725_Registered_Images

All modalities were aligned to IMS data on a per pixel basis, by linking of the theoretical pixel location in each image to the laser ablation marks made by the IMS laser in a raster across the tissue. Both single-section multi-image, and multi-section multi-image workflows were used in registration16. Images were exported as high-resolution jpeg, tiff, mha, or png files. All images were registered to a pixel map, or theoretical grid, generated from a coordinate extraction from flex imaging, using a padding of 10 to add area to the images around the edges (Supplemental Fig. 6, Supplemental Table 4). We used external fiducial marks (rectangular regions external to the tissue) to reduce inaccuracy that could be introduced in determination of the exact center of the ablation mark for alignment to the theoretical pixel map of tissue at high spatial resolution (Supplemental Table 4). These fiducial benchmarks were acquired at half the spatial resolution and were used to enable high accuracy alignment with the center of the theoretical pixel map obtained from the spot list map. Laser ablation sites post-acquisition as well as external fiducial marks were recorded in the FITC channel. Masks in FIJI were used to increase accuracy of registration 43, and image figures were created using FIJI, using its pre-programmed macros.