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{
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{
            
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  "authors": "Ahmad Ahsan Nawaz, Marta Urbanska, Maik Herbig, Martin 
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  "authors": "Ahmad Ahsan Nawaz, Marta Urbanska, Maik Herbig, Martin 
            
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N\u00f6tzel, Martin Kr\u00e4ter, Philipp Rosendahl, Christoph Herold, 
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N\u00f6tzel, Martin Kr\u00e4ter, Philipp Rosendahl, Christoph Herold, 
            
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Nicole T\u00f6pfner, Marketa Kubankova, Ruchi Goswami, Shada 
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Nicole T\u00f6pfner, Marketa Kubankova, Ruchi Goswami, Shada 
            
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Abuhattum, Felix Reichel, Paul M\u00fcller, Anna Taubenberger, 
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Abuhattum, Felix Reichel, Paul M\u00fcller, Anna Taubenberger, 
            
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Salvatore Girardo, Angela Jacobi, Jochen Guck",
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Salvatore Girardo, Angela Jacobi, Jochen Guck",
            
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  "creator_user_id": "d76d937b-3601-4559-92da-585a47c257f5",
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  "creator_user_id": "d76d937b-3601-4559-92da-585a47c257f5",
            
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  "doi": "10.6084/m9.figshare.11302595.v1",
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  "doi": "10.6084/m9.figshare.11302595.v1",
            
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  "groups": [],
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  "groups": [],
            
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  "id": "e61bb335-3523-e539-78d1-0720ad5a99f1",
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  "id": "e61bb335-3523-e539-78d1-0720ad5a99f1",
            
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  "isopen": true,
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  "isopen": true,
            
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  "license_id": "CC0-1.0",
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  "license_id": "CC0-1.0",
            
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  "license_title": "Creative Commons Public Domain Dedication",
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  "license_title": "Creative Commons Public Domain Dedication",
            
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  "license_url": "https://creativecommons.org/publicdomain/zero/1.0/",
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  "license_url": "https://creativecommons.org/publicdomain/zero/1.0/",
            
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  "metadata_created": "2020-09-16T15:59:26.382535",
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  "metadata_created": "2020-09-16T15:59:26.382535",
            
n
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  "metadata_modified": "2020-09-16T16:33:09.296899",
n
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  "metadata_modified": "2020-09-16T16:33:10.170143",
            
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  "name": "figshare-11302595-v1",
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  "name": "figshare-11302595-v1",
            
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  "notes": "This dataset contains results presented in _Nawaz, 
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  "notes": "This dataset contains results presented in _Nawaz, 
            
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Urbanska, Herbig_ et al\nmanuscript entitled \u2018 **Using real-time 
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Urbanska, Herbig_ et al\nmanuscript entitled \u2018 **Using real-time 
            
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fluorescence and deformability\ncytometry and deep learning to 
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fluorescence and deformability\ncytometry and deep learning to 
            
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transfer molecular specificity to label-free\nsorting** \u2019 
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transfer molecular specificity to label-free\nsorting** \u2019 
            
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available on bioRxiv (https://doi.org/10.1101/862227) 
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available on bioRxiv (https://doi.org/10.1101/862227) 
            
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and\nincludes:\n\n(i) 29 '*.rtdc' files containing measurements 
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and\nincludes:\n\n(i) 29 '*.rtdc' files containing measurements 
            
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performed with real time\nfluorescence and deformability cytometry 
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performed with real time\nfluorescence and deformability cytometry 
            
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[1,2]\n\n(ii) 1 '*.fcs' file containing a measurement performed with a 
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[1,2]\n\n(ii) 1 '*.fcs' file containing a measurement performed with a 
            
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standard flow\ncytometer (BD LSR II, BD Biosciences)\n\n(iii) 1 
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standard flow\ncytometer (BD LSR II, BD Biosciences)\n\n(iii) 1 
            
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'*.xlsx' file containing results from AFM-based 
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'*.xlsx' file containing results from AFM-based 
            
