The TissueFAXS Plus (Tissue Gnostics, Vienna, Austria) automated microscopy workstation, which contains an eight‐slide ultra‐precise motorized stage and utilizes a Zeiss Z2 Axioimager microscope (Zeiss, Oberkocken, Germany), was used for automated image acquisition. First, a DAPI preview image is captured with a ×10 objective to allow for appropriate orientation. Next, the TMA spots are identified and images are captured with a ×40 oil objective using the DAPI, GFP, Cy3, and Cy5 filters. An autofocus algorithm in the DAPI filter and the extended focus parameter by capturing three steps above and below (step size = 0.8 μm) was utilized. An entire TMA with 400 spots can be imaged in ~14 h, which is faster than other current imaging modalities [13 (link)]. For image analysis, a separate high‐performance workstation with the TissueQuest software module to analyze the fluorescent images with precise nuclear segmentation was used [12 (link), 14 (link)]. A region of interest is set (e.g. stroma or cancer) and processed for nuclear segmentation. If required, exclusion regions were set to exclude benign prostate glands.