Immunohistochemistry

Multi-label microscopy without spectral or spatial cross-talk

Until now, double- or triple-staining of samples on a single slide in standard brightfield (non-fluorescence) microscopy using colored labels (chromagens) was generally unsatisfactory because of the potential for both spatial overlapping and muddy mixtures of colors. The conventional approach to multiplexing in brightfield has been to cut serial sections and stain each one with a different antibody and a single colored label. However with this approach, information on multiple markers is not available on a cell-by-cell basis. Our Nuance EX and Nuance FX enables single-cell-level multiplexed imaging of standard IHC chromogenic labels – such as DAB, Fast Red, and others – in the same cellular compartment, even in the presence of a general counterstain. Each label can be unmixed into its own channel without bleed-through. Multispectral imaging has utility even for single-stained samples, by helping to accurately separate the specific label signals from the background counterstain. Imaging time for a brightfield multispectral acquisition is only a few seconds at most, and the unmixing can be performed in less than one second. Thus, the Nuance multispectral camera – easily mounted onto virtually all existing microscopes – can deliver quantitative results almost instantly.