Unfortunately, hematoxylin does not have similar properties. Since eosin is fluorescent and compatible with our C圓 filter set, it is simple to image within the existing system. Similar to another report, we leave the coverslip in place on the instrument and use the fluid well created by the coverslip, gaskets, and coverslip holder to stain with eosin. We therefore developed our own protocol for creating images using CODEX coverslips that closely resemble traditional H&E-stained tissue sections (“virtual H&E” images, see for examples ). However, their report lacked further details regarding their approach. Other investigators have reported staining and imaging the coverslips with eosin at the end of the regular CODEX imaging process while the coverslips are still in place (i.e., still between the gaskets).
#IMAGEJ QUANTIFY HE STAINING CRACKED#
In our experiments, half or more of the coverslips cracked in the process of removing them from the gaskets. A coverslip is required since the tissue is imaged on an inverted microscope, and microscope slides would be too thick for the optical objectives used in the usual system. The coverslip is sandwiched between two gaskets (or between a gasket and microscope stage adapter in the updated version) and serves as the bottom of a well, through which fluids are passed during the cyclical staining and imaging process that makes highly multiparametric imaging possible. In the CODEX system, tissue is attached to a glass coverslip rather than a microscope slide. As our work involves a commercial CODEX instrument, we had hoped to perform regular H&E staining on the same tissue used for fluorescence imaging. In order to incorporate multiparametric fluorescence imaging in clinical use, it is therefore desirable to, in addition to the fluorescence images, have H&E-stained images to compare with fluorescence images. As machine learning algorithms for automated image interpretation in pathology have thus far also focused primarily on H&E-stained slides, transfer of some of that learning is desirable and could be facilitated by co-registered H&E staining in addition to the multiparametric biomarker data. Given the critical importance of proper interpretation of histology for clinical care, pathologists are understandably hesitant when asked to interpret histology using unfamiliar staining and imaging approaches. Pathologists, however, are trained to interpret histology using primarily hematoxylin and eosin (H&E) stained slides and immunohistochemically stained slides using 3,3⍰-diaminobenzidine (DAB) and hematoxylin counterstain. Some academic pathology labs, and in particular, hematopathology divisions, are also taking notice, with the possibility of translation to clinical use becoming more likely as the methods are further developed. CODEX imaging is available as a commercial system from Akoya and is one such multiparametric imaging system. Highly multiparametric fluorescence imaging is gaining increased use in research laboratories, particularly those involved in tumor microenvironments and related immunological research wherein the accurate co-localization of many markers within individual and adjacent cells is important. We also output images derived from fluorescent nuclear staining and autofluorescence images for additional evaluation. While there are many ways to achieve virtual H&E images, including use of a fluorescent nuclear stain and tissue autofluorescence to simulate eosin staining, we opted to combine fluorescent nuclear staining (via DAPI) with actual eosin staining. 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.
#IMAGEJ QUANTIFY HE STAINING PLUS#
Having the fluorescence data plus co-registered H&E or similar data could facilitate adoption of multiparametric imaging into regular workflows, as well as facilitate the transfer of algorithms and machine learning previous developed around H&E slides. 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., H&E slides and DAB-stained immunohistochemistry slides). Multiparametric fluorescence imaging via CODEX allows the simultaneous imaging of many biomarkers in a single tissue section.