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indentation\nexperiments.\n\nSorting real-time fluorescence and 
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indentation\nexperiments.\n\nSorting real-time fluorescence and 
            
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deformability cytometry (soRT-FDC) is a\nrobust sorting platform, 
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deformability cytometry (soRT-FDC) is a\nrobust sorting platform, 
            
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combining image-based morphological cell analysis\nwith mechanical 
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combining image-based morphological cell analysis\nwith mechanical 
            
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characterisation and subsequent active sorting by feeding\nreal-time 
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characterisation and subsequent active sorting by feeding\nreal-time 
            
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fluorescence and deformability cytometry (RT-FDC) [2] information 
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fluorescence and deformability cytometry (RT-FDC) [2] information 
            
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to\na down-stream SSAW-based cell sorter [3].\n\nEach filename of this 
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to\na down-stream SSAW-based cell sorter [3].\n\nEach filename of this 
            
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dataset starts with the name of the figure to which the\ndatafile 
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dataset starts with the name of the figure to which the\ndatafile 
            
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corresponds. The measured samples are briefly described below. 
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corresponds. The measured samples are briefly described below. 
            
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The\n'Initial' measurements refer to the samples used for sorting, and 
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The\n'Initial' measurements refer to the samples used for sorting, and 
            
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the 'Target'\nmeasurements to the samples collected in the target 
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the 'Target'\nmeasurements to the samples collected in the target 
            
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outlet after sorting. For\nmore detailed description of samples please 
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outlet after sorting. For\nmore detailed description of samples please 
            
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refer to the manuscript.\n\nThe '*.rtdc' files are HDF5 files and can 
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refer to the manuscript.\n\nThe '*.rtdc' files are HDF5 files and can 
            
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be analysed using a Python library\ncalled dclab [4], or a software 
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be analysed using a Python library\ncalled dclab [4], or a software 
            
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called Shape-Out [5]. They can also be opened\nusing other HDF viewer 
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called Shape-Out [5]. They can also be opened\nusing other HDF viewer 
            
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programs.\n\n[1] Otto et al., \"Real-time deformability cytometry: 
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programs.\n\n[1] Otto et al., \"Real-time deformability cytometry: 
            
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on-the-fly cell\nmechanical phenotyping\". Nature Methods, 
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on-the-fly cell\nmechanical phenotyping\". Nature Methods, 
            
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12(3):199\u2013202, 2015.\ndoi:10.1038/nmeth.3281.\n\n[2] Rosendahl et 
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12(3):199\u2013202, 2015.\ndoi:10.1038/nmeth.3281.\n\n[2] Rosendahl et 
            
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al., \"Real-time fluorescence and deformability cytometry\".\nNature 
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al., \"Real-time fluorescence and deformability cytometry\".\nNature 
            
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Methods, 15(5):355\u2013358, 2018. doi:10.1038/nmeth.4639.\n\n[3] 
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Methods, 15(5):355\u2013358, 2018. doi:10.1038/nmeth.4639.\n\n[3] 
            
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Nawaz et al., \u201cAcoustofluidic Fluorescence Activated Cell 
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Nawaz et al., \u201cAcoustofluidic Fluorescence Activated Cell 
            
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Sorter\u201d.\nAnalitycial Chemistry 87(24): 12051\u201312058, 2015. 
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Sorter\u201d.\nAnalitycial Chemistry 87(24): 12051\u201312058, 2015. 
            
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doi:\ndoi.org/10.1021/acs.analchem.5b02398\n\n[4] 
49
doi:\ndoi.org/10.1021/acs.analchem.5b02398\n\n[4] 
            
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https://github.com/ZellMechanik-Dresden/dclab\n\n[5] 
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https://github.com/ZellMechanik-Dresden/dclab\n\n[5] 
            
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https://github.com/ZellMechanik-Dresden/ShapeOut\n\n  
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https://github.com/ZellMechanik-Dresden/ShapeOut\n\n  
            
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\n\n**_Figure1d_01_Beads_FL_Initial.rtdc, 
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\n\n**_Figure1d_01_Beads_FL_Initial.rtdc, 
            
