Multiplexed tissue biomarker imaging.

Description of the technology The detection of structural and functional proteins in cells within the tumor microenvironment in tissue samples is achieved by immunolabeling with specific antibodies. These target proteins are visualized with the subsequent application of either an enzymatic reaction that induces chromogen precipitation at the site of antibody-antigen binding (immunoenzyme method) or by using fluorescent dyes (e.g., fluorophores, fluorescent quantum dot nanocrystals) conjugated either to primary or secondary antibodies (direct or indirect immunofluorescence, respectively). In order to maximize the amount of information that can be acquired from the intact tumor anatomy as well as to delineate the spatial and temporal expression information, multiplexed staining approaches are required. Serial sections lack sufficiency due to the changing tissue morphology, which prevents the accurate identification of co-expression and lacks contextual assessment [1–3]. Advanced multiplexed immunofluorescence involving iterative stain and strip procedures can offer significant multiplexing of up to 30 markers [2] and through linear alignment can report on a single region of interest. Through a more standard immunoenzyme method, the Tyramide Signal Amplification (TSA) technique allows for serial application of multiple TSA fluorophores on a single tissue section. This technique can result in multiplexes of up to 7 fluorescent dyes, which can be effectively interrogated using a multispectral microscope [3], Fig. 1.