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. CRi's Nuance 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.
Click on images to enlarge
Image 1: ER-PR co-localization with histogram.
Image 2: Multiple cell-cycle markers in breast tissue. (C Van der Loos, Univ Amsterdam)
Image 3: Unmixed overlapping chromogenic dyes. (Sample courtesy P Lee, Stanford Univ)
Spectral unmixing of DAB from hematoxylin-and-eosin-stained specimen. Tonsil (FFPE, 20X) stained for T-cells (CD3) with a brown chromogen (DAB) and counterstained with both hematoxylin AND eosin. The DAB signal is unmixed, recolored green and shown superimposed on the H&E stain, and then removed, to reveal the underlying higher-contrast H&E-stained morphology unobscured by the presence of the DAB layer.
Sample courtesy C. van der Loos, AMC, Amsterdam.
