Fig. 1.

Outline of the workflow used to delineate area occupied by USPIO and cell granularity, based on calculated cellular features on the Amnis ImageStreamX Mark II. A: Single cells were selected by plotting area against aspect ratio assuming that most non-adherent macrophages were round in shape. Focused cells were selected by plotting gradient against contrast and selecting cells with highest values of both features. SSC was measured to assess granularity. B: Both custom-generated (green) and the ‘adaptive erode’ mask (no. 83) were used to identify USPIO-free intracellular area. USPIOs were identified using ‘intensity’ mask with the pixel intensity range (0–600). Intracellular USPIOs were identified by selecting overlapping cell- and USPIO-occupied areas. C: Images from two BF channels were included in the final calculation, given the slight difference between these images (white arrows). D: Calculation used to identify the relative areas of the cells occupied by USPIO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)