Journal: Nature Communications

Article Title: iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays

doi: 10.1038/s41467-023-40522-4

Figure Lengend Snippet: Designed for use with fluorescence microscopy images such that multiple components of a cell suspension can be simultaneously monitored, users adjust threshold values for any image color channel(s) where immunofluorescence signal is present. a Line graphs representing occlusion and accumulation over time are automatically generated. Here, CD41+ platelets and CD45+ white blood cells from sickle cell disease patient whole blood accumulate on an ibidi chamber device coated with collagen at a faster rate than healthy control whole blood ( n = 3 replicates). Data taken at ×20 magnification, scale bar represents 50 μm. b The application is designed to automatically generate a map of all signals present, e.g., the dimensions of a microfluidic device, shown here with a microvasculature-on-a-chip device designed to investigate the effect of crizanlizumab on sickle cell disease whole blood samples ( n = 1 experiment). Data taken at ×20, scale bar represents 200 μm. Percent occlusion ( c ) and accumulation rate ( d ) changes over the course of an experiment, showing microvascular occlusion instability. e A microchannel-specific application is available for spatial analysis of one or many straight microchannel portions of a microfluidic device. Data taken at ×20, scale bar represents 50 μm. f Spatial quantification of microvascular occlusion is automatically performed by calculating an occlusion percentage for each pixel point along the length of each microchannel. ML algorithms enable further analysis in microchannels by treating each x-coordinate and corresponding occlusion measurement from each channel as a data point. At the initial time course timepoint, t = 7 min, CD45+ white blood cells in SCD whole blood ( g ) and CD45+ white blood cells in SCD whole blood treated with drug crizanlizumab ( h ) occlude endothelialized microchannels to variable degrees at each point along the 32 analyzed microchannels. i CD45+ white blood cells in SCD whole blood predominantly occlude distal ends of microchannels at early timepoints, while CD45+ WBCs in SCD whole blood treated with crizanlizumab occlude proximal entry points of microchannels at early timepoints. Source data are provided as a Source data file.

Article Snippet: In order to facilitate perfusion, syringes were loaded with whole blood treated with 40 µg/ml corn trypsin inhibitor (CTI; Haematologic Technologies), Anti-CD45 Mouse Monoclonal Antibody (VWR) at a ratio of 1:100, Integrin alpha 2b/CD41 Antibody (VWR) at a ratio of 1:200, and 6 mM CaCl 2 .

Techniques: Fluorescence, Microscopy, Immunofluorescence, Generated

Journal: Nature Communications

Article Title: iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays

doi: 10.1038/s41467-023-40522-4

Figure Lengend Snippet: a Analysis windows are designed to be intuitively followed from left (inputs) to right (outputs), with the image processing steps as applied displayed in the center. Here, the microchannel analysis application from iCLOTS’ suite of multiscale microfluidic accumulation tools is shown with whole blood perfused through an in vitro microvasculature-on-a-chip microfluidic model. b The user uploads the desired number of microscopy images, time course microscopy series, or videomicroscopy files as inputs. These files are then automatically displayed on the screen. c Depending on the application and file type, users are guided through a series of windows facilitating the analyses of their data, such as choosing a region of interest (ROI, shown) or indicating immunofluorescence staining color channels present in a file. iCLOTS applications designed for fluorescence microscopy can accommodate up to three stains in separate channels: here, red indicates CD41+ platelets, green indicates CD45+ white blood cells, and blue indicates the endothelial cell layer. Data in this example is taken at ×100 magnification, scale bars represent 50 μm (left) and 10 μm (right). d Parameters, numerical factors that define how image processing algorithms should be applied, are typically simple, e.g., minimum and maximum cell area, or fluorescence signal threshold, as shown here. All parameters are adjusted interactively from a default value to best match the researcher’s specific dataset. In iCLOTS, pixel intensity values are understood to be arbitrary units. Effects of changing parameters are shown in real time to assist in gauging the appropriateness of selected values. e A button initiates the finalized analysis with algorithms customized by the selected parameters. Upon completion, graphical results appropriate for the application are automatically displayed, such as line graphs representing quantitative accumulation and occlusion values at each time point for each channel of interest, as seen in this example. f Users may export any of the outputs generated by iCLOTS, including tabular data as an Excel file, graphical results as .png images, or the initial imaging dataset as transformed by the image processing algorithms and/or labeled with indices.

Article Snippet: In order to facilitate perfusion, syringes were loaded with whole blood treated with 40 µg/ml corn trypsin inhibitor (CTI; Haematologic Technologies), Anti-CD45 Mouse Monoclonal Antibody (VWR) at a ratio of 1:100, Integrin alpha 2b/CD41 Antibody (VWR) at a ratio of 1:200, and 6 mM CaCl 2 .

Techniques: In Vitro, Microscopy, Immunofluorescence, Staining, Fluorescence, Generated, Imaging, Transformation Assay, Labeling