Creating Virtual Hematoxylin and Eosin Images using Samples Imaged on a Commercial CODEX Platform

Multiparametric fluorescence imaging through CODEX allows the simultaneous imaging of many biomarkers in a single tissue section. While the digital fluorescence data thus obtained can provide highly specific characterizations of individual cells and microenvironments, the images obtained are different from those usually interpreted by pathologists (i.e., hematoxylin and eosin [H&E] slides and 3,3′-diaminobenzidine-stained immunohistochemistry slides). Having the fluorescence data plus coregistered H&E or similar data could facilitate the adoption of multiparametric imaging into regular workflows, as well as facilitate the transfer of algorithms and machine learning previously developed around H&E slides. Since commercial CODEX instruments do not produce H&E-like images by themselves, we developed a staining protocol and associated image processing to make “virtual H&E” images that can be incorporated into the CODEX workflow. While there are many ways to achieve virtual H&E images, including the use of a fluorescent nuclear stain and tissue autofluorescence to simulate eosin staining, we opted to combine fluorescent nuclear staining (through 4′,6-diamidino-2-phenylindole) with actual eosin staining. We also output images derived from fluorescent nuclear staining and autofluorescence images for additional evaluation.