Fig. 1: Overview of Hyperplexed Immunofluorescence Imaging (HIFI) Workflow.

Experimental workflow of cyclic immunofluorescence staining, followed by image processing, alignment, and registration to create 45+ dimensional images across whole-slide sections. Specific domains within HIFI images of tumors were automatically annotated using trained machine-learning classifiers, and individual cells were segmented with deep-learning object detection. Single-cell objects were annotated as individual cell types with semi-supervised classification and mapped back onto images to create highly-annotated digital pathology images. Images were analyzed for regional cellular composition and spatial organizational analysis. The HIFI workflow is scalable to over 100 simultaneous sections for high-throughput spatial experiments.