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Figure1d_02_Beads_FL_Target.rtdc_**\n\nsample: 1:5 mixture of 
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Figure1d_02_Beads_FL_Target.rtdc_**\n\nsample: 1:5 mixture of 
            
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polyacrylamide microgel beads labeled with\nAlexaFluor488 and 
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polyacrylamide microgel beads labeled with\nAlexaFluor488 and 
            
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unlabeled ones, these beads were produced in house; sorted\nfor 
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unlabeled ones, these beads were produced in house; sorted\nfor 
            
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fluorescence\n\nsorting gates: FL-1 maximum 1000 \u2013 10000, area 
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fluorescence\n\nsorting gates: FL-1 maximum 1000 \u2013 10000, area 
            
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ratio: 1.0 - 1.1\n\n  \n\n**_Figure1d_03_Beads_Size_Initial.rtdc, 
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ratio: 1.0 - 1.1\n\n  \n\n**_Figure1d_03_Beads_Size_Initial.rtdc, 
            
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Figure1d_04_Beads_Size_Target.rtdc_**\n\nsample: 1:6 mixture of 13.79 
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Figure1d_04_Beads_Size_Target.rtdc_**\n\nsample: 1:6 mixture of 13.79 
            
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\u00b1 0.59 \u03bcm silica beads (SiO2-F-L3519-1;\nMicroparticles, 
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\u00b1 0.59 \u03bcm silica beads (SiO2-F-L3519-1;\nMicroparticles, 
            
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Germany) and 17.23 \u00b1 0.24 \u03bcm poly(methyl methacrylate) 
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Germany) and 17.23 \u00b1 0.24 \u03bcm poly(methyl methacrylate) 
            
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beads\n(PMMM-F-B1423; Microparticles); sorted for size\n\nsorting 
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beads\n(PMMM-F-B1423; Microparticles); sorted for size\n\nsorting 
            
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gates: size 220 \u2013 300 \u03bcm2, def 0.00 \u2013 0.01, are ratio 
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gates: size 220 \u2013 300 \u03bcm2, def 0.00 \u2013 0.01, are ratio 
            
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1.0 \u2013 1.1\n\n  
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1.0 \u2013 1.1\n\n  
            
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ize_Initial.rtdc,\nFigure1d_06_Beads_DefSize_Target.rtdc_**\n\nsample: 
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ize_Initial.rtdc,\nFigure1d_06_Beads_DefSize_Target.rtdc_**\n\nsample: 
            
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a mixture of two polyacrylamide microgel bead populations of 
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a mixture of two polyacrylamide microgel bead populations of 
            
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different\nstiffness, these beads were produced in house; sorted for 
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different\nstiffness, these beads were produced in house; sorted for 
            
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deformation and size\n\nsorting gates: size 95 \u2013 105 \u03bcm2, 
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deformation and size\n\nsorting gates: size 95 \u2013 105 \u03bcm2, 
            
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def 0.000 \u2013 0.016, are ratio 1.0 \u2013 1.1\n\n  
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def 0.000 \u2013 0.016, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_Figure2b_Blood_Initial_Inlet.rtdc_**\n\nsample: RBC-depleted 
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\n\n**_Figure2b_Blood_Initial_Inlet.rtdc_**\n\nsample: RBC-depleted 
            
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blood sample analysed in the inlet region of the chip\n\n  
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blood sample analysed in the inlet region of the chip\n\n  
            
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\n\n**_Figure2c_Blood_Initial_Channel.rtdc_**\n\nsample: RBC-depleted 
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\n\n**_Figure2c_Blood_Initial_Channel.rtdc_**\n\nsample: RBC-depleted 
            
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blood sample analysed in the usual ROI at the end of the\nconstricting 
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blood sample analysed in the usual ROI at the end of the\nconstricting 
            
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channel\n\n  \n\n**_Figure2d_Blood_Target_RBCs.rtdc_**\n\nsample: 
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channel\n\n  \n\n**_Figure2d_Blood_Target_RBCs.rtdc_**\n\nsample: 
            
