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Over the past 2 decades, hyperspectral imaging technologies have been adapted to address the need for molecule-specific identification in the biomedical imaging field. Applications have ranged from single-cell microscopy to whole-animal in vivo imaging and from basic research to clinical systems. Enabling this growth has been the availability of faster, more effective hyperspectral filtering technologies and more sensitive detectors. Hence, the potential for growth of biomedical hyperspectral imaging is high, and many hyperspectral imaging options are already commercially available. However, despite the growth in hyperspectral technologies for biomedical imaging, little work has been done to aid users of hyperspectral imaging instruments in selecting appropriate analysis algorithms. Here, we present an approach for comparing the effectiveness of spectral analysis algorithms by combining experimental image …