<a href="https://doi.org/10.5281/zenodo.7875937"> </a><strong> 10.5281/zenodo.11281609 .</strong></p>
Imaging Mass Cytometry Images (APP1) from: A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially resolved tissue phenotyping at single-cell resolution.
<p>Four µm-thick sections were cut from the APP1 FFPE block with a microtome and used for staining with a panel of 26 antibodies targeting the main immune, stromal and epithelial cell populations of the gastrointestinal tract (Supplementary Table 2). The optimal dilution of each antibody in the panel was identified by staining and ablating FFPE appendix sections. The resulting images were reviewed by a mucosal immunologist (J.S.) and the dilution giving the best signal to background ratio was selected for each antibody (Supplementary Table 2). To perform the staining for IMC, slides were dewaxed after a one-hour incubation at 60°C, rehydrated and heat-induced antigen retrieval was performed with a pressure cooker in Antigen Retrieval Reagent-Basic (R&D Systems). Slides were incubated in a 10% BSA (Sigma), 0.1% Tween (Sigma), and 2% Kiovig (Shire Pharmaceuticals) Superblock Blocking Buffer (Thermo Fisher) blocking solution at room temperature for two hours. Each antibody was added to a primary antibody mix at the selected concentration in blocking solution and incubated overnight at 4°C. After two washes in PBS and PBS-0.1% Tween, the slides were treated with the DNA intercalator Cell-ID™ Intercalator-Ir (Fluidigm) (containing the two iridium isotopes 191Ir and 193Ir) 1.25 mM in a PBS solution. After a 30-minute incubation, the slides were washed once in PBS and once in MilliQ water and air-dried. The stained slides were then loaded in the Hyperion Imaging System (Fluidigm) imaging module to obtain light-contrast high resolution images of approximately four mm<sup>2</sup>. These images were used to select the ROI in each slide. For APP1, a one mm<sup>2</sup> ROI containing a lymphoid follicle in its whole depth alongside a portion of lamina propria and of epithelium was selected. ROIs were ablated at a o µm/pixel resolution and 200 Hz frequency.</p>
Processed single cell data from CODEX multiplexed imaging of the human intestine
<p>We performed CODEX (co-detection by indexing) multiplexed imaging on 64 sections of the human intestine (~16 mm2) from 8 donors (B004, B005, B006, B008, B009, B010, B011, and B012) using a panel of 57 oligonucleotide-barcoded antibodies. Subsequently, images underwent standard CODEX image processing (tile stitching, drift compensation, cycle concatenation, background subtraction, deconvolution, and determination of best focal plane), single cell segmentation, and column marker z-normalization by tissue. The outputs of this process were data frames of 2.6 million cells with 57 antibody fluorescence values quantified from each marker. Each cell has its cell type, cellular neighborhood, community of neighborhooods, and tissue unit defined with x, y coordinates representing pixel location in the original image. This is from a total of 25 cell types, 20 multicellular neighborhoods, 10 communities of neighborhoods, and 3 tissue segments that could be used to understand the cellular interactions, composition, and structure of the human intestine from the duodenum to the sigmoid colon and understand differences between different areas of the intestine. This data could be used as a healthy baseline to compare other single-cell datasets of the human intestine, particularly multiplexed imaging ones. </p> <p>The overall structure of the datasets is individual cells segmented out in each row. Columns MUC2 through CD161 are the markers used for clustering the cell types. These are the columns that are the values of the antibody staining the target protein within the tissue quantified at the single-cell level. This value is the per cell/area averaged fluorescent intensity that has subsequently been z normalized along each column as described above. OLFM4 through MUC6 were captured in the quantification but not used within the clustering of cell types. Other columns are explained in the table in the Usage Notes section below.</p> <p>Along with this main data table, there is also a donor metadata table that links the donor ids to clinical metadata such as: age, sex, race, BMI, history of diabetes, history of cancer, history of hypertension, and history of gastorintestinal disease.</p> <p>The raw imaging data can be found at (<a href="https://portal.hubmapconsortium.org/">https://portal.hubmapconsortium.org/</a>). We have created a landing page with links to all the raw dataset IDs and the HuBMAP ID for this Collection is HBM692.JRZB.356 and the DOI is:10.35079/HBM692.JRZB.356. This can be used to also pair it with the matched snRNAseq and snATACseq for each section of tissue.</p>
Dataset: CODEX highly multiplexed tissue imaging in pancreas
<p><strong>Human pancreas</strong></p> <p>This dataset was acquired using CODEX, multiplexed single-cell imaging technology for spatial profiling, where all image data is in .tif format and it includes an associated imaging metadata .csv file. The combination of the targets present in this experiment define some of the main cell types and anatomical structures in human pancreas tissue.</p> <p>This dataset is a 12-highly multiplexed experiment performed on a human pancreas 5 μm section including the nuclear marker Hoechst and antibodies conjugated with oligo-sequences directed against the individual markers. Images were acquired using a Leica DMi8 widefield microscope, a digital CMOS camera (Hamamatsu, ORCA-Flash4.0 V3), and a 20x (0.75) NA dry objective. The light source was a SOLA-SM-II. All images were captured at a 16-bit depth with the following dimensions: x (0.325 μm), y (0.325 μm), and z (1.5 μm). In addition, images were processed, tiled and merged using the CODEX® Processor application (CODEX Processor 1.7.0.6).</p>
scProAtlas: an atlas of multiplexed single-cell spatial proteomics imaging in human tissues
<p>All analysis results for the spatial proteomics imaging techniques in the scProAtlas database are stored in compressed files named accordingly. Within each compressed file, the folders are organized in a fixed storage structure in the following order: Analysis module > Imaging Technique > Dataset > Tissue > ROI.</p> <p>Each folder contains the corresponding metadata (including original sample information, cell type annotations, and neighborhood annotations) stored in a file named <code>cells.tsv</code>. Additionally, the module used to identify spatial pattern genes includes an <code>anndata</code> format file, named <code>adata_moran.h5ad</code>, which stores the integrated results of scRNA-seq and spatial proteomics.</p> <p>scProAtlas_analysis_code.tar.gz contains example codes for all analysis modules in scProAtlas. Here, we provide the example using <strong>SCP_CODEX1 - Large intestine. </strong>The codes include all the scripts used for the entire workflow, from image segmentation to scRNA-spatial proteomics integration, and spatial analysis.</p> <p>We have also uploaded the raw protein channel matrices with AnnData format in <strong>version 3 and 4.</strong></p>
Human intestine processed CODEX multiplexed images for donors B004-6, B008 (Part 1/2)
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