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RBC-depleted blood, sorted RBCs\n\nsorting gates: size 25 \u2013 65 
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RBC-depleted blood, sorted RBCs\n\nsorting gates: size 25 \u2013 65 
            
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\u03bcm2, def 0.16 \u2013 0.40, are ratio 1.0 \u2013 1.1\n\n  
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\u03bcm2, def 0.16 \u2013 0.40, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_Figure2e_Blood_Target_ly.rtdc_**\n\nsample: RBC-depleted blood, 
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\n\n**_Figure2e_Blood_Target_ly.rtdc_**\n\nsample: RBC-depleted blood, 
            
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sorted lymphocytes\n\nsorting gates: size 25 \u2013 45 \u03bcm2, def 
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sorted lymphocytes\n\nsorting gates: size 25 \u2013 45 \u03bcm2, def 
            
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0.00 \u2013 0.10, are ratio 1.0 \u2013 1.1\n\n  
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0.00 \u2013 0.10, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_Figure2f_Blood_Target_my.rtdc_**\n\nsample: RBC-depleted blood, 
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\n\n**_Figure2f_Blood_Target_my.rtdc_**\n\nsample: RBC-depleted blood, 
            
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sorted myeloid cells\n\nsorting gates: size 53 \u2013 120 \u03bcm2, 
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sorted myeloid cells\n\nsorting gates: size 53 \u2013 120 \u03bcm2, 
            
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def 0.00 \u2013 0.15, are ratio 1.0 \u2013 1.1\n\n  
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def 0.00 \u2013 0.15, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_Figure3_Blood_Initial.rtdc, 
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\n\n**_Figure3_Blood_Initial.rtdc, 
            
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Figure3_Blood_Target_neuBr.rtdc_**\n\nsample: RBC-depleted blood, 
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Figure3_Blood_Target_neuBr.rtdc_**\n\nsample: RBC-depleted blood, 
            
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brightness-based neutrophils sorting*\n\nsorting gates: size 56 \u2013 
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brightness-based neutrophils sorting*\n\nsorting gates: size 56 \u2013 
            
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100 \u03bcm2, brightness 75 \u2013 78, are ratio 1.0 \u2013 1.1\n\n  
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100 \u03bcm2, brightness 75 \u2013 78, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_Figure4_Blood_DNN_Training(1).rtdc, 
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\n\n**_Figure4_Blood_DNN_Training(1).rtdc, 
            
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Figure4_Blood_DNN_Training(2).rtdc_**\n\nsample: RBC-depleted blood, 
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Figure4_Blood_DNN_Training(2).rtdc_**\n\nsample: RBC-depleted blood, 
            
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datasets for training of deep neural network\n(DNN)*\n\n  
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datasets for training of deep neural network\n(DNN)*\n\n  
            
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\n\n**_Figure4_Blood_DNN_Validation.rtdc_**\n\nsample: RBC-depleted 
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\n\n**_Figure4_Blood_DNN_Validation.rtdc_**\n\nsample: RBC-depleted 
            
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blood, dataset for validation of trained DNN*\n\n  
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blood, dataset for validation of trained DNN*\n\n  
            
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\n\n**_Figure4d_Blood_Initial.rtdc, 
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\n\n**_Figure4d_Blood_Initial.rtdc, 
            
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Figure4e_Blood_Target_neuDNN.rtdc_**\n\nsample: RBC-depleted blood, 
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Figure4e_Blood_Target_neuDNN.rtdc_**\n\nsample: RBC-depleted blood, 
            
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DNN-based neutrophil sorting*\n\n  \n\n*for identification of 
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DNN-based neutrophil sorting*\n\n  \n\n*for identification of 
            
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neutrophils (CD66+/CD14\u2212), these samples were stained with 
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neutrophils (CD66+/CD14\u2212), these samples were stained with 
            
