Fig. 1.

MxIF data acquisition, image processing, and data analysis scheme. (A) In the laboratory, background autofluorescence (AF) tissue images are acquired before subsequent application of fluorescent dye-conjugated primary antibodies. Stained images are then acquired, followed by dye inactivation and restaining with new directly conjugated antibodies. New images are acquired, and the cycle is repeated until all target antigens are exhausted. Times associated with each step are indicated. (B) Stained images are registered, background AF is removed from each stained image, and images are segmented into epithelial and stromal regions, followed by identification of individual cells and corresponding plasma membrane, cytoplasm, and nuclear regions. Pixel-level data are summarized in cellular features, which is subsequently queried in data analysis (C). Data analysis can consist of a variety of statistical and visual explorations. In this work, we use K-median clustering to group cells with similar mTOR activity.