Fig. 2: All iCLOTS applications follow a common, easy-to-use interactive format.

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.