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APC-conjugated anti-human CD14 (dilution 1:20, #17- 809 0149-42, 
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APC-conjugated anti-human CD14 (dilution 1:20, #17- 809 0149-42, 
            
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eBioscience, CA, USA; recorded with FL-3 channel) and PE-conjugated 
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eBioscience, CA, USA; recorded with FL-3 channel) and PE-conjugated 
            
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anti-human CD66a/c/e (dilution 1:40, 810 #34303, BioLegend, CA, USA; 
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anti-human CD66a/c/e (dilution 1:40, 810 #34303, BioLegend, CA, USA; 
            
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recorded with FL-2 channel); for cross-talk compensation of the 
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recorded with FL-2 channel); for cross-talk compensation of the 
            
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fluorescence signal in channels FL-2 and FL-3 for these samples 
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fluorescence signal in channels FL-2 and FL-3 for these samples 
            
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introduce the following correction factor in the ShapeOut software: 
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introduce the following correction factor in the ShapeOut software: 
            
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spill from channel 2 to 3 = 0.025, spill from channel 3 to 2 = 
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spill from channel 2 to 3 = 0.025, spill from channel 3 to 2 = 
            
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0.100\n\n  \n\n**_SFigure2a_BeadMix_RT-FDC.rtdc, 
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0.100\n\n  \n\n**_SFigure2a_BeadMix_RT-FDC.rtdc, 
            
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igure2b_BeadMix_soRT-FDC.rtdc,\nSFigure2c_BeadMix_FC.fcs_**\n\nsample: 
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igure2b_BeadMix_soRT-FDC.rtdc,\nSFigure2c_BeadMix_FC.fcs_**\n\nsample: 
            
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a mixture of 4 bead types with different diameter, each made 
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a mixture of 4 bead types with different diameter, each made 
            
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of\ndifferent material:\n\n9.78 \u00b1 0.15 \u03bcm melamine beads 
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of\ndifferent material:\n\n9.78 \u00b1 0.15 \u03bcm melamine beads 
            
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(MF-FluoBlau-L948),\n\n13.79 \u00b1 0.59 \u03bcm silica beads 
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(MF-FluoBlau-L948),\n\n13.79 \u00b1 0.59 \u03bcm silica beads 
            
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(SiO2-F-L3519-1),\n\n15.21 \u00b1 0.31 \u03bcm polystyrene beads 
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(SiO2-F-L3519-1),\n\n15.21 \u00b1 0.31 \u03bcm polystyrene beads 
            
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(PS/Q-F-KM194),\n\n17.23 \u00b1 0.24 \u03bcm poly(methyl methacrylate) 
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(PS/Q-F-KM194),\n\n17.23 \u00b1 0.24 \u03bcm poly(methyl methacrylate) 
            
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beads (PMMM-F-B1423),\n\nall purchased from Microparticles, 
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beads (PMMM-F-B1423),\n\nall purchased from Microparticles, 
            
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Germany.\n\n  \n\n**_SFigure3a_Beads_FLDef_Initial.rtdc, 
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Germany.\n\n  \n\n**_SFigure3a_Beads_FLDef_Initial.rtdc, 
            
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SFigure3a_Beads_FLDef_Target.rtdc_**\n\nsample: a mixture of 
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SFigure3a_Beads_FLDef_Target.rtdc_**\n\nsample: a mixture of 
            
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fluorescent and non-fluorescent polyacrylamide beads with\ndifferent 
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fluorescent and non-fluorescent polyacrylamide beads with\ndifferent 
            
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mechanical properties, these beads were produced in house; sorted\nfor 
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mechanical properties, these beads were produced in house; sorted\nfor 
            
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fluorescence and deformation\n\nsorting gates: FL-1 maximum 1000 
114
fluorescence and deformation\n\nsorting gates: FL-1 maximum 1000 
            
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\u2013 10000, def 0.01 \u2013 0.02, are ratio 1.0 \u2013 1.1\n\n  
115
\u2013 10000, def 0.01 \u2013 0.02, are ratio 1.0 \u2013 1.1\n\n  
            
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\n\n**_SFigure3b_Beads_BrSize_Initial.rtdc, 
116
\n\n**_SFigure3b_Beads_BrSize_Initial.rtdc, 
            
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SFigure3b_Beads_BrSize_Target.rtdc_**\n\nsample: a mixture of 4 bead 
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SFigure3b_Beads_BrSize_Target.rtdc_**\n\nsample: a mixture of 4 bead 
            
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types as in SFigure2; sorted for brightness and\nsize\n\nsorting 
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types as in SFigure2; sorted for brightness and\nsize\n\nsorting 
            
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gates: size 160 \u2013 240 \u03bcm2, brightness 75 \u2013 85, are 
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gates: size 160 \u2013 240 \u03bcm2, brightness 75 \u2013 85, are 
            
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ratio 1.0 \u2013 1.1\n\n  
120
ratio 1.0 \u2013 1.1\n\n  
            
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ize_Initial.rtdc,\nSFigure4b_Kc167_HL60_Size_Target.rtdc_**\n\nsample: 
121
ize_Initial.rtdc,\nSFigure4b_Kc167_HL60_Size_Target.rtdc_**\n\nsample: 
            
122
a mixture of Kc167 _Drosophila_ cells and HL60/S4 human 
122
a mixture of Kc167 _Drosophila_ cells and HL60/S4 human 
            
123
promyelocytic\nleukaemia cells, sorted for size\n\nsorting gates: size 
123
promyelocytic\nleukaemia cells, sorted for size\n\nsorting gates: size 
            
124
25 \u2013 77 \u03bcm2, def 0.00 \u2013 0.15, are ratio 1.0 \u2013 
124
25 \u2013 77 \u03bcm2, def 0.00 \u2013 0.15, are ratio 1.0 \u2013 
            
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1.1\n\n  \n\n**_SFigure5a_RBC_Def_Initial.rtdc, 
125
1.1\n\n  \n\n**_SFigure5a_RBC_Def_Initial.rtdc, 
            
126
5b_RBC_Def_Target1.rtdc,\nSFigure5c_RBC_Def_Target2.rtdc_**\n\nsample: 
126
5b_RBC_Def_Target1.rtdc,\nSFigure5c_RBC_Def_Target2.rtdc_**\n\nsample: 
            
127
blood anticoagulated with citrate, RBCs sorted for 
127
blood anticoagulated with citrate, RBCs sorted for 
            
128
deformation\n\nsorting gates: def 0.15 \u2013 0.40 (Target1) / def 
128
deformation\n\nsorting gates: def 0.15 \u2013 0.40 (Target1) / def 
            
129
0.00 \u2013 0.10 (Target2), are\nratio 1.0 \u2013 1.2\n\n  
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0.00 \u2013 0.10 (Target2), are\nratio 1.0 \u2013 1.2\n\n  
            
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\n\n**_SFigure7_AFM_AfterSorting.xlsx_**\n\nsample: HL60/S4 cells 
130
\n\n**_SFigure7_AFM_AfterSorting.xlsx_**\n\nsample: HL60/S4 cells 
            
131
collected after sorting experiment; the initial sample\nwas prepared 
131
collected after sorting experiment; the initial sample\nwas prepared 
            
132
as for the soRT-FDC experiment but not loaded onto the chip, 
132
as for the soRT-FDC experiment but not loaded onto the chip, 
            
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the\ndefault sample was collected in the default outlet, i.e., run 
133
the\ndefault sample was collected in the default outlet, i.e., run 
            
134
through the\nsorting chip but not exposed to SSAW, and the target 
134
through the\nsorting chip but not exposed to SSAW, and the target 
            
135
sample corresponds to\ncells exposed to SSAW; reported values 
135
sample corresponds to\ncells exposed to SSAW; reported values 
            
136
correspond to apparent Young\u2019s modulus\nestimated from AFM 
136
correspond to apparent Young\u2019s modulus\nestimated from AFM 
            
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indentation experiments\n\n  \n\n",
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indentation experiments\n\n  \n\n",
            
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